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Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine
Author and Curator: Larry H Bernstein, MD, FCAP
and
Curator: Aviva Lev-Ari, PhD, RN
This document is entirely devoted to medical and surgical therapies that have made huge strides in
simplification of interventional procedures,
reduced complexity, resulting in procedures previously requiring surgery are now done, circumstances permitting, by medical intervention.
This revolution in cardiovascular interventional therapy is regenerative medicine. It is regenerative because it is largely driven by
the introduction into the impaired vasculature of an induced pleuripotent cell, called a stem cell, although
the level of differentiation may not be a most primitive cell line.
There is also a very closely aligned development in cell biology that extends beyond and including vascular regeneration that is called synthetic biology. These developments have occurred at an accelerated rate in the last 15 years. The methods of interventional cardiology were already well developed in the mid 1980s. This was at the peak of cardiothoracic bypass surgery.
Research on the endothelial cell,
endothelial cell proliferation,
shear flow in small arteries, especially at branch points, and
endothelial-platelet interactions
led to insights about plaque formation and vessel thrombosis.
Much was learned in biomechanics about the shear flow stresses on the luminal surface of the vasculature, and there was also
the concomitant discovery of nitric oxide,
oxidative stress, and
the isoenzymes of nitric oxide synthase (eNOS, iNOS, and nNOS).
It became a fundamental tenet of vascular biology that
atherogenesis is a maladjustment to oxidative stress not only through genetic, but also
non-genetic nutritional factors that could be related to the balance of omega (ω)-3 and omega (ω)-6 fatty acids,
a pro-inflammatory state that elicits inflammatory cytokines, such as, interleukin-6 (IL6) and c-reactive protein(CRP),
insulin resistance with excess carbohydrate associated with type 2 diabetes and beta (β) cell stress,
excess trans- and saturated fats, and perhaps
the now plausible colonic microbial population of the gastrointestinal tract (GIT).
There is also an association of abdominal adiposity,
including the visceral peritoneum, with both T2DM and with arteriosclerotic vessel disease,
which is presenting at a young age, and has ties to
the effects of an adipokine, adiponectin.
Much important work has already been discussed in the domain of cardiac catheterization and research done to
prevent atheroembolization.and beyond that,
research done to implant an endothelial growth matrix.
Even then, dramatic work had already been done on
the platelet structure and metabolism, and
this has transformed our knowledge of platelet biology.
The coagulation process has been discussed in detailed in a previous document. The result was the development of a
new class of platelet aggregation inhibitors designed to block the activation of protein on the platelet surface that
is critical in the coagulation cascade.
In addition, the term long used to describe atherosclerosis, atheroma notwithstanding, is “hardening of the arteries”. This is particularly notable with respect to mid-size arteries and arterioles that feed the heart and kidneys. Whether it is preceded by or develops concurrently with chronic renal insufficiency and lowered glomerular filtration rate is perhaps arguable. However, there is now a body of evidence that points to
a change in the vascular muscularis and vessel stiffness, in addition to the endothelial features already mentioned.
This has provided a basis for
targeted pharmaceutical intervention, and
reduction in salt intake.
So we have a group of metabolic disorders, which may alone or in combination,
lead to and be associated with the long term effects of cardiovascular disease, including
congestive heart failure.
This has been classically broken down into forward and backward failure,
depending on decrease outflow through the aorta (ejection fraction), or
decreased venous return through the vena cava,
which involves increased pulmonary vascular resistance and decreased return into the left atrium.
This also has ties to several causes, which may be cardiac or vascular. This document, as the previous, has four pats. They are broadly:
Stem Cells in Cardiovascular Diseases
Regenerative Cell and Molecular Biology
Therapeutics Levels In Molecular Cardiology
Research Proposals for Endogenous Augmentation of circulating Endothelial Progenitor Cells (cEPCs)
As in the previous section, we start with the biology of the stem cell and the degeneration in cardiovascular diseases, then proceed to regeneration, then therapeutics, and finally – proposals for augmenting therapy with circulating endogenous endothelial progenitor cells (cEPCs).
neurohumoral activity and vesicles vital and essential for all functions related to
cell movement,
migration, and
contraction.
Calmodulin and Protein Kinase C Increase Ca–stimulated Secretion by Modulating
Membrane-attached Exocytic Machinery
YA Chen, V Duvvuri, H Schulmani, and RH.Scheller‡
From the ‡Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology,
and the Department of Neurobiology, Stanford University School of Medicine, Stanford, CA
The molecular mechanisms underlying the Ca2+ regulation of hormone and neurotransmitter release
are largely unknown.
Using a reconstituted [3H]norepinephrine release assay in permeabilized PC12 cells, we found
essential proteins that support the triggering stage of Ca2+-stimulated exocytosis
are enriched in an EGTA extract of brain membranes.
Fractionation of this extract allowed purification of two factors that stimulate secretion
in the absence of any other cytosolic proteins.
These are calmodulin and protein kinase Ca (PKCa). Their effects on secretion were
confirmed using commercial and recombinant proteins.
Calmodulin enhances secretion
in the absence of ATP, whereas
PKC requires ATP to increase secretion, suggesting that
phosphorylationis involved in PKC-mediated stimulation
but not calmodulin mediated stimulation.
Both proteins modulate
The half-maximal increase was elicited by 3 nM PKC and 75 nM calmodulin.
These results suggest that calmodulin and PKC increase Ca2+-activated exocytosis by
directly modulating the membrane- or cytoskeleton-attached exocytic machinery downstream of Ca2+ elevation.
The abbreviations used are:
NE, norepinephrine; PKC, protein kinase C; CaM, calmodulin; SNAP-25, synaptosome-associated protein of 25 kDa; CAPS, calcium-dependent activator protein for secretion; SNARE, SNAP (soluble N-ethylmaleimide-sensitive factor attachment proteins) receptor; CaMK, Ca2+/calmodulin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; AMP-PNP, adenosine 59-(b,g- imido) triphosphate; HA, hydroxyapatite
*This work was supported in part by Conte Center Grant MH48108. The costs of publication of
this article were defrayed in part by the payment of page charges. This article has been marked
“advertisement” in accordance with 18 U.S.C. Section 1734.
The molecular mechanisms of presynaptic vesicle release have been extensively examined
by a combination of
biochemical,
genetic, and
electrophysiological techniques.
A series of protein-protein interaction cascadeshave been proposed to lead to vesicle
docking and fusion (1–3). The SNARE protein family, including
syntaxin, SNAP-25, and vesicle-associated membrane protein
(VAMP, also called synaptobrevin),
plays an essential role in promoting membrane fusion, and
is thought to comprise the basic fusion machinery (4, 5).
In Ca2+-stimulated exocytosis, many additional proteins are important in the Ca2+ regulation
of the basic membrane trafficking apparatus.Calcium
not only triggers rapid fusion of release-competent vesicles, but is also involved in
earlier processes which replenish the pool of readily releasable vesicles (6).
Furthermore, it appears to be critical in initiating several forms of synaptic plasticity including
post-tetanic potentiation (7).
The molecular mechanisms by which Ca2+ regulates these processes is not well understood.
PC12 cells have often been utilized to study Ca2+-activated exocytosis, as
they offer a homogeneous cell population that possesses the same basic exocytic machinery as neurons (8).
In this study, we used an established cracked cell assay, in which
[3H]norepinephrine (NE)1 labeled PC12 cells are
permeabilized by mechanical “cracking” and
then reconstituted for secretion of NE in the presence of test proteins (9).
Transmitter-filled vesicles and intracellular cytoskeletal structures
remain intact in these cells,
while cytosolic proteins leak out (10).
These cracked cells readily release NE upon addition of
ATP,
brain cytosol, and
1 mM free Ca2+
at an elevated temperature.
We term this a “composite assay,” as
all essential components are added into one reaction mixture.
Alternatively, cracked cells can be
first primed with cytosol and ATP, washed, then
reconstituted for NE release with cytosol and Ca2+ (11).
This sequential priming-triggering protocol is useful
for determining whether a protein acts early or late in the exocytic pathway, and
whether its effect is dependent on Ca2+ or ATP.
This semi-intact cell system serves as
a bridge between an in vitro system comprised of purified components, and
electro-physiological systems that monitor release in vivo.
It provides information on protein functions in a cell with an intact membrane infrastructure while being easily manipulatable.
Ca2+ regulation by membrane depolarization is no longer a concern, as intra-cellular Ca2+ concentrationcan be controlled by a buffered solution.
Indirect readout of neurotransmitter release using a postsynaptic cell is replaced by
direct readout of [3H]NEreleased into the buffer.
Complications associated with interpreting overlapping
exo- and endocytotic signals are also eliminated as only one round of exocytosis is measured.
Finally, concentration estimates are likely to be accurate, since
added compounds do not need to diffuse long distances along axons and dendrites to their sites of action.
Using this assay, several proteins required for NE release have been purified from rat brain cytosol, including
phosphatidyl-inositol transfer protein (12),
phosphatidylinositol-4-phosphate 5-kinase (13), and
calcium-dependent activator protein for secretion (CAPS) (9).
The validity of the cracked cell system is confirmed by the finding that
phosphatidylinositol transfer protein and CAPS are mammalian homologues of
yeast SEC14p (12) and
nematode UNC31p, respectively (14),
both proteins involved in membrane trafficking (15, 16).
Calmodulin is the most ubiquitous calcium mediator in eukaryotic cells, yet its involvement in membrane trafficking has not been well established. Some early studies showed
that calmodulin inhibitors (17–19), anti-calmodulin antibodies (20,21),
However, in other studies, calmodulin-binding peptides and an anti-calmodulin antibody led to the conclusionthat
calmodulin is only involved in endocytosis,
not exocytosis (23).
More recently, it was reported that
Ca2+/ calmodulin signals the completion of docking and
triggers a late step of homotypic vacuole fusion in yeast,
thus suggesting an essential role for Ca2+/calmodulin in constitutive intracellular membrane fusion (24).
If calmodulin indeed plays an important role in exocytosis,
a likely target of calmodulin is
Ca2+/calmodulin-dependent protein kinase II (CaMKII),
a multifunctional kinase that is found on synaptic vesicles (25) and
has been shown to potentiate neurotransmitter release (26, 27).
Another Ca2+ signaling molecule, PKC, has also been implicated in regulated exocytosis.
In various cell systems, it has been shown that
the phorbol esters stimulate secretion (28, 29).
It is usually assumed that phorbol esters effect on exocytosis is
through activation of PKC,
but Munc13-1 was recently shown to be a presynaptic phorbol ester receptor that enhances neurotransmitter release (30, 31),
which complicates the interpretation of some earlier reports. The mode of action of PKC remains controversial. There is evidence
that PKC increases the intracellular Ca2+ levels by modulating plasma membrane Ca2+ channels (32, 33),
that it increases the size of the release competent vesicle pool (34, 35), or
that it increases the Ca2+ sensitivity of the membrane trafficking apparatus (36).
no consensus on these issues has been reached.
PKC substrates that have been implicated in exocytosis include
SNAP-25 (37),
synaptotagmin (28),
CAPS (38), and
nsec1 (39).
It is believed that upon phosphorylation, these PKC substrates might
interact differently with their binding partners, which, in turn,
leads to the enhancement of exocytosis.
In addition, evidence is accumulating that PKC and calmodulin interfere with each others actions, as
PKC phosphorylation sites are embedded in the calmodulin-binding domains of substrates such as
neuromodulin and
neurogranin (40).
It is therefore possible that PKC could modulate exocytosis via
a calmodulin-dependent pathway by synchronously releasing calmodulin from storage proteins.
In this study, we fractionated an EGTA extract of brain membranes in order to identify active components that could reconstitute release in the cracked cell assay system. We identified calmodulin and PKCas two active factors. Thus, we demonstrate that
calmodulin and PKC play a role in the Ca2+ regulation of exocytosis, and provide further insight into the mechanisms of their action.
DISCUSSION
In this study, we first identified an EGTA extract of brain membranes as a protein source
capable of reconstituting Ca2+- activated exocytosis in cracked PC12 cells.
EGTA only extracts a small pool of Ca2+-dependent membrane-associating proteins,
it served as an efficient initial purification step.
Further protein chromatography led to the identification of two active factors in the starting extract,
calmodulin and PKC,
which together accounted for about half of the starting activity.
Upon confirmation with commercially obtained proteins, this result unambiguously demonstrated
that calmodulin and PKC mediate aspects of Ca2+-dependent processes in exocytosis.
The finding that brain membrane EGTA extract alone is able
to replace cytosol in supporting Ca2+-triggered NE secretion
in PC12 cells is somewhat surprising. We suggest that the likely explanation is 2-fold.
some cytosolic proteins essential for exocytosis have a membrane-bound pool within permeabilized cells, whose activity might be sufficient for a normal level of exocytosis.
although the 100,000 3 g membrane pellet was washed to remove as many cytosolic proteins as possible,
some cytosolic proteins that associate with membranes in a
Ca2+-independent manner are probably present in the membrane EGTA extract.
these proteins likely constitute only a small percentage of the proteins in the extract, as
the characteristics of the activity triggered by the membrane extract
are quite different to that of cytosol (Fig. 2).
Using an unbiased biochemical purification method, we demonstrated that
calmodulin and PKC directly modulate the exocytotic machinery downstream of Ca2+ entry
they signal through membrane-attached molecules to increase exocytosis.
These targets include integral and peripheral membrane proteins, and cytosolic proteins that have a significant
membrane-bound pool. The modest stimulation by calmodulin and PKC on secretion might suggest a regulatory
role. However, it is also possible that some intermediates in their signaling pathways are in limiting amounts in the cell ghosts, so that their full effects were not observed. Half-maximal stimulation was obtained at
about 3 nM for PKC and
at about 75 nM for calmodulin.
This is consistent with an enzymatic role for PKC, and predicts a high-affinity interaction between
calmodulin and its substrate protein.
Ca2+ regulates exocytosis at many different levels. Prior studies indicated that Ca2+ signaling occurs in
the priming steps as well
as in triggering steps (49, 50).
Our priming triggering protocol
does not allow Ca2+-dependent priming events to be assayed, as EGTA is present in the priming reaction.
a different approach revealed the existence of both high and low Ca2+-dependent processes (Fig. 2).
this analysis indicated that late triggering events require high [Ca2+], whereas
early priming events require low [Ca2+]. If, as proposed, there is
a pronounced intracellular spatial and temporal [Ca2+] gradient from
the point of Ca2+ entry during depolarization (51),
perhaps triggered events occur closer to the point of Ca2+ entry,
while Ca2+-dependent priming events occur further away from the point of Ca2+ entry.
Distinct Ca2+ sensors at these stages might be appropriately tuned to different [Ca2+] to handle different tasks.
By analyzing the Ca2+ sensitivity of calmodulin-and PKC-stimulated release, we addressed the question of
whether calmodulin and PKC plays an early or a late role in vesicle release.
they both require relatively high [Ca2+] (Fig. 8B),
implying that calmodulin and PKC both mediate late triggering events, consistent with some earlier reports
(34, 52, 53).
In addition, it is interesting to note that PKC does not alter the calcium sensitivity of release in cracked cells, in contrast
to observations from the chick ciliary ganglion (36). Therefore, in contrast to previous electrophysiological studies (28),
we are able to limit the possible modes of PKC action in our system to an increase in the readily releasable vesicle pool or
release sites, or an enhancement of the probability of release of individual vesicles upon Ca2+ influx.
The experiments assaying the calcium sensitivity of release (Figs. 2, 5, and 8) demonstrated
a drop in release at very high [Ca2+].
This decline in release at high [Ca2+] has been previously reported (49, 51), and may represent
the true Ca2+ sensitivity of the Ca2+-sensing mechanism inside cells.
However, in our system, it could also be due to the activation of a variety of Ca2+ -activated proteases, as experiments are usually performed in the presence of crude extracts, which include unsequestered proteases.
What might the molecular targets of PKC and calmodulin be? An obvious calmodulin target molecule is CaMKII.
but calmodulin’s effect on exocytosis is ATP-independent, rendering the involvement of a kinase unlikely.
Calmodulin has also been shown to associate with
synaptic vesicles in a Ca2+-dependent fashion through synaptotagmin (54),
probably by binding to its C-terminal tail (55), and to promote Rab3A dissociation from synaptic vesicles (56).
However, there was little calcium-dependent binding of calmodulin to synaptotagmin
either on synaptic vesicles, in a bead binding assay with recombinant proteins,
or in a calmodulin overlay (data not shown).
In addition, using immobilized calmodulin, we did not see
significant Ca2+-dependent pull-down of synaptotagmin or Rab3A from rat brain extract (data not shown).
Recent work has suggested three other candidate targets for calmodulin, Munc13, Pollux, and CRAG (57).
Pollux has similarity to a portion of a yeast Rab GTPase-activating protein, while
CRAG is related to Rab3 GTPase exchange proteins.
Further work is required to investigate the role of their interactions with calmodulin in vivo.
The recent report that calmodulin mediates yeast vacuole fusion (24) is intriguing, as it raises the possibility that
calmodulin, a highly conserved ubiquitous molecule,
may mediate many membrane trafficking events.
It is not yet known if
the effector molecule of calmodulin is conserved or variable across species and different trafficking steps.
It is enticing to propose a model for Ca2+ sensing whereby
calmodulin is a high affinity Ca2+ sensor for both constitutive and regulated membrane fusion.
In the case of constitutive fusion, calmodulin may be the predominant Ca2+ sensor.
In the case of slow, non-local exocytosis of large dense core granules, an additional requirement for
the concerted actions of other molecule(s) that are better tuned to intermediate rises in [Ca2+] might exist.
At the highly localized sites of fast exocytosis of small clear vesicles where high [Ca2+] is reached,
specialized low affinity sensor(s) are likely required
in addition to calmodulin to achieve membrane fusion.
Therefore, although calmodulin participates in multiple types of vesicle fusion,
the impact of Ca2+ sensing by calmodulin on vesicle release likely varies.
Due to the fact that calmodulin binding to some proteins can be modulated by PKC phosphorylation, one might suspect
PKC action on exocytosis proceeds through a calmodulin-dependent pathway.
but the effects of calmodulin and PKC are additive within our system,
suggesting that PKC does not act by releasing calmodulin from a substrate
that functions as a calmodulin storage protein.
How Ca2+ regulates presynaptic vesicle release has been an open question for many years. By
identifying calmodulin and PKC as modulators of Ca21-regulated exocytosis and clarifying their functions,
we have extended our knowledge of the release process.
While the basic machinery of membrane fusion is becoming better understood,
the multiple effects of Ca2+ on exocytosis remain to be elucidated at the molecular level.
In addition, the ways that Ca2+ regulation may be important to
the mechanisms of synaptic plasticity in the central nervous system
EXPERIMENTAL PROCEDURES
Materials Rat Brain Cytosol Preparation Membrane EGTA Extract Preparation
Cracked Cell Assay
PC12 cells were maintained and [3H]NE labeled as described previously (11). Labeled cells were harvested by pipetting with ice-cold potassium glutamate buffer (50 mM Hepes, pH 7.2, 105 mM potassium glutamate, 20 mM potassium acetate, 2 mM EGTA) containing 0.1% bovine serum albumin. Subsequent manipulations were carried out at 0–4 °C. Labeled cells (1–1.5 ml/dish) were mechanically permeabilized passage through a stainless steel homogenizer. The cracked cells were adjusted to 11 mM EGTA and
incubated on ice for 0.5–3 h, followed by three washes in which
the cells were centrifuged at 800 3 g for 5 min and
resuspended in potassium glutamate buffer containing 0.1% bovine serum albumin.
Composite Assay
Each release reaction contains 0.5–1 million cracked cells, 1.5 mM free Ca2+, 2 mM MgATP,
and the protein solution to be tested in potassium glutamate buffer. Release reactions were initiated
by incubation at 30 °C and terminated by returning to ice. The supernatant of each reaction was
isolated by centrifugation at 2,500 3 g for 30 min at 4 °C, and the
released [3H]NE was quantified by scintillation counting (Beckman LS6000IC).
Cell pellets were dissolved in 1% Triton X-100, 0.02% azide and similarly counted. NE release
was calculated as a percentage of total [3H] in the supernatant.
Priming Assay
A priming reaction contains about
1–2 million cracked cells,
2 mM MgATP, and
the protein solution to be tested.
Ca2+ is omitted.
The primed cells were spun down, washed once with fresh potassium glutamate buffer, and
distributed into two triggering reactions, each containing
rat brain cytosol and free Ca2+
The triggering reaction was performed at 30 °C for 3 min, and
the NE release was measured
as in a composite assay.
Triggering Assay
Cracked cells were primed …, centrifuged, washed …, and
distributed into triggering reactions containing
1.5 mM free Ca2+ and the protein solution
To inhibit any ATP dependent activity in the triggering reaction, an
ATP depletion system of
hexokinase
MgCl2,
glucose or
a non-hydrolyzable ATP analogue AMPPNP
was added into the triggering reaction. NE release was measured as above.
Free Ca2+ Concentration Determination
The range of Ca2+free in the release reaction (Fig. 2B) was achieved
by adding Ca2+ into potassium glutamate buffer to reach final [Ca2+] total values of
0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, and 2.0 mM.
The pH of the reaction was 7.24 when no Ca2+ was added and
7.04 when 2.0 mM Ca2+ was added,
in the absence ofprotein extracts or cracked cells.
Fig. 2B. The range of [Ca21]free in the release reaction (Fig. 2B)
Free Ca2+ concentrations were determined using video microscopic
measurements of fura-2 fluorescence (41). [Ca2+]free was calculated from the equation
[Ca2+]free 5 Kd*3 (R 2 Rmin)/(Rmax 2 R)(42).
The values of Rmin, Rmax, and Kd* were determined in the following solutions:
potassium glutamate buffer (PGB) containing
8 x 3 10^6 cracked cells/ml, 2 mM MgATP (PGB+CC)
1) Rmin: PGB+CC and 10 mM additional EGTA;
2) Rmax: PBG+CC, and 10 mM total Ca2+;
3) Kd*: PGB+CC, 28 mM additional EGTA, and 18 mM total Ca2+, pH 7.2
([Ca2+]free 5 = 169 nM, determined in the absence of cells and MgATP
based on fura-2 calibration in cell-free solutions).
These solutions were
incubated at 37 °C ,
mixed with fura-2 pentapotassium salt
(100 mM; Molecular Probes, Eugene, OR), and
imaged.
This procedure allowed us to take into account
changes in fura-2 properties
caused by the presence of
permeabilized cells.
Duplicate measurements of the above range of [Ca2+] total gave
the following average [Ca2+] free values:
106, 146, 277, 462, 971, 1468, 1847, and 2484 nM.
Purification of Active Proteins
All procedures were carried out at 4 °C or on ice. Membrane EGTA extract of one or two bovine brain(s) was
filtered through cheesecloth and
loaded overnight onto a column packed with DEAE-Sepharose
CL-6B beads (Amersham Pharmacia Biotech).
The column was then
washed with (20 mM Hepes, pH 7.5, 0.25 mM sucrose, 2 mM EGTA, 1 mM dithiothreitol) and
step eluted with 10 column volumes of elution buffer
(20 mM Hepes, pH 7.5, 2 mM EGTA, 400 mM KCl, 1 mM dithiothreitol). 100 ml of every other fraction was
dialyzed overnight into PGB, and
tested in a composite release assay for activity.
The active fractions were pooled and dialyzed into zero salt buffer (20 mM Hepes, pH 7.5, 2 mM EGTA) and
batch bound to 10 ml of Affi-Gel Blue beads (Bio-Rad) or DyeMatrex-Green A beads (Amicon)
Blue beads were used in earlier experiments, and Green beads were used later to
specifically deplete CAPS, which was known to bind to Green beads (9).
The unbound material was
collected,
concentrated to about 2 ml using a Centriprep-10 (Amicon), and
loaded onto a 120-ml HiPrep Sephacryl S-200 gel filtration column
(Amersham Pharmacia Biotech).
Samples were run on the S-200 column in PGB at a flow rate of 7 ml/h.
10–50 ml of every other fraction was tested for
activity in the cracked cell composite assay, and
two peaks of activity were observed (Fig. 3).
The first peak of activity had a predicted molecular mass of 85 kDa.
The corresponding material was
adjusted to 10 mM potassium phosphate concentration (pH 7.2) and
loaded onto a 1-ml column packed with hydroxyapatite Bio-Gel HT (Bio-Rad).
The bound material was
eluted with a linear K-PO4 gradient from 10 to 500 mM (pH 7.2)
at a flow rate of about 0.1 ml/min, and
0.4–0.5-ml fractions were collected.
each fraction was dialyzed into PGB and
tested for activity.
The fractions were also analyzed by
SDS-PAGE and silver staining (Sigma silver stain kit).
The active material was concentrated and resolved
on an 8% poly-acrylamide gel.
Two Coomassie-stained protein bands that matched the activity profile (Fig. 6)
were excised from the gel,
sequenced by the Stanford PAN facility.
The two polypeptide sequences obtained from the upper band were:
LLNQEEGEYYNVPIXEGD
IRSTLNPRWDESFT.
The only bovine protein that contains both polypeptides is PKCa.
The four polypeptide sequences obtained from the lower band were:
YELTGKFERLIVGLMRPPAY,
LIEILASRTNEQIHQLVAA,
MLVVLLQGTREEDDVVSEDL, and
EMSGDVRDVFVAIVQSVK.
Based on these sequences, the protein band was
unambiguously identified to be bovine annexin VI.
The second S-200 peak has a predicted molecular mass of 25 kDa.
The corresponding material was
dialyzed into zero salt buffer
(20 mM Tris, pH 7.5, 1 mM EGTA) and
injected onto a Mono-Q HR 5/5 FPLC column
(Pharmacia).
The FPLC runwas performed at 18 °C at 1 ml/min and
1-ml fractions were collected
with a linear salt gradient from 0 to 1 M KCl over 71 ml.
The fractions containing proteins (determined by A280) were
dialyzed into PGB and
tested in the cracked cell assay.
Western Blot
Anti-calmodulin antibody and anti-PKC antibody were used, and
ECL (Amersham) was used for detection.
RESULTS
A Membrane EGTA Extract Supports NE Release
Brain cytosol, prepared as the supernatant of the brain homogenate,
effectively stimulates NE release
in the cracked cell assay (Fig. 1) as previously shown (9).
We wondered whether crude extracts other than cytosol
could support NE release, and we focused on
extractable peripheral membrane proteins.
We found that a salt or EGTA extract of brain membranes,
membranes defined as the
100,000 3 g pellet of the crude homogenate,
reconstituted secretion in the absence of cytosol.
the salt extract only slightly enhanced NE release
above background (data not shown), the
EGTA extract not only stimulated NE release to a high level,
similar to that supported by cytosol, but also
had a higher specific activity than cytosol (Fig. 1).
FIG. 1. The EGTA extract of brain membranes can support NE release in the absence of cytosol. Rat brain membrane EGTA extract (closed triangles) and rat brain cytosol (closed squares) were prepared as described under “Experimental Procedures.” NE release was measured in a composite reaction mixture of cracked cells, MgATP, Ca2+, and the indicated amount of crude extracts.
The ability of the membrane EGTA extract to support secretion is consistent with the fact that
following cracking, the cells are immediately extracted with EGTA, and are presumably
devoid of most membrane EGTA-extractable factors.
This also suggests that these factors, some of which are probably
Ca2+-dependent membrane-associating proteins,
participate in Ca2+- triggered exocytosis.
The Membrane EGTA Extract Is Enriched in Triggering Fators
NE release in cracked cells can be resolved into two sequential stages,
an ATP-dependent priming stage and
an ATP-independent Ca21-dependent triggering stage (11), and
proteins can be tested for activity in either stage.
An effect in priming indicates
an early role for the protein, and
an effect in triggering a late ATP-independent role.
Since the protein composition of the
membrane EGTA extract and cytosol are different,
we tested whether they had different activities
in the priming stage versus the triggering stage.
We found that the membrane EGTA extract is enrichedin factors that
act during triggering stage of NE release, as
the same amount of protein from the membrane EGTA extract as cytosol
gave a higher stimulation in the triggering assay, but
not in the priming assay (Fig. 2A).
Regular cytosol is prepared in a buffer containing 2 mM EGTA, and thus
presumably contains some of the proteins present in the membrane EGTA extract.
Cytosol prepared in the absence of EGTA showed an even lower specific activity
in the triggering assay compared with regular cytosol (Fig. 2A).
Identification of Calmodulin as an Active Triggering Factor in the EGTA Extract
Biochemical fractionation of the bovine brain membrane EGTA extract was carried out
to identify the active components capable of reconstituting NE release.
Activity was assayed in a composite reaction mixture containing
cracked cells,
ATP,
Ca2+, and
the test protein(s).
Except for the presence of bovine serum albumin in the basal buffer,
no other proteins were added to the cell ghosts except for the test protein(s).
Initial tests indicated that at least
part of the activity in the membrane EGTA extract binds to and
can be efficiently eluted from an anion exchanger and hydroxyapatite resin,
but does not bind to Amicon color resins.
The starting material was, therefore, sequentially purified using
DEAE, Affi-Gel Blue (or Matrex Green-A), and gel filtration chromotography.
Gel filtration fractionation indicated the presence of two peaks of activity with
predicted molecular masses of 25 and 85 kDa, respectively (Fig. 3).
FIG. 3. Gel filtration chromatography reveals two stimulatory factors in the membrane EGTA extract.
In order to purify the active component(s) in the membrane EGTA extract, the crude extract from one bovine brain was fractionated chromatographically (see Experimental Procedures” for details). Fractions from a Sephacryl S-200 gel filtration column were tested for their activity in stimulating NE release in the composite assay. The two activity peaks have predicted molecular masses of 85 and 25 kDa, respectively. The arrows indicate the retention volume of standard proteins run on the same column.
The low molecular weight active factor was purified to homogeneity, as judged by a
Coomassie-stained SDS-PAGE gel, after a subsequent Mono-Q fractionation (Fig. 4).
FIG. 4. The low molecular wen.ight active factor is calmodulin
A, the membrane EGTA extract from one bovine brain (Start) was subjected to sequential fractionation on DEAE, Blue A, and
Sephacryl S-200 columns. The pooled material containing the activity after each chromotographic step was analyzed by SDS-
PAGE and Coomassie staining. The arrowheads indicate the presence of calmodulin in all the lanes. Calmodulin shows a
mobility shift depending on whether or not Ca2+ is present during electrophoresis (see panel C). B, the active material pooled from Sephacryl S-200 was fractionated on a Mono-Q FPLC column and the fractions
(5 ml/fraction) were tested for activity in a composite assay. The activity peak is shown. C,the active Mono-Q fractions (5 ml/fraction) were subjected to SDS-PAGE in the presence of 1 mM EGTA or 0.1 mM Ca2+,
and the gels stained with Coomassie Blue. D, fraction 47 (1 ml) was probed by Western blotting with a monoclonal anti-calmodulin antibody. No Ca2+ or EGTA was
added during SDS-PAGE.
We reasoned that the protein might be calmodulin (43) based on the following:
1) It is a relatively small protein (14–18 kDa) that is abundant in the
starting extract (Fig. 4A).
2) It elutes at a very high salt concentration (0.41 M KCl) on the
Mono-Q column.
3) It stains negatively in silver stain (data not shown).
4) Its electrophoretic mobility shifts depending on the presence or
absence of Ca21 (Fig. 4C).
A Western blot with an anti-calmodulin monoclonal antibody gave a
positive signal (Fig. 4D), confirming our prediction.
Properties of Calmodulin-stimulated Exocytosis
We used commercial calmodulin or bacterially expressed recombinant calmodulin to confirm our purification result; both sources of authentic calmodulin stimulated NE release as expected. Moreover, we found that calmodulin stimulates secretion in a triggering assay as well as in a composite assay (Fig. 5A).
The half-maximal increase was at 75 nM (250 ng/200 ml) final calmodulin concentration. This is within the broad
range of affinities between calmodulin and its various targets and suggests that the interaction between
calmodulin and its target molecule in exocytosis is in the physiological range. When the triggering reaction was
performed at different Ca2+ concentrations, calmodulin increased NE release only at high [Ca2+] (0.4 – 2 mM)
similar to the crude EGTA extract (Fig. 5B),
suggesting that calmodulin contributes to the triggering activity of the membrane EGTA extract. Calmodulin’s affinity for Ca2+ has
been reported to be around 1 mM (25),
consistent with the Ca2+ requirementfor
calmodulin-stimulated secretion that we observed.
FIG. 5. Calmodulin stimulates NE release in the triggering stage. A, calmodulin (obtained from Sigma) increased NE release in the triggering assay in a dose-dependent fashion, in the absence of ATP
or any other cytosolic proteins. In this particular experiment, the
maximal release achieved by addition of rat brain cytosol was 46.5%.
B,the triggering assay was performed with different concentrations
of free Ca2+. Calmodulin (3 mg bacterially expressed recombinant
protein; closed squares) increased NE release with a similar Ca2+
sensitivity to rat brain membrane EGTA extract (10 mg; closed
triangles), as compared with conditions in which no protein was
added (open squares).
Western analysis with commercial protein as standards indicated that calmodulin
constitutesabout 5% of total proteins in the rat brain membrane EGTA extract
and about 2% of total proteins in the rat brain cytosol(data not shown).
In addition, a significant amount of calmodulin appears to be left
in the washed cell ghosts (data not shown).
Based on the activity of saturating levels of
pure calmodulin (releasing 6–10% of total [3H]NE)
and crude EGTA extract (releasing ;45% of total [3H]NE),
we estimated that
calmodulin accounts for 13–22% of total activity of the extract.
Consistent with this,
a high affinity calmodulin-binding peptide (CaMKIIa(291–312) (44), used at 5 mM) and
an anti-calmodulin antibody (2 mg/200 ml)
inhibited about 20% of the membrane EGTA extract-stimulated release (6.7 mg of extract added; data not shown).
We showed that calmodulin increased NE release
in the triggering stage.
Since regular triggering reactions were performed
in the absence of any added ATP,
this suggests that
calmodulin enhanced secretion in an ATP-independent fashion.
Furthermore, residual ATP in the cell ghosts did not play a role, since
addition of a hexokinase ATP depletion system that
can deplete millimolar concentrations of ATP
within a few minutes (11) had little effect, as did
addition of 5 mM AMPPNP,
which blocks ATP-dependent enzymatic activity (Fig.8A).
Therefore, we ruled out the possibility that a kinase mediates calmodulin’s effect.
FIG. 8. PKC and calmodulin stimulate the late triggering reaction in
an ATP-dependent and ATP-independent manner respectively. A, triggering assays were performed to test the activity of calmodulin
(recombinant; black bars) and PKC (purified rat brain PKC from
Calbiochem; shaded bars) in the absence of ATP. A regular triggering
assay is done in the absence of ATP (2ATP). To deplete residual ATP
in the cells, hexokinase-based ATP depletion was employed (1Hexo).
Alternatively, 5 mM AMP-PNP (1AMP-PNP) was added in the triggering
reaction. Under all three conditions, calmodulin increased release
as compared with the background (buffer only; white bars), whereas
PKC did not. B, NE release in a composite assay was measured with varying
concentrations of free Ca2+ in the presence of 10 mg of calmodulin
(recombinant; closed triangles), 70 ng of PKC (purified rat brain PKC
from Calbiochem; closed squares), or buffer only (open squares).
A series of calmodulin mutants from Paramecium and chicken were tested
for their ability to enhance Ca2+-stimulated secretion, and
none of the mutations abolished the calmodulin effect (data not shown).
These mutations include
S101F, M145V, E54K, G40E/D50N, V35I/D50N within Paramecium
calmodulin (45), and M124Q, M51A/V55A, and M51A/V55A/L32A
within chicken calmodulin (46, 47).
The Paramecium calmodulin mutants are the result of
naturally occurring mutations that result in aberrations in their behavior.
These mutants can be grouped into two categories according to their
behavior, reflecting their loss of either
a Ca2+-dependent Na1 current (calmodulin N-terminal lobe mutants: E54K, G40E/D50N, and V35I/D50N) or
a Ca21-dependent K1 current (calmodulin C-terminal lobe mutants: S101F and M145V) (45).
The chicken calmodulin mutants have been shown to
differentially activate myosin light chain kinase (M124Q, M51A/V55A, and M51A/V55A/L32A),
CaMKII (M124Q), and CaMKIV (M124Q),
and the mutated residues are thought to be important in
Other related articles published in this Open Access Online Scientific Journal include the following:
The role of ion channels in Na(+)-K(+)-ATPase: regulation of ion transport across the plasma membrane has been studies by our Team in 2012 and 2013. Chiefly, our sources of inspiration were the following:
1. 2013 Nobel work on vesicles and calcium flux at the neuromuscular junction Machinery Regulating Vesicle Traffic, A Major Transport System in our Cells The 2013 Nobel Prize in Physiology or Medicine is awarded to Dr. James E. Rothman, Dr. Randy W. Schekman and Dr. Thomas C. Südhof
for their discoveries of machinery regulating vesicle traffic,
a major transport system in our cells.
This represents a paradigm shift in our understanding of how the eukaryotic cell, with its complex internal compartmentalization, organizes
the routing of molecules packaged in vesicles
to various intracellular destinations,
as well as to the outside of the cell
Specificity in the delivery of molecular cargo is essential for cell function and survival.
3. Professor David Lichtstein, Hebrew University of Jerusalem, Dean, School of Medicine
Lichtstein’s main research focus is the regulation of ion transport across the plasma membrane of eukaryotic cells.
His work led to the discovery that specific steroids that have crucial roles, as
the regulation of cell viability,
heart contractility,
blood pressure and
brain function.
His research has implications for the fundamental understanding of body functions,
as well as for several pathological states such as
heart failure, hypertension
and neurological and psychiatric diseases.
Physiologist, Professor Lichtstein, Chair in Heart Studies at The Hebrew University elected
Dean of the Faculty of Medicine at The Hebrew University of Jerusalem
4. Professor Roger J. Hajjar, MD at Mount Sinai School of Medicine
Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension
and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
6. Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
This study presents the possible correlation between Myocardial Ischemia (Coronary Artery Disease (CAD)) aka Ischemic Heart Disease (IHD) and single-nucleotide polymorphisms (SNPs) genes encoding several regulators involved in Coronary Blood Flow Regulation (CBFR), including
ion channels acting in vascular smooth muscle and/or
endothelial cells of coronary arteries.
They completely analyzed exon 3 of both KCNJ8 and KCNJ11 genes (Kir6.1 and Kir6.2 subunit, respectively) as well as
the whole coding region of KCN5A gene (Kv1.5 channel).
The work suggests certain genetic polymorphisms may represent a non-modifiable protective factor that could be
used to identify individuals at relatively low-risk for cardiovascular disease
an independent protective role of the
rs5215_GG against developing CAD and
a trend for rs5219_AA to be associated with protection against coronary microvascular dysfunction
Other related articles published on this Open Access Online Scientific Journal include the following:
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
CaKMII Inhibition in Obese, Diabetic Mice leads to Lower Blood Glucose Levels
Reporter: Larry H Bernstein, MD, FCAP
This recent publication was reported in MedPage today. It is different than, but highly suggestive of our recent report about the Univesity of Iowa discovery of “Oxidized CaKMII inhibition” as a therapeutic target for atrial arrhythmia.
This is a review of a recent work from the laboratory of Mark E. Anderson and associates at the University of Iowa. We have covered the role of CaMKII in calcium signaling and myocardiocyte contraction, as well as signaling in smooth muscle, skeletal muscle, and nerve transmission. There are tissue specific modus operandi, partly related to the ryanogen receptor, and also related to tissue specific isoenzymes of CaMKII. There is much ground that has been traversed in exploring these mechanisms, most recently, the discoverey of hormone triggering by the release from vesicles at the nerve muscle junction, and much remains open to investigation. The recently published work by Mark E. Anderson and associates in Mannheim and Heidelberg, Germany, clarifies the relationship between the oxidized form of CaMKII and the triggering of atrial fibrillation. The following studies show:
Ang II infusion increased the susceptibility of mice to AF induction by rapid right atrial pacing and established a framework for us to test the hypothesized role of ox-CaMKII in promoting AF. ox-CaMKII is critical for AF.
Established a critical role of ox-CaMKII in promoting AF
Ang II induced increases in ROS production seen in WT atria were absent in atria from MsrA TG mice suggesting that MsrA sensitive targets represent an important component of Ang II mediated atrial oxidation.
The protection from AF in MsrA TG mice appeared to be independent of pressor effects that are critical for the proarrhythmic actions.
These findings suggest that NADPH oxidase dependent ROS and elevated ox-CaMKII
drive Ang II -pacing-induced AF and that
targeted antioxidant therapy, by MsrA over-expression,
can reduce or prevent AF in Ang -II-infused mice.
Atrial myocytes from Ang II treated WT mice showed a significant (p<0.05) increase in spontaneous Ca2+ sparks compared to atrial myocytes from saline treated control mice
In contrast to findings in WT mice, the atrial myocytes isolated from Ang II treated MM-VV mice did not show an increase in Ca2+ sparks compared to saline treated MM-VV mice
These data to suggest that in ox–the proarrhythmic effects of Ang II infusion depend upon an increaseCaMKII, sarcoplasmic reticulum Ca2+ leak and DADs.
Enhanced CaMKII-mediated phosphorylation of serine 2814 on RyR2
is associated with an increased susceptibility to acquired arrhythmias, including AF
Mutant S2814A knock-in mice (lacking serine 2814) were highly resistant to Ang II mediated AF
AC3-I mice with transgenic myocardial expression of a CaMKII inhibitory peptide were also resistant to the proarrhythmic effects of Ang II infusion on pacing-induced AF
S2814A, AC3-I and WT mice, all developed similar BP increases and cardiac hypertrophy in response to Ang II, indicating that
these mice were not resistant to the hemodynamic effects of Ang II, but were nevertheless protected from AF.
selectively targeted antioxidant therapies could be effective in preventing or reducing AF
half of patients enrolled in the Mode Selection Trial (MOST) with sinus node dysfunction had a history of AF
Ang II and diabetes-induced CaMKII oxidation caused sinus node dysfunction by increased pacemaker cell death and fibrosis
ox-CaMKII increases susceptibility for AF via increased diastolic sarcoplasmic reticulum Ca2+ release
clinical association between sinus node dysfunction and AF might have a mechanistic basis because
sinus node dysfunction and AF are downstream consequences of elevated ox-CaMKII.
We refer the reader to the following related articles published in pharmaceutical Intelligence:
This article is a followup of the wonderful study of the effect of oxidation of a methionine residue in calcium dependent-calmodulin kinase Ox-CaMKII on stabilizing the atrial cardiomyocyte, giving protection from atrial fibrillation. It is also not so distant from the work reviewed, mostly on the ventricular myocyte and the calcium signaling by initiation of the ryanodyne receptor (RyR2) in calcium sparks and the CaMKIId isoenzyme.
Diabetes: Mouse Studies Point to Kinase as Treatment Target
Published: Nov 24, 2013
By Kristina Fiore, Staff Writer, MedPage Today
Targeting a pathway that plays a major role in both hepatic glucose production and insulin sensitivity may eventually help treat type 2 diabetes, researchers reported.
In a series of experiments in mice, researchers found that inhibition of the kinase CaKMII — or even some of its downstream components — lowered blood glucose and insulin levels, Ira Tabas, MD, PhD, of Columbia University Medical Center in New York City, and colleagues reported online in Cell Metabolism.
The pathway is activated by glucagon signaling in the liver, and appears to have roles in both insulin resistance as well as hepatic glucose production in the liver.
In an earlier study, Tabas and colleagues showed that inhibiting the CaKMII pathway lowered hepatic glucose production by suppressing p38-mediated FoxO1 nuclear localization.
In the current study, they found CaKMII inhibition suppresses levels of the pseudo-kinase TRB3 to improve Akt-phosphorylation, thereby improving insulin sensitivity.
Thus this single pathway targets “two cardinal features of type 2 diabetes — hyperglycemia and defective insulin signaling,” the researchers wrote.
“When we realized we had one common pathway that was responsible for these two disparate processes that, in essence, comprises all of type 2 diabetes, we though it would be an ideal target for new drug therapy,” Tabas told MedPage Today.
Tabas and colleagues conducted several experiments to evaluate the CaKMII pathway.
In one experiment in obese mice, they found that no matter how CaKMII was knocked out, it led to lower blood glucose levels and lower fasting plasma insulin levels in response to a glucose challenge.
The improvements also occurred when they
knocked out downstream processes, including p38 and MAPK-activating protein kinase 2 (MK2).
“Thus liver p38 and MK2, like CaKMII, play an important role in the development of hyperglycemia and hyperinsulinemia in obese mice,” they wrote.
In further analyses, the researchers discovered deleting or inhibiting any of these three elements ultimately
improved insulin-induced Akt-phosphorylation in obese mice —
an important part of improving insulin sensitivity.
And unlike the effects on hepatic glucose production,
these changes didn’t occur through effects on FoxO1.
Instead, inhibiting the CaKMII pathway suppressed levels of the pseudo-kinase TRB3, which likely occurred because of
suppression of ATF4 — all of which led to an
increase in Akt-phosphorylation and insulin sensitivity.
Indeed, when mice were made tooverexpress TRB3, the improvement in phosphorylation disappeared, “indicating that
the suppression of TRB3 by CaKMII deficiency is
causally important in the improvement in insulin signaling,
As a result, there “appear to betwo separate CaKMII pathways”,
one involved in CaKMII-p38-FoxO1 dependent hepatic glucose production, and
the other involved in defective insulin-induced p-Akt,
The findings suggest the possibility of a drug that can target
both hyperglycemia and insulin resistance in type 2 diabetes
The authors have started developing such an agent. Although kinases can act very generally, Tabas said he and colleagues are working on
an allosteric version that will more specifically target MK2
by binding to a site that is unique to this enzyme.
He said this should help to avoid problems with drugs that targeted p38but ultimately failed, with little efficacy and too many side effects.
The reason for this is now known at a very detailed level –
when you inhibit p38 by that mechanism, mainly by inhibiting MK2,
you avoid the adverse effects,
“When we realized all of this and had to make a choice [for further development],the obvious choice was MK2.”
CaKMII inhibitors are in development for heart failure and
MK2 inhibitors are being looked at as an alternative to p38 inhibitors for inflammatory diseases.
Tabas also said the drug may be valuable in treating prediabetes, since early data have suggested that
CaKMII is generally overactive in obese patients
who have not yet progressed to full blown diabetes, but is not overactive in lean people.
“One of the areas we’re most excited about in potential clinical use is in obese people before they get diabetic,” Tabas told MedPage Today. “There are hundreds of millions of people who are obese but not yet diabetic even though
they have the hallmarks that they’re going to get diabetes.”
This recent publication was reported in MedPage Today. [CaKMII overactivity in obesity] Tabas noted that his group’s early human data “suggest that our pathway is turned on in prediabetes. If we can block that pathway before people get diabetes, it would even be better.”
The study was supported by the NIH, the American Heart Association, the German Center for Cardiovascular Research, the German Ministry of Education and Research, and the European Union.
Tabas and a co-author are among the founders of Tabomedex Biosciences, which is developing MK2 inhibitors.
Primary source: Cell Metabolism
Source reference: Ozcan L, et al. “Activation of calcium/calmodulin-dependent protein kinase II in obesity mediates suppression of hepatic insulin signaling” Cell Metab 2013.
This article is a followup of the wonderful study of the effect of oxidation of a methionine residue in calcium dependent-calmodulin kinase Ox-CaMKII on stabilizing the atrial cardiomyocyte, giving protection from atrial fibrillation. It is also not so distant from the work reviewed, mostly on the ventricular myocyte and the calcium signaling by initiation of the ryanodyne receptor (RyR2) in calcium sparks and the CaMKII d isoenzyme.
We refer to the following related articles published in pharmaceutical Intelligence:
The material presented is very focused, and cannot be found elsewhere in Pharmaceutical Intelligence with respedt to genetics and heart disease. However, there are other articles that may be of interest to the reader.
PART 3. Determinants of Cardiovascular Diseases: Genetics, Heredity and Genomics Discoveries
3.2 Leading DIAGNOSES of Cardiovascular Diseases covered in Circulation: Cardiovascular Genetics, 3/2010 – 3/2013
The Diagnoses covered include the following – relevant to this discussion
MicroRNA in Serum as Bimarker for Cardiovascular Pathologies: acute myocardial infarction, viral myocarditis, diastolic dysfunction, and acute heart failure
Genomics of Ventricular arrhythmias, A-Fib, Right Ventricular Dysplasia, Cardiomyopathy
Heredity of Cardiovascular Disorders Inheritance
3.2.1: Heredity of Cardiovascular Disorders Inheritance
The implications of heredity extend beyond serving as a platform for genetic analysis, influencing diagnosis,
prognostication, and
treatment of both index cases and relatives, and
enabling rational targeting of genotyping resources.
This review covers acquisition of a family history, evaluation of heritability and inheritance patterns, and the impact of inheritance on subsequent components of the clinical pathway.
3.2.2.1 MicroRNA in Serum as Biomarker for Cardiovascular Pathologies: acute myocardial infarction, viral myocarditis, diastolic dysfunction, and acute heart failure
Increased MicroRNA-1 and MicroRNA-133a Levels in Serum of Patients With Cardiovascular Disease Indicate Myocardial Damage
Y Kuwabara, Koh Ono, T Horie, H Nishi, K Nagao, et al.
SOURCE: Circulation: Cardiovascular Genetics. 2011; 4: 446-454 http://dx.doi.org/10.1161/CIRCGENETICS.110.958975
3.2.2.2 Circulating MicroRNA-208b and MicroRNA-499 Reflect Myocardial Damage in Cardiovascular Disease
3.2.4.2 Large-Scale Candidate Gene Analysis in Whites and African Americans Identifies IL6R Polymorphism in Relation to Atrial Fibrillation
The National Heart, Lung, and Blood Institute’s Candidate Gene Association Resource (CARe) Project
RB Schnabel, KF Kerr, SA Lubitz, EL Alkylbekova, et al.
SOURCE: Circulation: Cardiovascular Genetics.2011; 4: 557-564 http://dx.doi.org/10.1161/CIRCGENETICS.110.959197
Weighted Gene Coexpression Network Analysis of Human Left Atrial Tissue Identifies Gene Modules Associated With Atrial Fibrillation
N Tan, MK Chung, JD Smith, J Hsu, D Serre, DW Newton, L Castel, E Soltesz, G Pettersson, AM Gillinov, DR Van Wagoner and J Barnard
From the Cleveland Clinic Lerner College of Medicine (N.T.), Department of Cardiovascular Medicine (M.K.C., D.W.N.), and Department of Thoracic & Cardiovascular Surgery (E.S., G.P., A.M.G.); and Department of Cellular & Molecular Medicine (J.D.S., J.H.), Genomic Medicine Institute (D.S.), Department of Molecular Cardiology (L.C.), and Department of Quantitative Health Sciences (J.B.), Cleveland Clinic Lerner Research Institute, Cleveland, OH
Circ Cardiovasc Genet. 2013;6:362-371; http://dx.doi.org/10.1161/CIRCGENETICS.113.000133 http://circgenetics.ahajournals.org/content/6/4/362The online-only Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.113.000133/-/DC1
Background—Genetic mechanisms of atrial fibrillation (AF) remain incompletely understood. Previous differential expression studies in AF were limited by small sample size and provided limited understanding of global gene networks, prompting the need for larger-scale, network-based analyses.
Methods and Results—Left atrial tissues from Cleveland Clinic patients who underwent cardiac surgery were assayed using Illumina Human HT-12 mRNA microarrays. The data set included 3 groups based on cardiovascular comorbidities: mitral valve (MV) disease without coronary artery disease (n=64), coronary artery disease without MV disease (n=57), and lone AF (n=35). Weighted gene coexpression network analysis was performed in the MV group to detect modules of correlated genes. Module preservation was assessed in the other 2 groups. Module eigengenes were regressed on AF severity or atrial rhythm at surgery. Modules whose eigengenes correlated with either AF phenotype were analyzed for gene content. A total of 14 modules were detected in the MV group; all were preserved in the other 2 groups. One module (124 genes) was associated with AF severity and atrial rhythm across all groups. Its top hub gene, RCAN1, is implicated in calcineurin-dependent signaling and cardiac hypertrophy. Another module (679 genes) was associated with atrial rhythm in the MV and coronary artery disease groups. It was enriched with cell signaling genes and contained cardiovascular developmental genes including TBX5.
Conclusions—Our network-based approach found 2 modules strongly associated with AF. Further analysis of these modules may yield insight into AF pathogenesis by providing novel targets for functional studies. (Circ Cardiovasc Genet. 2013;6:362-371.)
trial fibrillation (AF) is the most common sustained cardiac arrhythmia, with a prevalence of ≈1% to 2% in the general population.1,2 Although AF may be an isolated condition (lone AF [LAF]), it often occurs concomitantly with other cardiovascular diseases, such as coronary artery disease (CAD) and valvular heart disease.1 In addition, stroke risk is increased 5-fold among patients with AF, and ischemic strokes attributed to AF are more likely to be fatal.1 Current antiarrhythmic drug therapies are limited in terms of efficacy and safety.1,3,4 Thus, there is a need to develop better risk prediction tools as well as mechanistically targeted therapies for AF. Such developments can only come about through a clearer understanding of its pathogenesis.
Family history is an established risk factor for AF. A Danish Twin Registry study estimated AF heritability at 62%, indicating a significant genetic component.5 Substantial progress has been made to elucidate this genetic basis. For example, genome-wide association studies (GWASs) have identified several susceptibility loci and candidate genes linked with AF. Initial studies performed in European populations found 3 AF-associated genomic loci.6–9 Of these, the most significant single-nucleotide polymor-phisms (SNPs) mapped to an intergenic region of chromosome 4q25. The closest gene in this region, PITX2, is crucial in left-right asymmetrical development of the heart and thus seems promising as a major player in initiating AF.10,11 A large-scale GWAS meta-analysis discovered 6 additional susceptibility loci, implicating genes involved in cardiopulmonary development, ion transport, and cellular structural integrity.12
Differential expression studies have also provided insight into the pathogenesis of AF. A study by Barth et al13 found that about two-thirds of the genes expressed in the right atrial appendage were downregulated during permanent AF, and that many of these genes were involved in calcium-dependent signaling pathways. In addition, ventricular-predominant genes were upregulated in right atrial appendages of subjects with AF.13 Another study showed that inflammatory and transcription-related gene expression was increased in right atrial appendages of subjects with AF versus controls.14 These results highlight the adaptive responses to AF-induced stress and ischemia taking place within the atria.
Despite these advances, much remains to be discovered about the genetic mechanisms of AF. The AF-associated SNPs found thus far only explain a fraction of its heritability15; furthermore, the means by which the putative candidate genes cause AF have not been fully established.9,15,16 Additionally, previous differential expression studies in human tissue were limited to the right atrial appendage, had small sample sizes, and provided little understanding of global gene interactions.13,14 Weighted gene coexpression network analysis (WGCNA) is a technique to construct gene modules within a network based on correlations in gene expression (ie, coexpression).17,18 WGCNA has been used to study genetically complex diseases, such as metabolic syndrome,19 schizophrenia,20 and heart failure.21 Here, we obtained mRNA expression profiles from human left atrial appendage tissue and implemented WGCNA to identify gene modules associated with AF phenotypes.
Methods
Subject Recruitment
From 2001 to 2008, patients undergoing cardiac surgery at the Cleveland Clinic were prospectively screened and recruited. Informed consent for research use of discarded atrial tissues was obtained from each patient by a study coordinator during the presurgical visit. Demographic and clinical data were obtained from the Cardiovascular Surgery Information Registry and by chart review. Use of human atrial tissues was approved by the Institutional Review Board of the Cleveland Clinic.
Table S1: Clinical definitions of cardiovascular phenotype groups
Criterion Type
Mitral Valve (MV) Disease
Coronary Artery Disease (CAD)
Lone Atrial Fibrillation (LAF)
Inclusion Criteria
Surgical indication –
Surgical indication –
History of atrial fibrillation
mitral valve repair or replacement
coronary artery bypass graft
Surgical indication
– MAZE procedure
Preserved ejection fraction (≥50%)
Exclusion Criteria
Significant coronary artery disease:
Significant mitral valve disease:
Significant
coronary artery
– Significant (≥50%) stenosis
– Documented echocardiography
disease:
in at least
finding of
– Significant
one coronary artery
mitral regurgitation (≥3) or
(≥50%) stenosis in
via cardiac catheterization
mitral stenosis
at least one
– History of revascularization
– History of mitral valve
coronary artery via
(percutaneous coronary intervention or coronary artery bypass graft surgery)
repair or replacement
cardiac catheterization
– History of revascularization
(percutaneous coronary intervention or coronary artery bypass graft surgery)
Significant valvular heart disease:
-Documented echocardiography finding of valvular regurgitation (≥3) or stenosis
-History of valve repair or replacement
RNA Microarray Isolation and Profiling
Left atria appendage specimens were dissected during cardiac surgery and stored frozen at −80°C. Total RNA was extracted using the Trizol technique. RNA samples were processed by the Cleveland Clinic Genomics Core. For each sample, 250-ng RNA was reverse transcribed into cRNA and biotin-UTP labeled using the TotalPrep RNA Amplification Kit (Ambion, Austin, TX). cRNA was quantified using a Nanodrop spectrophotometer, and cRNA size distribution was assessed on a 1% agarose gel. cRNA was hybridized to Illumina Human HT-12 Expression BeadChip arrays (v.3). Arrays were scanned using a BeadArray reader.
Expression Data Preprocessing
Raw expression data were extracted using the beadarray package in R, and bead-level data were averaged after log base-2 transformation. Background correction was performed by fitting a normal-gamma deconvolution model using the NormalGamma R package.22 Quantile normalization and batch effect adjustment with the ComBat method were performed using R.23 Probes that were not detected (at a P<0.05 threshold) in all samples as well as probes with relatively lower variances (interquartile range ≤log2[1.2]) were excluded.
The WGCNA approach requires that genes be represented as singular nodes in such a network. However, a small proportion of the genes in our data have multiple probe mappings. To facilitate the representation of singular genes within the network, a probe must be selected to represent its associated gene. Hence, for genes that mapped to multiple probes, the probe with the highest mean expression level was selected for analysis (which often selects the splice isoform with the highest expression and signal-to-noise ratio), resulting in a total of 6168 genes.
Defining Training and Test Sets
Currently, no large external mRNA microarray data from human left atrial tissues are publicly available. To facilitate internal validation of results, we divided our data set into 3 groups based on cardiovascular comorbidities: mitral valve (MV) disease without CAD (MV group; n=64), CAD without MV disease (CAD group; n=57), and LAF (LAF group; n=35). LAF was defined as the presence of AF without concomitant structural heart disease, according to the guidelines set by the European Society of Cardiology.1 The MV group, which was the largest and had the most power for detecting significant modules, served as the training set for module derivation, whereas the other 2 groups were designated test sets for module reproducibility. To minimize the effect of population stratification, the data set was limited to white subjects. Differences in clinical characteristics among the groups were assessed using Kruskal–Wallis rank-sum tests for continuous variables and Pearson x2 test for categorical variables.
Weight Gene Coexpression Network Analysis
WGCNA is a systems-biology method to identify and characterize gene modules whose members share strong coexpression. We applied previously validated methodology in this analysis.17 Briefly, pair-wise gene (Pearson) correlations were calculated using the MV group data set. A weighted adjacency matrix was then constructed. I is a soft-thresholding parameter that provides emphasis on stronger correlations over weaker and less meaningful ones while preserving the continuous nature of gene–gene relationships. I=3 was selected in this analysis based on the criterion outlined by Zhang and Horvath17 (see the online-only Data Supplement).
Next, the topological overlap–based dissimilarity matrix was computed from the weighted adjacency matrix. The topological overlap, developed by Ravasz et al,24 reflects the relative interconnectedness (ie, shared neighbors) between 2 genes.17 Hence, construction of the network dendrogram based on this dissimilarity measure allows for the identification of gene modules whose members share strong intercon-nectivity patterns. The WGCNA cutreeDynamic R function was used to identify a suitable cut height for module identification via an adaptive cut height selection approach.18 Gene modules, defined as branches of the network dendrogram, were assigned colors for visualization.
Network Preservation Analysis
Module preservation between the MV and CAD groups as well as the MV and LAF groups was assessed using network preservation statistics as described in Langfelder et al.25 Module density–based statistics (to assess whether genes in each module remain highly connected in the test set) and connectivity-based statistics (to assess whether connectivity patterns between genes in the test set remain similar compared with the training set) were considered in this analysis.25 In each comparison, a Z statistic representing a weighted summary of module density and connectivity measures was computed for every module (Zsummary). The Zsummary score was used to evaluate module preservation, with values ≥8 indicating strong preservation, as proposed by Langfelder et al.25 The WGCNA R function network preservation was used to implement this analysis.25
Table S2: Network preservation analysis between the MV and CAD groups – size and Zsummary scores of gene modules detected.
Module
Module Size
ZSummary
Black
275
15.52
Blue
964
44.79
Brown
817
12.80
Cyan
119
13.42
Green
349
14.27
Green-Yellow
215
19.31
Magenta
239
15.38
Midnight-Blue
83
15.92
Pink
252
23.31
Purple
224
16.96
Red
278
17.30
Salmon
124
13.84
Tan
679
28.48
Turquoise
1512
44.03
Table S3: Network preservation analysis between the MV and LAF groups – size and Zsummary scores of gene modules detected
Module
Module Size
ZSummary
Black
275
13.14
Blue
964
39.26
Brown
817
14.98
Cyan
119
11.46
Green
349
14.91
Green-Yellow
215
20.99
Magenta
239
18.58
Midnight-Blue
83
13.87
Pink
252
19.10
Purple
224
8.80
Red
278
16.62
Salmon
124
11.57
Tan
679
28.61
Turquoise
1512
42.07
Clinical Significance of Preserved Modules
Principal component analysis of the expression data for each gene module was performed. The first principal component of each module, designated the eigengene, was identified for the 3 cardiovascular disease groups; this served as a summary expression measure that explained the largest proportion of the variance of the module.26 Multivariate linear regression was performed with the module ei-gengenes as the outcome variables and AF severity (no AF, paroxysmal AF, persistent AF, permanent AF) as the predictor of interest (adjusting for age and sex). A similar regression analysis was performed with atrial rhythm at surgery (no AF history, AF history in sinus rhythm, AF history in AF rhythm) as the predictor of interest. The false discovery rate method was used to adjust for multiple comparisons. Modules whose eigengenes associated with AF severity and atrial rhythm were identified for further analysis.
In addition, hierarchical clustering of module eigengenes and selected clinical traits (age, sex, hypertension, cholesterol, left atrial size, AF state, and atrial rhythm) was used to identify additional module–trait associations. Clusters of eigengenes/traits were detected based on a dissimilarity measure D, as given by
D=1−cor(Vi,Vj),i≠j (3)
where V=the eigengene or clinical trait.
Enrichment Analysis
Gene modules significantly associated with AF severity and atrial rhythm were submitted to Ingenuity Pathway Analysis (IPA) to determine enrichment for functional/disease categories. IPA is an application of gene set over-representation analysis; for each dis-ease/functional category annotation, a P value is calculated (using Fisher exact test) by comparing the number of genes from the module of interest that participate in the said category against the total number of participating genes in the background set.27 All 6168 genes in the current data set served as the background set for the enrichment analysis.
Hub Gene Analysis
Hub genes are defined as genes that have high intramodular connectivity17,20
Alternatively, they may also be defined as genes with high module membership21,25
Both definitions were used to identify the hub genes of modules associated with AF phenotype.
To confirm that the hub genes identified were themselves associated with AF phenotype, the expression data of the top 10 hub genes (by intramodular connectivity) were regressed on atrial rhythm (adjusting for age and sex). In addition, eigengenes of AF-associated modules were regressed on their respective (top 10) hub gene expression profiles, and the model R2 indices were computed.
Membership of AF-Associated Candidate Genes From Previous Studies
Previous GWAS studies identified multiple AF-associated SNPs.8,9,12,15,28 We selected candidate genes closest to or containing these SNPs and identified their module locations as well as their closest within-module partners (absolute Pearson correlations).
Sensitivity Analysis of Soft-Thresholding Parameter
To verify that the key results obtained from the above analysis were robust with respect to the chosen soft-thresholding parameter (I=3), we repeated the module identification process using I=5. The eigen-genes of the detected modules were computed and regressed on atrial rhythm (adjusting for age and sex). Modules significantly associated with atrial rhythm in ≥2 groups of data set were compared with the AF phenotype–associated modules from the original analysis.
Results
Subject Characteristics
Table 1 describes the clinical characteristics of the cardiac surgery patients who were recruited for the study. Subjects in the LAF group were generally younger and less likely to be a current smoker (P=2.0×10−4 and 0.032, respectively). Subjects in the MV group had lower body mass indices (P=2.7×10−6), and a larger proportion had paroxysmal AF compared with the other 2 groups (P=0.033).
Table 1. Clinical Characteristics of Study Subjects
A total of 14 modules were detected using the MV group data set (Figure 1), with module sizes ranging from 83 genes to 1512 genes; 38 genes did not share similar coexpression with the other genes in the network and were therefore not included in any of the identified modules
Figure 1. Network dendrogram (top) and colors of identified modules (bottom). The dendrogram was constructed using the topological overlap matrix as the similarity measure. Modules corresponded to branches of the dendrogram and were assigned colors for visualization.
Network Preservation Analysis Revealed Strong Preservation of All Modules Between the Training and Test Sets
All 14 modules showed strong preservation across the CAD and LAF groups in both comparisons, with Z [summary] scores of >10 in most modules (Figure 2). No major deviations in the Z [summary] score distributions for the 2 comparisons were noted, indicating that modules were preserved to a similar extent across the 2 groups
Figure 2. Preservation of modules between mitral valve (MV) and coronary artery disease (CAD) groups (left), and MV and lone atrial fibrillation (LAF) groups (right). A Zsummary statistic was computed for each module as an overall measure of its preservation relating to density and connectivity. All modules showed strong preservation in both comparisons with Zsummary scores >8 (red dotted line).
Regression Analysis of Module Eigengene Profiles Identified 2 Modules Associated With AF Severity and Atrial Rhythm
Table IV in the online-only Data Supplement summarizes the proportion of variance explained by the first 3 principal components for each module. On average, the first principal component (ie, the eigengene) explained ≈18% of the total variance of its associated module. For each group, the module eigengenes were extracted and regressed on AF severity (with age and sex as covariates). The salmon module (124 genes) eigengene was strongly associated with AF severity in the MV and CAD groups (P=1.7×10−6 and 5.2×10−4, respectively); this association was less significant in the LAF group (P=9.0×10−2). Eigengene levels increased with worsening AF severity across all 3 groups, with the greatest stepwise change taking place between the paroxysmal AF and persistent AF categories (Figure 3A). When the module eigen-genes were regressed on atrial rhythm, the salmon module eigengene showed significant association in all groups (MV: P=1.1×10−14; CAD: P=1.36×10−6; LAF: P=2.1×10−4). Eigen-gene levels were higher in the AF history in AF rhythm category (Figure 3B).
Table S4:Proportion of variance explained by the principal components for each module.
Dataset
Group
Principal
Component
Black
Blue
Brown
Cyan
Green
Green-
Yellow
Magenta
Mitral
1
20.5%
22.2%
20.1%
21.8%
21.4%
22.8%
19.6%
2
4.1%
3.6%
4.8%
5.7%
4.5%
5.9%
3.9%
3
3.4%
3.1%
3.8%
4.4%
3.9%
3.7%
3.7%
CAD
1
12.5%
18.6%
7.1%
16.8%
12.2%
20.3%
12.8%
2
6.0%
5.5%
5.0%
7.0%
5.5%
6.1%
6.4%
3
4.9%
4.1%
4.4%
6.5%
4.8%
4.4%
4.8%
LAF
1
14.0%
16.6%
11.7%
14.3%
14.7%
20.8%
20.2%
2
8.9%
8.5%
7.6%
9.3%
7.3%
11.1%
6.9%
3
6.5%
6.3%
5.5%
8.2%
6.1%
5.3%
6.2%
Dataset
Group
Principal
Component
Midnight- Blue
Pink
Purple
Red
Salmon
Tan
Turquoise
Mitral
1
28.5%
22.6%
18.7%
20.5%
22.3%
19.0%
25.8%
2
4.6%
6.0%
4.7%
4.1%
6.9%
4.0%
3.5%
3
4.2%
4.2%
4.2%
3.5%
4.0%
3.6%
3.3%
CAD
1
23.4%
17.1%
15.5%
15.0%
18.0%
14.6%
18.2%
2
7.4%
8.6%
6.0%
6.4%
7.2%
5.8%
6.6%
3
5.1%
5.4%
5.3%
5.4%
6.2%
5.1%
4.5%
LAF
1
23.5%
18.4%
12.0%
15.9%
16.9%
13.7%
16.5%
2
7.9%
8.5%
9.8%
9.4%
9.5%
9.1%
9.6%
3
6.7%
7.0%
6.6%
6.0%
6.9%
6.8%
6.3%
Figure 3. Boxplots of salmon module eigengene expression levels with respect to atrial fibrillation (AF) severity (A) and atrial rhythm (B).
A, Eigengene expression correlated positively with AF severity, with the largest stepwise increase between the paroxysmal AF and permanent AF categories. B, Eigengene expression was highest in the AF history in AF rhythm category in all 3 groups. CAD indicates coronary artery disease; LAF, lone AF; and MV, mitral valve.
The regression analysis also revealed statistically significant associations between the tan module (679 genes) eigengene and atrial rhythm in the MV and CAD groups (P=5.8×10−4 and 3.4×10−2, respectively). Eigengene levels were lower in the AF history in AF rhythm category compared with the AF history in sinus rhythm category (Figure 4); this trend was also observed in the LAF group, albeit with weaker statistical evidence (P=0.15).
Figure 4. Boxplots of tan module eigengene expression levels with respect to atrial rhythm. Eigengene expression levels were lower in the atrial fibrillation (AF) history in AF rhythm category compared with the AF history in sinus rhythm category. CAD indicates coronary artery disease; LAF, lone AF; and MV, mitral valve
Hierarchical Clustering of Eigengene Profiles With Clinical Traits
Hierarchical clustering was performed to identify relationships between gene modules and selected clinical traits. The salmon module clustered with AF severity and atrial rhythm; in addition, left atrial size was found in the same cluster, suggesting a possible relationship between salmon module gene expression and atrial remodeling (Figure 5A). Although the tan module was in a separate cluster from the salmon module, it was negatively correlated with both atrial rhythm and AF severity (Figure 5B).
Figure 5. Dendrogram (A) and correlation heatmap (B) of module eigengenes and clinical traits.
A, The salmon module eigengene but not the tan module eigengene clustered with atrial fibrillation (AF) severity, atrial rhythm, and left atrial size. B, AF severity and atrial rhythm at surgery correlated positively with the salmon module eigengene and negatively with the tan module eigengene. Arhythm indicates atrial rhythm at surgery; Chol, cholesterol; HTN, hypertension; and LASize, left atrial size.
IPA Enrichment Analysis of Salmon and Tan Modules
The salmon module was enriched in genes involved in cardiovascular function and development (smallest P=4.4×10−4) and organ morphology (smallest P=4.4×10−4). In addition, the top disease categories identified included endocrine system disorders (smallest P=4.4×10−4) and cardiovascular disease (smallest P=2.59×10−3).
The tan module was enriched in genes involved in cell-to-cell signaling and interaction (smallest P=8.9×10−4) and cell death and survival (smallest P=1.5×10−3). Enriched disease categories included cancer (smallest P=2.2×10−4) and cardiovascular disease (smallest P=4.5×10−4).
We identified hub genes in the 2 modules based on intramod-ular connectivity and module membership. For the salmon module, the gene RCAN1 exhibited the highest intramodular connectivity and module membership. The top 10 hub genes (by intramodular connectivity) were significantly associated with atrial rhythm, with false discovery rate–adjusted P values ranging from 1.5×10−5 to 4.2×10−12. These hub genes accounted for 95% of the variation in the salmon module eigengene.
In the tan module, the top hub gene was CPEB3. The top 10 hub genes (by intramodular connectivity) correlated with atrial rhythm as well, although the statistical associations in the lower-ranked hub genes were relatively weaker (false discovery rate–adjusted P values ranging from 1.1×10−1 to 3.4×10−4). These hub genes explained 94% of the total variation in the tan module eigengene.
The names and connectivity measures of the hub genes found in both modules are presented in Table 2.
Table 2. Top 10 Hub Genes in the Salmon (Left) and Tan (Right) Modules as Defined by Intramodular Connectivity and Module Membership
Salmon Module
Tan Module
Gene
IMC
Gene
MM
Gene
IMC
Gene
MM
RCAN1
8.2
RCAN1
0.81
CPEB3
43.3
CPEB3
0.85
DNAJA4
7.7
DNAJA4
0.81
CPLX3
42.4
CPLX3
0.84
PDE8B
7.7
PDE8B
0.80
NEDD4L
40.8
NEDD4L
0.83
PRKAR1A
6.9
PRKAR1A
0.77
SGSM1
40.7
SGSM1
0.82
PTPN4
6.7
PTPN4
0.75
UCKL1
39.0
UCKL1
0.81
SORBS2
6.0
FHL2
0.69
SOSTDC1
37.2
SOSTDC1
0.79
ADCY6
5.7
ADCY6
0.69
PRDX1
35.5
RCOR2
0.78
FHL2
5.7
SORBS2
0.68
RCOR2
35.4
EEF2K
0.77
BVES
5.4
DHRS9
0.67
NPPB
35.3
PRDX1
0.76
TMEM173
5.3
LAPTM4B
0.65
LRRN3
34.6
MMP11
0.76
A visualization of the salmon module is shown using the Cytoscape tool (Figure 6). A full list of the genes in the salmon and tan modules is provided in the online-only Data Supplement.
Figure 6. Cytoscape visualization of genes in the salmon module.
Nodes representing genes with high intramodu-lar connectivities, such as RCAN1 and DNAJA4, appear larger in the network. Strong connections are visualized with darker lines, whereas weak connections appear more translucent
Membership of AF-Associated Candidate Genes From Previous Studies
The tan module contained MYOZ1, which was identified as a candidate gene from the recent AF meta-analysis. PITX2 was located in the green module (n=349), and ZFHX3 was located in the turquoise module (n=1512). The locations of other candidate genes (and their closest partners) are reported in the online-only Data Supplement.
Sensitivity Analysis of Key Results
We repeated the WGCNA module identification approach using a different soft-thresholding parameter (β=5). One module (n=121) was found to be strongly associated with atrial rhythm at surgery across all 3 groups of data set, whereas another module (n=244) was associated with atrial rhythm at surgery in the MV and CAD groups. The first module overlapped significantly with the salmon module in terms of gene membership, whereas most of the second modules’ genes were contained within the tan module. The top hub genes found in the salmon and tan modules remained present and highly connected in the 2 new modules identified with the different soft-thresholding parameter.
Discussion
To our knowledge, our study is the first implementation of an unbiased, network-based analysis in a large sample of human left atrial appendage gene expression profiles. We found 2 modules associated with AF severity and atrial rhythm in 2 to 3 of our cardiovascular comorbidity groups. Functional analyses revealed significant enrichment of cardiovascular-related categories for both modules. In addition, several of the hub genes identified are implicated in cardiovascular disease and may play a role in AF initiation and progression.
In our study, WGCNA was used to construct modules based on gene coexpression, thereby reducing the net-work’s dimensionality to a smaller set of elements.17,21 Relating modulewise changes to phenotypic traits allowed statistically significant associations to be detected at a lower false discovery rate compared with traditional differential expression studies. Furthermore, shared functions and pathways among genes in the modules could be inferred via enrichment analyses.
We divided our data set into 3 groups to verify the reproducibility of the modules identified by WGCNA; 14 modules were identified in the MV group in our gene network. All were strongly preserved in the CAD and LAF groups, suggesting that gene coexpression patterns are robust and reproducible despite differences in cardiovascular comorbidities.
The use of module eigengene profiles as representative summary measures has been validated in a number of studies.20,26 Additionally, we found that the eigengenes accounted for a significant proportion (average 18%) of gene expression variability in their respective modules. Regression analysis of the module eigengenes found 2 modules associated with AF severity and atrial rhythm in ≥2 groups of data set. The association between the salmon module eigengene and AF severity was statistically weaker in the LAF group (adjusted P=9.0×10−2). This was probably because of its significantly smaller sample size compared with the MV and CAD groups. Despite this weaker association, the relationship between the salmon module eigengene and AF severity remained consistent among the 3 groups (Figure 3A). Similarly, the lack of statistical significance for the association between the tan module eigengene and atrial rhythm at surgery in the LAF group was likely driven by the smaller sample size and (by definition) lack of samples in the no AF category.
A major part of our analysis focused on the identification of module hub genes. Hubs are connected with a large number of nodes; disruption of hubs therefore leads to widespread changes within the network. This concept has powerful applications in the study of biology, genetics, and disease.29,30 Although mutations of peripheral genes can certainly lead to disease, gene network changes are more likely to be motivated by changes in hub genes, making them more biologically interesting targets for further study.17,29,31 Indeed,
the hub genes of the salmon and tan modules accounted for the vast majority of the variation in their respective module eigengenes, signaling their importance in driving gene module behavior.
The hub genes identified in the salmon and tan modules were significantly associated with AF phenotype overall. It was noted that this association was statistically weaker for the lower-ranked hub genes in the tan module. This highlights an important aspect and strength of WGCNA—to be able to capture module-wide changes with respect to disease despite potentially weaker associations among individual genes.
The implementation of WGCNA necessitated the selection of a soft-thresholding parameter 13. Unlike hard-thresholding (where gene correlations below a certain value are shrunk to zero), the soft-thresholding approach gives greater weight to stronger correlations while maintaining the continuous nature of gene–gene relationships. We selected a 13 value of 3 based on the criteria outlined by Zhang and Horvath.17 His team and other investigators have demonstrated that module identification is robust with respect to the 13 parameter.17,19–21 In our data, we were also able to reproduce the key findings reported with a different, larger 13 value, thereby verifying the stability of our results relating to 13.
The salmon module (124 genes) was associated with both AF phenotypes; furthermore, IPA analysis of its gene contents suggested enrichment in cardiovascular development as well as disease. Its eigengene increased with worsening AF severity, with the largest stepwise change occurring between the paroxysmal AF and persistent AF categories (Figure 3). Hence,
the gene expression changes within the salmon module may reflect the later stages of AF pathophysiology.
The top hub gene of the salmon module was RCAN1 (regulator of calcineurin 1). Calcineurin is a cytoplasmic Ca2+/ calmodulin-dependent protein phosphatase that stimulates cardiac hypertrophy via its interactions with NFAT and L-type Ca2+ channels.32,33RCAN1 is known to inhibit calcineurin and its associated pathways.32,34 However, some data suggest that RCAN1 may instead function as a calcineurin activator when highly expressed and consequently potentiate hypertrophic signaling.35 Thus,
perturbations in RCAN1 levels (attributable to genetic variants or mutations) may cause an aberrant switching in function, which in turn triggers atrial remodeling and arrhythmogenesis.
Other hub genes found in the salmon module are also involved in cardiovascular development and function and may be potential targets for further study.
DNAJA4 (DnaJ homolog, subfamily A, member 4) regulates the trafficking and maturation of KCNH2 potassium channels, which have a prominent role in cardiac repolarization and are implicated in the long-QT syndromes.36
FHL2 (four-and-a-half LIM domain protein 2) interacts with numerous cellular components, including
actin cytoskeleton,
transcription machinery, and
ion channels.37
FHL2 was shown to enhance the hypertrophic effects of isoproterenol, indicating that
FHL2 may modulate the effect of environmental stress on cardiomyocyte growth.38
FHL2 also interacts with several potassium channels in the heart, such as KCNQ1, KCNE1, and KCNA5.37,39
Additionally, blood vessel epicardial substance (BVES) and other members of its family were shown to be highly expressed in cardiac pacemaker cells. BVES knockout mice exhibited sinus nodal dysfunction, suggesting that BVES regulates the development of the cardiac pacemaking and conduction system40 and may therefore be involved in the early phase of AF development.
The tan module (679 genes) eigengene was negatively correlated with atrial rhythm in the MV and CAD groups (Figure 4); this may indicate a general decrease in gene expression of its members in fibrillating atrial tissue. IPA analysis revealed enrichment in genes involved in cell signaling as well as apoptosis. The top-ranked hub gene, cytoplasmic polyade-nylation element binding protein 3 (CPEB3), regulates mRNA translation and has been associated with synaptic plasticity and memory formation.41 The role of CPEB3 in the heart is currently unknown, so further exploration via animal model studies may be warranted.
Natriuretic peptide-precursor B (NPPB), another highly interconnected hub gene, produces a precursor peptide of brain natriuretic peptide, which
regulates blood pressure through natriuresis and vasodilation.42
(NPPB) gene variants have been linked with diabetes mellitus, although associations with cardiac phenotypes are less clear.42 TBX5 and GATA4, which play important roles in the embryonic heart development,43 were members of the tan module. Although not hub genes, they may also contribute toward developmental susceptibility of AF. In addition, TBX5 was previously reported to be near an SNP associated with PR interval and AF in separate large-scale GWAS studies.12,28 MYOZ1, another candidate gene identified in the recent AF GWAS meta-analysis, was found to be a member as well; it associates with proteins found in the Z-disc of skeletal and cardiac muscle and may suppress calcineurin-dependent hypertrophic signaling.12
Some, but not all, of the candidate genes found in previous GWAS studies were located in the AF-associated modules. One possible explanation for this could be the difference in sample sizes. The meta-analysis involved thousands of individuals, whereas the current study had <100 in each group of data set, which limited the power to detect significant differences between levels of AF phenotype even with the module-wise approach. Additionally, transcription factors like PITX2 are most highly expressed during the fetal phase of development. Perturbations in these genes (attributable to genetic variants or mutations) may therefore initiate the development of AF at this stage and play no significant role in adults (when we obtained their tissue samples).
Limitations in Study
We noted several limitations in this study. First, no human left atrial mRNA data set of adequate size currently exists publicly. Hence, we were unable to validate our results with an external, independent data set. However, the network preservation assessment performed within our data set showed strong preservation in all modules, indicating that our findings are robust and reproducible.
Although the module eigengenes captured a significant proportion of module variance, a large fraction of variability did remain unaccounted for, which may limit their use as representative summary measures.
We extracted RNA from human left atrial appendage tissue, which consists primarily of cardiomyocytes and fibroblasts. Atrial fibrosis is known to occur with AF-associated remodeling.44 As such, the cardiomyocyte to fibroblast ratio is likely to change with different levels of AF severity, which in turn influences the amount of RNA extracted from each cell type. Hence, true differences in gene expression (and coexpression) within cardiomyocytes may be confounded by changes in cellular composition attributable to atrial remodeling. Also, there may be significant regional heterogeneity in the left atrium with respect to structure, cellular composition, and gene expression,45 which may limit the generaliz-ability of our results to other parts of the left atrium.
All subjects in the study were whites to minimize the effects of population stratification. However, it is recognized that the genetic basis of AF may differ among ethnic groups.9 Thus, our results may not be generalizable to other ethnicities.
Finally, it is possible for genes to be involved in multiple processes and functions that require different sets of genes. However, WGCNA does not allow for overlapping modules to be formed. Thus,
this limits the method’s ability to characterize such gene interactions.
Conclusions
In summary, we constructed a weighted gene coexpression network based on RNA expression data from the largest collection of human left atrial appendage tissue specimens to date. We identified 2 gene modules significantly associated with AF severity or atrial rhythm at surgery. Hub genes within these modules may be involved in the initiation or progression of AF and may therefore be candidates for functional studies.
Refererences
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13. Barth AS, Merk S, Arnoldi E, Zwermann L, Kloos P, Gebauer M, et al. Reprogramming of the human atrial transcriptome in permanent atrial fibrillation: expression of a ventricular-like genomic signature. Circ Res. 2005;96:1022–1029.
Atrial fibrillation is the most common sustained cardiac arrhythmias in the United States. The genetic and molecular mechanisms governing its initiation and progression are complex, and our understanding of these mechanisms remains incomplete despite recent advances via genome-wide association studies, animal model experiments, and differential expression studies. In this study, we used weighted gene coexpression network analysis to identify gene modules significantly associated with atrial fibrillation in a large sample of human left atrial appendage tissues. We further identified highly interconnected genes (ie, hub genes) within these gene modules that may be novel candidates for functional studies. The discovery of the atrial fibrillation-associated gene modules and their corresponding hub genes provide novel insight into the gene network changes that occur with atrial fibrillation, and closer study of these findings can lead to more effective targeted therapies for disease management.
This is a review of a recent work from the laboratory of Mark E. Anderson and associates at the University of Iowa. WE have covered the role of CaMKII in calcium signaling and myocardiocyte contraction, as well as signaling in smooth muscle, skeletal muscle, and nerve transmission. There are tissue specific modus operandi, partly related to the ryanogen receptor, and also related to tissue specific isoenzymes of CaMKII. There is much ground that has been traversed in exploring these mechanisms, most recently, the discoverey of hormone triggering by the release from vesicles at the nerve muscle junction, and much remains open to investigation. The recently published work by Mark E. Anderson and associates in Mannheim and Heidelberg, Germany, clarifies the relationship between the oxidized form of CaMKII and the triggering of atrial fibrillation. The following studies show:
Ang II infusion increased the susceptibility of mice to AF induction by rapid right atrial pacing and established a framework for us to test the hypothesized role of ox-CaMKII in promoting AF. ox-CaMKII is critical for AF.
Estalished a critical role of ox-CaMKII in promoting AF
Ang II induced increases in ROS production seen in WT atria were absent in atria from MsrA TG mice suggesting that MsrA sensitive targets represent an important component of Ang II mediated atrial oxidation.
The protection from AF in MsrA TG mice appeared to be independent of pressor effects that are critical for the proarrhythmic actions.
These findings suggest that NADPH oxidase dependent ROS and elevated ox-CaMKII drive Ang II -pacing-induced AF and that
targeted antioxidant therapy, by MsrA over-expression, can reduce or prevent AF in Ang -II-infused mice.
Atrial myocytes from Ang II treated WT mice showed a significant (p<0.05) increase in spontaneous Ca2+ sparks compared to atrial myocytes from saline treated control mice
In contrast to findings in WT mice, the atrial myocytes isolated from Ang II treated MM-VV mice did not show an increase in Ca2+ sparks compared to saline treated MM-VV mice
These data to suggest that in ox–the proarrhythmic effects of Ang I I infusion depend upon an increaseCaMKII, sarcoplasmic reticulum Ca2+ leak and DADs.
Enhanced CaMKII-mediated phosphorylation of serine 2814 on RyR2 is associated with an increased susceptibility to acquired arrhythmias, including AF
Proarrhythmic actions of ox-CaMKII require access to RyR2 serine 2814.
Mutant S2814A knock-in mice (lacking serine 2814) were highly resistant to Ang II mediated AF
AC3-I mice with transgenic myocardial expression of a CaMKII inhibitory peptide were also resistant to the proarrhythmic effects of Ang II infusion on pacing-induced AF
S2814A, AC3-I and WT mice, all developed similar BP increases and cardiac hypertrophy in response to Ang II, indicating that these mice were not resistant to the hemodynamic effects of Ang II, but were nevertheless protected from AF.
selectively targeted antioxidant therapies could be effective in preventing or reducing AF
half of patients enrolled in the Mode Selection Trial (MOST) with sinus node dysfunction had a history of AF
Ang II and diabetes-induced CaMKII oxidation caused sinus node dysfunction by increased pacemaker cell death and fibrosis
ox-CaMKII increases susceptibility for AF via increased diastolic sarcoplasmic reticulum Ca2+ release
clinical association between sinus node dysfunction and AF might have a mechanistic basis because sinus node dysfunction and AF are downstream consequences of elevated ox-CaMKII.
We refer to the following related articles published in pharmaceutical Intelligence:
Anil Purohit, Adam G. Rokita, Xiaoqun Guan, Biyi Chen, Olha M. Koval, Niels Voigt, Stefan Neef, Thomas Sowa, Zhan Gao, Elizabeth D. Luczak, Hrafnhildur Stefansdottir, Andrew C. Behunin, Na Li, Ramzi N. El Accaoui, Baoli Yang, Paari Dominic Swaminathan, Robert M. Weiss, Xander H. T. Wehrens, Long-Sheng Song, Dobromir Dobrev, Lars S. Maier and Mark E. Anderson
1Dept of Internal Medicine, Division of Cardiovascular Medicine and Cardiovascular Research Center, Carver College of Medicine, University of Iowa, Iowa City, IA; 2Institute of Pharmacology, Faculty of Medicine, University Duisburg-Essen, Essen, Germany, and Division of Experimental Cardiology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; 3Cardiology and Pneumology, German Heart Center, University Hospital Goettingen, Goettingen, Germany; 4Dept of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX; 5Dept of Obstetrics and Gynecology; 6Dept of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
Background—Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKII(ox-CaMKII) could contribute to AF.Methods and Results—We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIId (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion. Conclusions—Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.
Key words: atrial fibrillation, calcium/calmodulin-dependent protein kinase II, angiotensin II, reactive oxygen species, arrhythmia (mechanisms)
Introduction
Atrial fibrillation (AF) is the most common sustained arrhythmia. AF produces lifestyle-limiting symptoms and increases the risk of stroke and death,1 but current therapies have limited efficacy. The renin-angiotensin-system is upregulated in cardiovascular disease and elevated Angiotensin II (Ang II) favors AF.2,3 Ang II activates NADPH oxidase, leading to increased ROS and fibrillating atria are marked by increased reactive oxygen species (ROS).4,5 We recently identified the multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) as a ROS sensor6 and proarrhythmic signal.7 Oxidation of critical methionines (281/282) in the CaMKII regulatory domain lock CaMKII into a constitutively active, Ca2+ and calmodulinin-dependent conformation that is associated with cardiovascular disease.8 Based on this information, we asked if oxidized CaMKII (ox-CaMKII) could be a biomarker and proarrhythmic signal for connecting increased atrial ROS to AF. We found that ox-CaMKII was increased in atrial tissue from patients with AF compared to patients in sinus rhythm, and in atrial tissue from Ang II-infused, compared to saline-infused, mice. We used a validated mouse model of AF induction by rapid right atrial pacing9,10 and found that mice with prior Ang II infusion were at significantly higher risk of AF compared to vehicle-infused mice. We tested AF induction in Ang II and vehicle-infused mice with genetically engineered resistance to CaMKII oxidation by knock-in replacement of methionines 281/282 with valines in CaMKIId (MM-VV), the isoform associated with cardiovascular disease11-14 or by myocardial-targeted antioxidant therapy by transgenic over-expression of methionine sulfoxide reductase A (MsrA), an enzyme that reduces ox-CaMKII.15,16 Collectively, our results support a view that Ang II promotes AF induction by increasing ROS, ox-CaMKII, CaMKII activity, sarcoplasmic reticulum Ca2+ leak and delayed after-depolarizations (DADs). Our findings provide novel insights into a ROS and Ang II-dependent mechanism of AF by linking oxidative stress to dysfunctional intracellular Ca2+ signaling via ox-CaMKII and identify a potential new approach for treating AF by targeted antioxidant therapy.
Methods
Human samples and immunodetection of ox-CaMKII.
The human samples were provided by the Georg-August-University Goettingen and the University of Heidelberg after approval by the local ethics committee of the Georg-August-University Göttingen and the Medical Faculty Mannheim, University of Heidelberg (#2011-216N-MA).
Right atrial appendage tissue samples were obtained from patients undergoing thoracotomy with sinus rhythm or with AF (Table 1) as published previously.17 For immunostaining experiments a total of 9 samples were studied including 5 patients with sinus rhythm and 4 patients with AF ( Table 1A). For immunob lotting a total of 51 samples were studied including 25 patients with SR and 26 patients with AF (Table 1B). The pat ei nt charts were reviewed by the authors to obtain relevant clinical information.
Mouse Models and Experimental Methods
All mice used in the study were available to us in C57Bl6 background. All experiments were performed in male mice 8-12 weeks of age. In total we studied 262 mice. Numbers for each experimental group are provided in the figures or figure legends. See Supplemental Material for detailed methods.
Statistics
Data are presented as mean ± SEM. P values were assessed with a Student’s t-test (2-tailed), ANOVA or two-way ANOVA, as appropriate, for continuous data. The effect of Ang II compared to saline on ox-CaMKII, CaMKII, and ox-CaMKII/CaMKII ratio was tested within each mouse genotype (strain) and compared among the four genotypes using the two-way analysis of variance (ANOVA). The factors that were tested in the ANOVA model were genotype (WT, MM-VV, p47-/- and MsrA TG), treatment (Ang II versus saline), and genotype treatment interaction effect. A significant genotype treatment interaction (*) indicated that the effect of Ang II (versus saline) differed significantly among the strains. Post hoc comparisons after ANOVA were performed using the Bonferroni test. Discrete variables were analyzed by Fisher’s exact test.
Results
Oxidized CaMKII is increased in AF
Patients with AF have increased atrial CaMKII activity18,19 and high circulating levels of serum markers for oxidative stre ss. 4, 5 We first obtained right atrial tissue from patients undergoing cardiac surgery (Table 1) and measured ox-CaMKII using a validated antiserum against oxidized Met 281/282 in the CaMKII regulatory domains.6 These pilot immunofluorescence studies on atrial tissue samples made available upon consent by patients with AF or normal sinus rhythm (Table 1A) showed significantly (p<0.05) higher (~2.5 fold) ox-CaMKII levels in patients with AF (Figure 1A and B). Based on these initial findings, we measured ox-CaMKII in atrial tissue from a larger cohort of patients (Table 1B; for complete gels see supplementary Figure 1) in sinus rhythm (N = 25) or AF (N = 26) using Western blots, and confirmed that AF patients have significantly elevated expression of ox-CaMKII, while there was no difference in total CaMKII (Figure 1C-F). The patient characteristics in the two groups (Table 1) were similar in terms of age, presence of hypertension, diabetes and left ventricular ejection fraction, recognized risk factors for AF.20 The subgroup of AF patients that were not treated with angiotensin converting enzyme inhibitor (ACE-i) or angiotensin receptor blockers (ARB) showed the highest levels of ox-CaMKII and total CaMKII (Supplementary Figure 1A and B). Taken together, these findings showed a positive association between AF and increased expression of atrial ox-CaMKII and a loss of this association in AF patients treated with ACE-i or ARBs.
Ang II treatment enhances AF susceptibility
To test the hypothesis that ox-CaMKII contributes to AF we developed a mouse model of AF by infusing wild type (WT) mice with Ang II (2000 ng/kg/min) or an equal volume of normal saline via osmotic mini-pumps for three weeks. We previously established that this dose of Ang II caused a significant increase in atrial ox-CaMKII7 and resulted in serum Ang II levels similar to those measured in heart failure patients.21
In order to test if Ang II treatment can promote AF we performed burst pacing in the right atrium of anesthetized mice, using an established method ( Figure 2A-C). 10 Mice treated wit Ang II showed significantly higher AF induction rates compared to saline treated mice (64% [9/14] versus 18% [2/14], p=0.018 Fisher’s exact test) (Figure 2D). Ang II is known to contribute to hypertension, left ventricular hypertrophy and heart failure, all established clinical risk factors for AF.20 Therefore, we measured blood pressure (BP) by tail-cuff and assessed left ventricular size and systolic function by echocardiography. As expected, Ang II treatment significantly increased systolic BP (Figure 2E; p<0.01) and left ventricular mass (Figure 2F; p<0.001). Ang II treated mice maintained a normal left ventricular ejection fraction, similar to saline-infused control mice (Figure 2G). These data showed that Ang II infusion increased the susceptibility of mice to AF induction by rapid right atrial pacing and established a framework for us to test the hypothesized role of ox-CaMKII in promoting AF. ox-CaMKII is critical for AF.
In order to test if ox-CaMKII was required for AF induction in our model we used oxidation resistant knock in MM-VV mice (Supplementary Figure 2).22 CaMKII with the MM-VV mutation is resistant to oxidative activation but retains normal Ca2+ and calmodulin dependent activation and is capable of transitioning into a Ca2+ and calmodulin independent enzyme after threonine 287 autophosphorylation.6 The MM-VV mice were significantly resistant to AF induction after Ang II infusion, compared to WT controls (Figure 3A), suggesting that ox-CaMKII is required for increased AF susceptibility in Ang II infused mice. WT mice treated with Ang II showed significantly higher (~2.7 fold; 95% confidential interval, CI: 1.4, 5.1) ) levels of mice. When indexed to total CaMKII levels (Supplementary Figure 3A and B) this increase in ox-CaMKII was much higher (~14. 2 fold; 95% confidential interval, CI: 1.4, 5.1) in Ang II treated WT mice (figure 4C). The residual increase in ox–CaMKII in the -MM-VV mice likely results from expression of atrial ox-CaMKII compared to saline treated mice. As expected, Ang II infusion increased ox-CaMKII less in -MM-VV (~2.1 fold; 95% CI: 1.1, 4.0) than in control WT. ox-CaMKII was much higher (~14.2 fold; 95% CI: 5.9, 34.5) in Ang II treated WT mice.
CaMKIILI, a myocardial CaMKII isoform not affected by the MM-VV mutation.23 However, despite the greater increase in ox-CaMKII in WT compared to MM-VV mice, Ang II-related ROS production was increased in both WT and MM-VV mice to a similar degree (Supplementary Figure 4). Interestingly, Ang II treated WT mice showed a significant decrease in total CaMKII levels (Supplementary Figure 3A and B) suggesting feedback inhibition of total CaMKII expression.
Atrial lysates from MM-VV mice showed significantly less Ca2+ and calmodulin-independent activity after Ang II treatment, but retained WT level CaMKII activity increases in response to isoproterenol (Supplementary Figure 2A). At 8 weeks MM-VV mice had body weight (Supplementary Figure 2B) and BP (Figure 3B) that were similar to WT mice, suggesting CaMKIIį methionine 281/282 oxidation did not affect basal BP or developmentally appropriate growth. CaMKII is known to regulate the chronotropic response to stress and mice with CaMKII inhibition have a smaller increase in heart rate with isoproterenol treatment compared to controls.24 Isolated Langendorff-perfused hearts from WT and MM-VV mice had similar resting heart rates (Supplementary Figure 2C) and comparable heart rate increases after isoproterenol treatment (Supplementary Figure 2D), suggesting that CaMKII dependent physiological heart rate increases do not require CaMKIIį methionine oxidation. L-type Ca2+ currents were similar in MM-VV and WT mice, and L-type Ca2+ current facilitation, a CaMKII-dependent phenotype, was also preserved in MM-VV mice.25,26 KN-93, a small molecule CaMKII inhibitor,27 significantly reduced facilitation in WT and -MM-VV mice (Supplementary Figure 5). MM-VV mice and WT controls showed similar increases in systolic BP (Figure 3B) and heart weight (Figure 3C) or left ventricular mass estimated by echocardiography after Ang II infusion ( Supplementary Figure 6), suggesting that -ox-CaMK IIį is dispensable for hypertensive and myocardial hypertrophic actions of Ang II. Taken together, these findings indicate loss of methionines 281/282 in CaMKIIį selectively reduce the pro-arrhythmic actions of Ang II in a pacing-induced model of AF.
NADPH oxidase and MsrA regulate ox-CaMKII and AF susceptibility.
Ang II increases intracellular ROS in myocardium by activating NADPH oxidase and
p47-/-mice28, lacking functional NADPH oxidase, are resistant to Ang II dependent increases in ROS and ox-CaMKII.6
Atrial lysates from Ang II treated p47-/- mice did not show an increase in ox-CaMKII (Figure 4), and
the p47-/- mice were also resistant to Ang II-mediated increases in AF
However, there were similar increases in BP (Figure 3B) effects of Ang II. This was observed with MsrA TG and WT mice (Figure 3A), showing similar increases in BP (Figure 3B), overall heart weight (Figure 3C) and estimated left ventricular mass (Supplementary Figure 6) after Ang II treatment compared to WT controls. ox-CaMKII is reduced by MsrA15 and transgenic mice with myocardial-delimited MsrA overexpression (MsrA TG) have increased atrial MsrA protein (Supplementary Figure 3C) and
are resistant to ROS induced myocardial injury.16
We found that Ang II treated MsrA TG mice showed decreased AF induction compared to Ang II-treated WT mice (Figure 3A) and
had similar atrial ox-CaMKII expression compared to saline treated controls (Figure 4).
Ang II induced increases in ROS production seen in WT atria were absent in atria from MsrA TG mice (Supplementary Figure 4),
suggesting that MsrA sensitive targets represent an important component of Ang II mediated atrial oxidation. The protection from AF in MsrA TG mice appeared to be independent of pressor effects that are critical for the proarrhythmic actions. Taken together, these findings suggest that
NADPH oxidase dependent ROS and elevated ox-CaMKII drive Ang II -pacing-induced AF and that
targeted antioxidant therapy, by MsrA over-expression, can reduce or prevent AF in Ang -II-infused mice.
Ang II increases Ca2+ sparks and triggered action potentials
CaMKII contributes to increased sarcoplasmic reticulum Ca2+ leak in mice with a RyR2 mutation modeled after a human arrhythmia syndrome, catecholaminergic polymorphic ventricular tachycardia,9 in a goat model of AF and in atrial myocytes isolated from patients with AF.18,29 Atrial myocytes from patients with AF
show increased CaMKII activity and increased CaMKII-dependent ryanodine receptor phosphorylation at serine 2814.29
CaMKII inhibition with KN-93 reduced the open probability of single RyR2 channels and
prevented the increased frequency of sarcoplasmic reticulum Ca2+ sparks in atrial myocardium biopsied from AF patients.18,29
Based on this knowledge, we asked if increased RyR2 Ca2+ leak also contributed to the mechanism of AF in WT Ang II infused mice and measured diastolic Ca2+ sparks, a marker of RyR2 Ca2+ leak.30
Atrial myocytes from Ang II treated WT mice showed a significant (p<0.05) increase in spontaneous Ca2+ sparks compared to atrial myocytes from saline treated control mice (Figure 5A and B).
Other Ca2+ spark parameters and sarcoplasmic reticulum Ca2+ content were not different between the saline and Ang II treated WT mice (Supplementary Figure 7). In contrast to findings in WT mice,
the atrial myocytes isolated from Ang II treated MM-VV mice did not show an increase in Ca2+ sparks compared to saline treated MM-VV mice (Figure 5A and B).
A significantly greater proportion of atrial myocytes isolated from Ang II treated WT mice showed DADs, compared to atrial myocytes from saline treated mice (Figure 5C and D, p=0.03; Fisher’s exact test).
atrial myocytes from Ang II infused MM-VV mice did not show a significant increase in DADs compared to the atrial myocytes from saline treated MM-VV mice.
We interpret these data to suggest that the proarrhythmic effects of Ang I I infusion depend upon an increase in ox–CaMKII, sarcoplasmic reticulum Ca2+ leak and DADs.
Mice with CaMKII-resistant RyR2 are protected from AF after Ang II infusion
Enhanced CaMKII-mediated phosphorylation of serine 2814 on RyR2 is associated with an increased susceptibility to acquired arrhythmias, including AF.31 Based on our findings
that atrial myocytes from Ang II infused WT mice developed more Ca2+ sparks than atrial myocytes from saline-infused mice,
we hypothesized that the proarrhythmic actions of ox-CaMKII require access to RyR2 serine 2814. We tested this hypothesis by treating mutant S2814A knock-in mice (lacking serine 2814)9 with Ang II or saline and performing right atrial burst pacing.
The S2814A mice were highly resistant to Ang II mediated AF (Figure 6A). Similarly,
AC3-I mice with transgenic myocardial expression of a CaMKII inhibitory peptide32 were also resistant to the proarrhythmic effects of Ang II infusion on pacing-induced AF (Figure 6A). S2814A,
AC3-I and WT mice, all developed similar BP increases (Figure 6B) and cardiac hypertrophy (Figure 6C) in response to Ang II, indicating that
these mice were not resistant to the hemodynamic effects of Ang II, but were nevertheless protected from AF.
Discussion
AF usually develops in patients with underlying structural heart disease, such as left ventricular hypertrophy, coronary artery disease, valve disease and congestive heart failure.20 Elevated ROS is a common feature of these conditions.33 The dose of Ang II used in our model produces a fourfold increase in plasma Ang II compared to saline controls,7 similar to increases in Ang II observed in heart failure patients evidence of elevated ROS in structural heart disease, clinical trials with antioxidants have generally been unsatisfactory.34-36 One potential obstacle to developing effective antioxidant therapies is lack of detailed understanding of molecul ra pathways that are affected by ROS. The renin-angiotensin-system is one of the best understood pathways that contributes to ROS production in AF patients.37 In the current study, we created a model of AF by infusing mice with Ang II for three weeks and assembled a cohort of genetically altered mice to rigorously test a novel molecular pathway that links oxidative stress to AF (Figure 7). Our current study provides strong evidence that CaMKII is a critical ROS sensor for transducing increased ROS into enhanced AF susceptibility in mice and suggests that atrial ox-CaMKII could contribute to AF in patients.
CaMKII and increased ROS are now widely recognized to contribute to cardiac arrhythmias.8,38,39 Recent studies suggest that patients with persistent AF have elevated markers of oxidative stress in serum4 and depleted levels of atrial glutathione.40 Under increased oxidative stress CaMKII is activated by oxidation ofmethionines (M281/282),6 which lock it into a constitutively active conformation, suggesting a possible role for ox-CaMKII as a ROS activated proarrhythmic signal in AF.39 Our laboratory recently demonstrated that
ox-CaMKII plays a major role in sinus node dysfunction,7,22
adverse post-myocardial infarct remodeling6 and
cardiac rupture16.
In the current study, we investigated the role of ox-CaMKII in AF. Human atria (Figure 1) and Ang II treated WT mouse atria showed significantly elevated ox-CaMKII (Figure 4).
Atrial myocytes from Ang II treated WT mice had a higher frequency of spontaneous Ca2+ sparks and DADs compared to controls (Figure 5).
Based on these findings we hypothesized that oxidation of methionines 281/282 on CaMKII į causes diastolic sarcoplasmic reticulum Ca2+ leak and DADs, both cellular AF triggers. However,resistant to oxidative activation,22
Ang II, the myocardial CaMKII a recently developed knock-in mouse (MM-VV) where CaMKII isoform implicated in myocardial disease,1,2 13 treatment
did not increase Ca2+ and calmodulin independent CaMKII activity (Supplementary Figure 2A), Ca2+ sparks (Figure 5A and B), DADs (Figure 5C and D) or enhance AF susceptibility in MM-VV mice (Figure 3A).
It is important to note that the MM-VV mutant form of CaMKIIį selectively ablates the response to oxidation while retaining other aspects of CaMKII molecular physiology, such as
activation by Ca2+ and calmodulin and
constitutive activation by threonine 287 autophosphorylation.6
Thus, the residual AF observed in Ang II infused MM-VV mice could be a result of non-oxidation-dependent mechanisms for CaMKIIį activation in our model. We found that atrial tissue from AF patients treated with ACE-i or ARBs did not show elevated ox-CaMKII, suggesting that Ang II stimulation oxidizes CaMKII in human atria and that ox-CaMKII independent pathways are operative in AF patients. AF in patients is more complex than AF in our Ang II infused mice. In particular, patients present with variable chronicity, tissue and structural changes. In contrast the triggers for our mice are uniform (i.e. Ang II infusion and rapid right atrial pacing) and result in a similar, modest degree of hypertrophy. We interpret the data showing that an increase in ox-CaMKII in AF patients is reduced or eliminated by clinical antagonist drugs that reduce Ang II signaling to validate our findings in mice that Ang II increases ox-CaMKII. However, we suppose that the presence of AF in patients on ACE-i or ARBs means that other pathways also result in AF. Our sample is not powered to ask if AF resistance to Ang II antagonist drugs represents later stage disease, but this is our hypothesis. Furthermore, CaMKII can be activated independently of oxidation, although oxidation appears to be the primay r pathway for activating CaMKII during Ang II infusion. Thus, it is unknown if CaMKII is also important for AF progression in the group of patients treated by Ang II antagonist drugs who exhibit normal levels of ox -CaMKII.
Although we did not see higher total CaMKII in AF patients (as compared with patients in sinus rhythm), the sub-group of AF patients who were not treated with ACE-i or ARBs did show significantly elevated CaMKII levels, supporting prior studies that reported elevated CaMKII activity in AF18,19. In contrast to the situation in patients, total CaMKII expression was reduced in mice after sub-acute Ang II infusion. While the mechanism(s) for the variable response of CaMKII expression in mice and patients is unclear, the change in expression in mice and in humans in response to manipulation of the Ang II pathway supports the idea that CaMKII is a fundamental component of Ang II signaling. The relatively small number of patient samples is not powered for analysis of AF subtypes, but human AF may transition from paroxysmal to persistent and permanent (chronic) forms.41 In contrast, our mouse model is simpler because it is triggered by a single upstream event (i.e. Ang II infusion) and elicited in a highly controlled environment by rapid atrial pacing. The resistance of MM-VV mice to AFprovides new evidence that oxidative activation of CaMKII delta (d) is important for initiation of AF, while the finding that ox-CaMKII is elevated in atrial tissue from AF patients and particularly in AF patients naive to Ang II antagonist therapies suggests this pathway may also participate in human AF.
Thus, our findings in MM-VV mice provide strong, mechanistic evidence that ox-CaMKII plays a critical role in proarrhythmic responses to Ang II. Our studies showed that mice deficient in NADPH oxidase (p47-/-) and mice expressing increased MsrA are also resistant to AF(Figure 3A), suggesting that
selectively targeted antioxidant therapies could be effective in preventing or reducing AF.
Half of patients enrolled in the Mode Selection Trial (MOST) with sinus node dysfunction had a history of AF48,
but a clear mechanistic link between increased risk of AF and sinus node dysfunction is unknown. In recent studies we showed that Ang II and diabetes-induced CaMKII oxidation caused sinus node dysfunction by increased pacemaker cell death and fibrosis,7 while MM-VV mice are resistant to sinus node dysfunctionevoked by hyperglycemia.22 Here we provide evidence that
ox-CaMKII increases susceptibility for AF via increased diastolic sarcoplasmic reticulum Ca2+ release, showing that
the proarrhythmic actions of ox-CaMKII may occur in cardiomyocytes by increasing sarcoplasmic reticulum Ca2+ leak or by enhanced cell death.
Our findings suggest that the clinical association between sinus node dysfunction and AF might have a mechanistic basis because sinus node dysfunction and AF are downstream consequences of elevated ox-CaMKII.
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18. Neef S, Dybkova N, Sossalla S, Ort KR, Fluschnik N, Neumann K, Seipelt R, Schöndube FA, Hasenfuss G, Maier LS. CaMKII-dependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels in right atrial myocardium of patients with atrial fibrillation. Circ Res. 2010;106:1134-1144.
19. Tessier S, Karczewski P, Krause EG, Pansard Y, Acar C, Lang-Lazdunski M, Mercadier JJ, Hatem SN. Regulation of the transient outward K+ current by Ca2+/calmodulin-dependent protein kinases II in human atrial myocytes. Circ Res. 1999;85:810-819.
22. Luo M, Guan X, Luczak ED, Lang D, Kutschke W, Gao Z, Yang J, Glynn P, Sossalla S, Swaminathan PD, Weiss RM, Yang B, Rokita AG, Maier LS, Efimov IR, Hund TJ, Anderson ME. Diabetes increases mortality after myocardial infarction by oxidizing CaMKII. J Clin Invest. 2013;123:1262-1274. 24. Wu Y, Gao Z, Chen B, Koval OM, Singh MV, Guan X, Hund TJ, Kutschke W, Sarma S, Grumbach IM, Wehrens XHT, Mohler PJ, Song L-S, Anderson ME. Calmodulin kinase II is required for fight or flight sinoatrial node physiology. Proc Natl Acad Sci USA. 2009;106:5972-5977. 25. Dzhura I, Wu Y, Colbran RJ, Balser JR, Anderson ME. Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels. Nat Cell Biol. 2000;2:173-177. 26. Koval OM, Guan X, Wu Y, Joiner ML, Gao Z, Chen B, Grumbach IM, Luczak ED, Colbran RJ, Song LS, Hund TJ, Mohler PJ, Anderson ME. CaV1.2 -subunit coordinates CaMKII triggered cardiomyocyte death and afterdepolarizations. Proc Natl Acad Sci USA. 2010;107:4996–5000. 44. Anderson ME. Multiple downstream proarrhythmic targets for calmodulin kinase II: moving beyond an ion channel-centric focus. Cardiovasc Res. 2007;73:657-666.
46. Chang HY, Lin YJ, Lo LW, Chang SL, Hu YF, Li CH, Chao TF, Yin WH, Chen SA. Sinus node dysfunction in atrial fibrillation patients: the evidence of regional atrial substrate remodelling. Europace. 2013;15:205-211.
47. Lee JMS, Kalman JM. Sinus node dysfunction and atrial fibrillation: two sides of the same coin? Europace. 2013;15:161-162.
Table 1. Summary of patient characteristics. A. Patient characteristics for immunofluorescence studies in Figure 1A and B. B. Patient characteristics for immunoblotting experiments in Figure 1C-F. http://dx.doi.org/10.1161/CIRCULATIONAHA.113.003313
Figures and/or Legends
The source of all the figures is from the circulation article – including supplementary. Obtaining the images and presenting them in a cropped form was difficult.
Figure 1. ox-CaMKII is increased in atria from patients with Atrial Fibrillation (AF).
A. Representative immunofluorescence images using antiserum against ox-CaMKII in fixed sections of right atrial tissue from patients with sinus rhythm (SR) or AF. B. Image quantification showing significantly higher ox-CaMKII in patients with AF compared to SR (*p<0.05, Student’s t-test). C. Representative immunoblots with ox-CaMKII antiserum in right atrial tissue homogenates from patients in SR or AF. D. Quantification of immunoblots showing significantly higher ox-CaMKII expression in patients with AF compared to SR (*p<0.05, Student’s t-test). The % value indicates the mean ox-CaMKII/GAPDH ratio as normalized to the mean ox-CaMKII/GAPDH ratio in the SR group. E. CaMKII antiserum in right atrial tissue homogenates from patients in SR or AF. F. Quantification of immunoblots showing similar total CaMKII expression in patients with AF and SR (p=0.3, Student’s t-tes )t . The % value indicates the mean CaMKII/GAPDH ratio as normalized to the me na CaMKII/GAPDH ratio in the SR group. The numerals shown in the bars indicate the sample size in each group, here and in subsequent figures.
Figure 2. Ang II treatment increases AF inducibility in WT mice. A. Representative atrial (A-EGM) and ventricular (V-EGM) intracardiac electrograms and lead II surface ECG immediately after burst pacing show AF or SR in WT mice treated with Ang II or saline for 3 weeks. B. Contrasting R-R interval variability in AF and SR (C). Blue bars indicate calculated values from lead II ECGs shown in panel A. D. Higher AF inducibility in the Ang II treatment group (*p<0.05, Fisher’s exact test). E. Increase in systolic blood pressure (sBP) in WT mice after 3
Figure 3. CaMKII oxidation is critical to Ang II mediated AF. A. MM-VV, p47-/- and MsrA TG mice were resistant to Ang II mediated AF (*p<0.05 versus Ang II treated MM-VV, p47-/- and MsrA TG mice, Fisher’s exact test). B. All mice in panel A (WT, MM-VV, p47-/- and MsrA TG) showed a pressor response to Ang II. C. Ang II treatment induced cardiac hypertrophy as assessed by heart weight normalized to body weight (all comparisons versus saline controls from each genotype after 3 weeks of Ang II treatment(p< 0.05) (**p<0.01, Student’s t-test). The numerals shown in the graph indicate the number of mice in each group. F. Significantly higher echocardiographically estimated left ventricular (LV) mass in Ang II treated mice compared to saline controls (***p<0.001, Student’s t-test). G. Similar LV ejection fraction (LVEF) in Ang II and saline treated mice. (** p<0.01 and ***p<0.001, Student’s t-test).
Figure 4. – ox-CaMKII in atria after Ang II or saline treatment A. Atrial lys ate immunoblots from WT, MM-VV, p47 -/- and MsrA TG mice treated with Ang II or saline for 3 weeks and probed with an antiserum for ox-CaMKII. For quantification, ox-CaMKII bands were normalized to the total protein loading as assessed with Coomassie staining of the membrane. B. Increase in ox-CaMKII with Ang II treatment expressed as relative to the saline treated group. From each genotype 4 saline treated mice were used as controls. *p<0.05, for WT Ang II versus WT saline (*), in all other genotypes Ang II versus saline p>0.05; in addition, p=0.02 for WT Ang II versus MsrA TG Ang II and p=0.05 for MM-VV Ang II versus MsrA TG Ang II. C. Fold change in ox-CaMKII (over total CaMKII) in Ang II as relative to saline treated mice of the same genotype. From each genotype 4 saline treated mice were used as controls. ***p<0.001 versus WT saline, *p<0.05 versus MM-VV saline, #p<0.05 versus MsrA TG saline. WT Ang II versus p47-/- Ang II, P = 0.001, WT Ang II versus MsrA TG Ang II, P<0.0001, MM-VV Ang II versus MsrA TG Ang II, P=0.001. Data were analyzed using two-way ANOVA (for treatment and genotype) with Bonferroni post-hoc comparisons.
Figure 5. Ang II promotes Ca2+ sparks and DADs.
A. Representative examples of Ca2+ sparks in atrial myocytes from Ang II and saline treated WT and MM-VV mice. B. Summary of Ca2+ spark frequency data in atrial myocytes from Ang II treated mice compared to saline treated mice (*p<0.05 versus saline; Student’s t-test); WT saline (N=23 cells from 5 mice), WT Ang II (N=30 cells from 4 mice), MM-VV saline (N=36 cells from 4 mice) and MM-VV Ang II (N=28 cells from 4 mice). C. Examples of stimulated action potentials and a spontaneous, DAD triggered action potential. D. Higher incidence of DADs in atrial myocytes from Ang II treated WT mice ( *p<0.05 versus saline, Fisher’s exact test) but not in Ang II treated MM-VV mice compared to saline controls. Numerals show cells with DADs/total cells studied for each group.
Figure 6. CaMKII activation and RyR2 serine 2814 are required for AF in Ang II infused mice.
A. AC3-I and S2814A mice were treated with Ang II for 3 weeks and then burst paced to induce AF. AC3-I and S2814A mice were resistant to Ang II mediated AF promotion compared to WT Ang II treated mice (*p<0.05 versus all, Fisher’s Exact test, N=number of mice tested in each group). B. AC3-I and S2814A mice show similar systolic blood pressure (sBP) elevation after treatment with Ang II. Final sBP measurements were performed on three consecutive days prior to AF induction as shown in panel A. The numerals in the graph indicate the number of mice in each group. C. Ang II treatment causes similar cardiac hypertrophy in AC3-I and S2814A mice compared to saline controls (***p<0.001 versus AC3-I saline and **p=0.01 versus S2814A saline).
Figure 7. Schematic to illustrate the proposed mechanism of AF in Ang II infused mice.
Ang II binding activates NADPH oxidase (NOX) to increase reactive oxygen species (ROS), leading to oxidation of methionines 281/282 in CaMKII (ox-CaMKII). Elevated ox-CaMKII phosphorylates serine 2814 on RyR2, causing enhanced diastolic Ca2+ leak that promotes AF triggering DADs. Genetically modified mice were used to test key steps of the proposed pathway.
Additional Comments
This paper might be considered and compared with other papers in this series.
I Contributions to cardiomyocyte interactions and signaling
Cardiomyocyte hypertrophy and degradation of connexin43 through spatially restricted autocrine/paracrine heparin-binding EGF
J Yoshioka, RN Prince, H Huang, SB Perkins, FU Cruz, C MacGillivray, DA Lauffenburger, and RT Lee *Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; and Biological Engineering Division, MIT, Cambridge, MA PNAS 2005; 302(30):10622-10627. http://pnas.org/cgi/doi/10.1073/pnas.0501198102
Growth factor signaling can affect tissue remodeling through autocrine/paracrine mechanisms. Recent evidence indicates that EGF receptor transactivation by heparin-binding EGF (HB-EGF) contributes to hypertrophic signaling in cardiomyocytes. Here, we show that HB-EGF operates in a spatially restricted circuit in the extracellular space within the myocardium, revealing the critical nature of the local microenvironment in intercellular signaling. This highly localized microenvironment of HB-EGF signaling demonstrated with 3D morphology, consistent with predictions from a computational model of EGF signaling. HB-EGF secretion by a given cardiomyocyte in mouse left ventricles led to cellular hypertrophy and reduced expression of connexin43 in the overexpressing cell and in immediately adjacent cells but not in cells farther away.
!!. Ca2+/calmodulin δ Dependent Protein Kinase Modulates Cardiac Ryanodine Receptor Phosphorylation and Sarcoplasmic Reticulum Ca2+ Leak in Heart Failure.
This contribution is unique in establishing a relationship between Ca2+ sparks in abnormal release from sarcoplasmic reticulum via the ryanodine receptor (RyR2) in contractile dysfunction and arrhythmogenesis in heart failure. This is based on decreased transient amplitude and SR Ca2+ load with increased Na+/Ca++ exchange, and in nonischemic heart failure in a rabbit model. In this case – with HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin–dependent protein kinase II (CaMKII) expression were increased 50% to 100%. In this study, the arrhythmogenesis appears to be ventricular.
Contractile dysfunction in HF is caused by diminished sarcoplasmic reticulum (SR) Ca load that could arise from enhanced activity of Na/Ca exchange (NCX), reduced SR Ca ATPase (SERCA) function, and increased diastolic SR Ca leak via ryanodine receptors (RyR), all of which we have demon¬strated to occur in our arrhythmogenic rabbit model of nonis-chemic HF. HF is also associated with a nearly 50% incidence of sudden cardiac death from ventricular tachycardia (VT) that degenerates to ventricular fibrillation (VF). In 3D cardiac mapping studies in our HF rabbit model, we showed that spontaneously occurring VT initiates by nonreentrant mechanisms associated with delayed afterdepolarizations. These arise from spontaneous SR Ca release that activates a transient inward current (Iti) carried primarily by NCX.2 Thus abnormal SR Ca release via RyR may contribute to both contractile dysfunction and arrhythmogenesis.
Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin–dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF. (Circ Res. 2005;97:1314-1322.)
Mark E. Andserson makes the point that CaMKII(δ) is the biggest calcium signaling channel, and it is pluripotent in the heart muscle.
The multifunctional Ca2+ and calmodulin (CaM)-dependent protein kinase II (CaMKII) is a serine threonine kinase that is abundant in heart where it phosphorylates Ca2+i homeostatic proteins. It seems likely that CaMKII plays an important role in cardiac physiology because these target proteins significantly overlap with the more extensively studied serine threonine kinase, protein kinase A (PKA), which is a key arbiter of catecholamine responses in heart. However, the physiological functions of CaMKII remain poorly understood, whereas the potential role of CaMKII in signaling myocardial dysfunction and arrhythmias has become an area of intense focus. CaMKII activity and expression are upregulated in failing human hearts and in many animal models of structural heart disease. CaMKII inhibitory drugs can pre-vent cardiac arrhythmias and suppress afterdepolarizations that are a probable proximate focal cause of arrhythmias in heart failure.
Cardiac contraction is initiated when Ca2+ current (ICa), through sarcolemmal L-type Ca2+ channels (LTCC), triggers RyR opening by a Ca2+-induced Ca2+ release (CICR) mechanism. LTCCs “face off” with RyRs across a highly ordered cytoplasmic cleft that delineates a kind of Ca2+ furnace during each CICR-initiated heart beat (Figure). CICR has an obvious need to function reliably, so it is astounding to consider how this feed forward process is intrinsically unstable. The increased instability of CICR in heart failure is directly relevant to arrhythmias initiated by afterdepolarizations. RyRs partly rely on a collaboration of Ca2+-sensing proteins in the SR lumen to grade their opening probability and the amount of SR Ca2+ release to a given ICa stimulus.
LTCCs and RyRs form the protein machinery for initiating contraction in cardiac and skeletal muscle, but in cardiac muscle communication between these proteins occurs without a requirement for physical contact. PKA is preassociated with LTCCs and RyRs, and PKA-dependent phosphorylation increases LTCC8 and RyR9opening. The resultant increase in Ca2+i is an important reason for the positive inotropic response to cathecholamines. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by increased Ca2+I, and so catecholamine stimulation activatesCaMKII in addition to PKA. In contrast to PKA, which is tightly linked to inotropy, CaMKII inhibition does not cause a reduction in fractional shortening during acute cate-cholamine stimulation in mice.
The key clinical phenotypes of contractile dysfunction and electrical instability in heart failure involve problems with Ca2+i homeostasis. Broad changes in Ca2+I-handling proteins can occur in various heart failure models, but in general heart failure is marked by a reduction in the capacity for SR Ca2+ uptake, enhanced activity of the sarcolemmal Na+-Ca2+ exchanger, and reduction in CICR-coordinated SR Ca2+ release. On the other hand, the opening probability of individual LTCCs is increased in human heart failure.
The Marks group pioneered the concept that RyRs are hyperphosphorylated by PKA in patients with heart failure and showed that successful therapies, ranging from beta blockers to left ventricular assist devices, reduce RyR phosphorylation in step with improved mechanical function. They have developed a large body of evidence in patients and in animal models that PKA phosphorylation of Ser2809 on cardiac RyRs destabilizes binding of FK12.6 to RyRs and promotes increased RyR opening that causes an insidious Ca2+ leak. This leak is potentially problematic because it can reduce SR Ca2+ content (to depress inotropy), engage pathological Ca2+-dependent transcriptional programs (to promote myocyte hypertrophy), and activate arrhythmia-initiating af-terdepolarizations (to cause sudden death).
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
In the first part, we discussed common MOTIFs across cell-types that are essential for cell division, embryogenesis, cancer metastasis, osteogenesis, musculoskeletal function, vascular compliance, and cardiac contractility. This second article concentrates on specific functionalities for cardiac contractility based on Ca++ signaling in excitation-contraction coupling. The modifications discussed apply specifically to cardiac muscle and not to skeletal muscle. Considering the observations described might raise additional questions specifically address to the unique requirements of smooth muscle, abundant in the GI tract and responsible for motility in organ function, and in blood vessel compliance or rigidity. Due to the distinctly different aspects of the cardiac contractility and contraction force, and the interactions with potential pharmaceutical targets, there are two separate articles on calcium signaling and cardiac arrhythmias or heart failure (Part 2 and Part 3). Part 2 focuses on the RYANODINE role in cardiac Ca(2+) signaling and its effect in heart failure. Part 3 takes up other aspects of heart failure and calcium signaling with respect to phosporylation/dephosphorylation. I add a single review and classification of genetic cardiac disorders of the same cardiac Ca(2+) signaling and the initiation and force of contraction. Keep in mind that the heart is a syncytium, and this makes a huge difference compared with skeletal muscle dynamics. In Part 1 there was some discussion of the importance of Ca2+ signaling on innate immune system, and the immunology will be further expanded in a fourth of the series.
SUMMARY:
This second article on the cardiomyocyte and the Ca(2+) cycling between the sarcomere and the cytoplasm, takes a little distance from the discussion of the ryanodine that precedes it. In this discussion we found that there is a critical phosphorylation/dephosphorylation balance that exists between Ca(+) ion displacement and it occurs at a specific amino acid residue on the CaMKIId, specific for myocardium, and there is a 4-fold increase in contraction and calcium release associated with this CAM kinase (ser 2809) dependent exchange. These events are discussed in depth, and the research holds promise for therapeutic application. We also learn that Ca(2+) ion channels are critically involved in the generation of arrhythmia as well as dilated and hypertrophic cardiomyopathy. In the case of arrhythmiagenesis, there are two possible manners by which this occurs. One trigger is Ca(2+) efflux instability. The other is based on the finding that when the cellular instability is voltage driven, the steady-state wavelength (separation of nodes in space) depends on electrotonic coupling between cells and the steepness of APD and CV restitution. The last article is an in depth review of the genetic mutations that occur in cardiac diseases. It is an attempt at classifying them into reasonable groupings. What are the therapeutic implications of this? We see that the molecular mechanism of cardiac function has been substantially elucidated, although there are contradictions in experimental findings that are unexplained. However, for the first time, it appears that personalized medicine is on a course that will improve health in the population, and the findings will allow specific targets designed for the individual with a treatable impairment in cardiac function that is identifiable early in the course of illness. This article is a continuation to the following articles on tightly related topics: Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton Larry H Bernstein, MD, FCAP http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/ Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/ Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD and Aviva Lev-Ari, PhD, RN http://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-exchange-mechanism-in-health-and-disease/ Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN http:/pharmaceuticalintelligence.com/2013.09.089/lhbern/The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Part V: Heart Smooth Muscle and Cardiomyocyte Cells: Excitation-Contraction Coupling & Ryanodine Receptor (RyR) type-1/type-2 in Cytoskeleton Cellular Dynamics and Ca2+ Signaling
an increase in the initial rates of Ca(2+) transport by SR vesicles
which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence.
The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which
can dephosphorylate both the CAMP-dependent and the calcium calmodulin-dependent sites on phospholamban.
Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases.
Regulation of the Cardiac Ryanodine Receptor Channel by Luminal Ca2+ involves Luminal Ca2+ Sensing Sites
I Györke, S Györke. Biophysical Journal 01/1999; 75(6):2801-10. · 3.65 Impact factor http:// www.researchgate.net/publication/13459335/Regulation_of_the_cardiac_ryanodine_receptor_channel_by_luminal_Ca2_involves_luminal_Ca2_sensing_sites The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca(2+) was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca(2+) (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca(2+) on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low [trans Ca(2+)] (20 microM), increasing [cis Ca(2+)] from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating [cis Ca(2+)] [cytosolic] above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca(2+)] [luminal] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] [luminal] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca(2+)
enhanced the sensitivity of the channel to activating cytosolic Ca(2+), and it
essentially reversed the inhibition of the channel by high cytosolic Ca(2+).
Potentiation of Po by increased luminal Ca(2+) occurred irrespective of whether the electrochemical gradient for Ca(2+) supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca(2+) through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca(2+) acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca(2+)-sensitive site(s) at the luminal face of the channel or associated protein.
Protein phosphatases Decrease Sarcoplasmic Reticulum Calcium Content by Stimulating Calcium Release in Cardiac Myocytes
D Terentyev, S Viatchenko-Karpinski, I Gyorke, R Terentyeva and S Gyorke Texas Tech University Health Sciences Center, Lubbock, TX J Physiol 2003; 552(1), pp. 109–118. http://dx.doi.org/10.1113/jphysiol.2003.046367 Phosphorylation/dephosphorylation of Ca2+ transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation–contraction coupling; furthermore,
abnormal protein kinase and phosphatase activities have been implicated in heart failure.
However, the precise mechanisms of action of these enzymes on intracellular Ca2+ handling in normal and diseased hearts remains poorly understood. We have investigated
the effects of protein phosphatases PP1 and PP2A on spontaneous Ca(2+) sparks and SR Ca(2+) load in myocytes permeabilized with saponin.
Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca(2+) sparks followed by a nearly complete disappearance of events, which were accompanied by depletion of the SR Ca(2+) stores, as determined by application of caffeine. These changes in
Ca(2+) release and
SR Ca(2+) load
could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1-medited RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti-RyR antibody. Our results suggest that
leading to depleted SR Ca(2+) stores in cardiac myocytes.
In heart muscle cells, the process of excitation–contraction (EC) coupling is mediated by
Ca(2+) influx through sarcolemmal L-type Ca(2+) channels
activating Ca(2+) release channels (ryanodine receptors, RyRs) in the sarcoplasmic reticulum (SR).
Once activated, the RyR channels allow Ca(2+) to be released from the SR into the cytosol to induce contraction. This mechanism is known as Ca(2+)-induced calcium release (CICR) (Fabiato, 1985; Bers, 2002). During relaxation, most of the Ca(2+) is resequestered into the SR by the Ca(2+)-ATPase. The amount of Ca(2+) released and the force of contraction depend on
the magnitude of the Ca(2+) trigger signal,
the functional state of the RyRs and
the amount of Ca(2+) stored in the SR.
F1.large calcium movement and RyR2 receptor calcium release calmodulin + ER Reversible phosphorylation of proteins composing the EC coupling machinery plays an important role in regulation of cardiac contractility (Bers, 2002). Thus, during stimulation of the b-adrenergic pathway, phosphorylation of several target proteins, including
the L-type Ca(2+) channels,
RyRs and
phospholamban,
by protein kinase A (PKA) leads to an overall increase in SR Ca2+ release and contractile force in heart cells (Callewaert et al. 1988, Spurgeon et al. 1990; Hussain & Orchard, 1997; Zhou et al. 1999; Song et al. 2001; Viatchenko-Karpinski & Gyorke, 2001). PKA-dependent phosphorylation of the L-type Ca(2+) channels increases the Ca2+ current (ICa), increasing both
the Ca2+ trigger for SR Ca2+ release and
the SR Ca(2+) content
(Callewaert et al. 1988; Hussain & Orchard, 1997; Del Principe et al. 2001). Phosphorylation of phospholamban (PLB) relieves the tonic inhibition dephosphorylated PLB exerts on the SR Ca(2+)-ATPase (SERCA) resulting in enhanced SR Ca(2+) accumulation and enlarged Ca(2+) release (Kranias et al. 1985; Simmermann & Jones, 1998). With regard to the RyR, despite clear demonstration of phosphorylation of the channel in biochemical studies (Takasago et al. 1989; Yoshida et al. 1992), the consequences of this reaction to channel function have not been clearly defined. RyR phosphorylation by PKA and Ca(2+)–calmodulin dependent protein kinase (CaMKII) has been reported to increase RyR activity in lipid bilayers (Hain et al. 1995; Marx et al. 2000; Uehara et al. 2002). Moreover, it has been reported that in heart failure (HF), hyperphosphorylation of RyR causes
the release of FK-506 binding protein (FKBP12.6) from the RyR,
rendering the channel excessively leaky for Ca(2+) (Marx et al. 2000).
However, other studies have reported no functional effects (Li et al. 2002) or even found phosphorylation to reduce RyR channel steady-state open probability (Valdivia et al. 1995; Lokuta et al. 1995). The action of protein kinases is opposed by dephosphorylating phosphatases. Three types of protein phosphatases (PPs), referred to as PP1, PP2A and PP2B (calcineurin), have been shown to influence cardiac performance (Neumann et al. 1993; Rusnak & Mertz, 2000). Overall, according to most studies phosphatases appear to downregulate SR Ca(2+) release and contractile performance (Neumann et al. 1993; duBell et al. 1996, 2002; Carr et al. 2002; Santana et al. 2002). Furthermore, PP1 and PP2A activities appear to be increased in heart failure (Neumann, 2002; Carr et al. 2002). However, again the precise mode of action of these enzymes on intracellular Ca(2+) handling in normal and diseased hearts remains poorly understood. In the present study, we have investigated the effects of protein phosphatases PP1 and PP2A on local Ca(2+) release events, Ca(2+) sparks, in cardiac cells. Our results show that
phosphatases activate RyR mediated SR Ca(2+) release
leading to depletion of SR Ca(2+) stores.
These results provide novel insights into the mechanisms and potential role of protein phosphorylation/dephosphorylation in regulation of Ca(2+) signaling in normal and diseased hearts.
RESULTS
Effects of PP1 and PP2A on Ca2+ sparks and SR Ca(2+) content.
[1] PP1 caused an early transient potentiation of Ca2+ spark frequency followed by a delayed inhibition of event occurrence. [2] PP1 produced similar biphasic effects on the magnitude and spatio-temporal characteristics of Ca(2+) sparks Specifically, during the potentiatory phase (1 min after addition of the enzyme), PP1 significantly increased
the amplitude,
rise-time,
duration and
width of Ca(2+) sparks;
during the inhibitory phase (5 min after addition of the enzyme),
all these parameters were significantly suppressed by PP1.
The SR Ca(2+) content decreased by 35 % or 69 % following the exposure of myocytes to either 0.5 or 2Uml_1 PP1, respectively (Fig. 1C). Qualitatively similar results were obtained with phosphatase PP2A. Similar to the effects of PP1, PP2A (5Uml_1) produced a transient increase in Ca(2+) spark frequency (~4-fold) followed by a depression of event occurrence and decreased SR Ca(2+) content (by 82 % and 65 %, respectively). Also similar to the action of PP1, PP2A increased
the amplitude and
spatio-temporal spread (i.e. rise-time, duration and width) of Ca(2+) sparks at 1 min
and suppressed the same parameters at 5 min of exposure to the enzyme (Table 1).
Together, these results suggest that phosphatases enhance spark-mediated SR Ca2+ release, leading to decreased SR Ca(2+) content.Preventive effects of calyculin A and okadaic acid Preventive effects of ryanodine
PP1-mediated RyR dephosphorylation
F3.large cardiomyocyte SR F2.large RyR and calcium coupled receptors The cardiac RyR is phosphorylated at Ser-2809 (in the rabbit sequence) by both PKA and CAMKII (Witcher et al. 1991; Marx et al. 2000). Although additional phosphorylation sites may exist on the RyR (Rodriguez et al. 2003), but Ser-2809 is believed to be the only site that is phosphorylated by PKA, and RyR hyperphosphorylation at this site has been reported in heart failure (Marx et al. 2000). To test whether indeed phosphatases dephosphorylated the RyR in our permeabilized myocyte experiments we performed quantitative immunoblotting using an antibody that specifically recognizes the phosphorylated form of the RyR at Ser-2809 (Rodriguez et al. 2003). Myocytes exhibited a significant level of phosphorylation under baseline conditions. Maximal phosphorylation was 201 % of control. When exposed to 2Uml_1 PP1, RyR phosphorylation was 58 % of the control basal condition. Exposing to a higher PP1 concentration (10Uml_1) further reduced RyR phosphorylation to 22% of control. Thus, consistent with the results of our functional measurements,
PP1 decreased RyR phosphorylation in cardiac myocytes.
Figure 1. Effects of PP1 on properties of Ca(2+) sparks and SR Ca(2+) content in rat permeabilized myocytes see . http://dx.doi.org/10.1113/jphysiol.2003.046367 A, spontaneous Ca(2+) spark images recorded under reference conditions, and 1 or 5 min after exposure of the cell to 2Uml_1 PP1. Traces below the images are Ca(2+) transients induced by application of 10 mM caffeine immediately following the acquisition of sparks before (3 min) and after (5 min) application of PP1 in the same cell. The Ca(2+) transients were elicited by a whole bath application of 10 mM caffeine. B, averaged spark frequency at early (1 min) and late (5 min) times following the addition of either 0.5 or 2Uml_1 of PP1 to the bathing solution. C, averaged SR Ca(2+) content for 0.5 or 2Uml_1 of PP1 measured before and 5 min after exposure to the enzyme. Data are presented as means ± S.E.M. of 6 experiments in different cells. Figure 2. Effects of PP2A on properties of Ca2+ sparks and SR Ca2+ content in rat permeabilized myocytes see . http://dx.doi.org/10.1113/jphysiol.2003.046367 A, spontaneous Ca(2+) spark images recorded under reference conditions, and 1 or 5 min after exposure of the cell to 5Uml_1 PP2A. Traces below the images are Ca(2+) transients induced by application of 10 mM caffeine immediately following the acquisition of sparks before (3 min) and after (5 min) application of PP2A in the same cell. B and C, averaged spark frequency (B) and SR Ca(2+) content (C) for the same conditions as in A. Data are presented as means ± S.E.M. of 6 experiments in different cells.
DISCUSSION
In the present study, we have investigated the impact of physiologically relevant exogenous protein phosphatases PP1 and PP2A on RyR-mediated SR Ca(2+) release (measured as Ca(2+) sparks) in permeabilized heart cells. Our principal finding is that
phosphatases stimulated RyR channels lead to depleted SR Ca(2+) stores.
These results have important ramifications for understanding the mechanisms and role of protein phosphorylation/dephosphorylation in
modulation of Ca(2+) handling in normal and diseased heart.
Modulation of SR Ca2+ release by protein phosphorylation/dephophorylation
Since protein dephosphorylation clearly resulted in increased functional activity of the Ca(+)release channel, our results imply that a reverse, phosphorylation reaction should reduce RyR activity. If indeed such effects take place, why do they not manifest in inhibition of Ca(+)sparks? One possibility is that enhanced Ca(+) uptake by SERCA
masks or overcomes the effects phosphorylation may have on RyRs.
In addition, the potential inhibitory influence of protein phosphorylation on RyR activity in myocytescould be countered by feedback mechanisms involving changes in luminal Ca(2+)(Trafford et al. 2002; Gyorke et al. 2002). In particular, reduced open probability of RyRs would be expected to lead to
increased Ca2+ accumulation in the SR;
and increased intra-SR [Ca(2+)], in turn would
increase activity of RyRs at their luminal Ca(2+) regulatory sites
as demonstrated for the RyR channel inhibitor tetracaine (Gyorke et al. 1997; Overend et al. 1997). Thus
potentiation of SERCA
combined with the intrinsic capacity of the release mechanism to self-regulate
could explain at least in part why PKA-mediated protein phoshorylation results in maintained potentiation of Ca(2+) sparks despite a potential initial decrease in RyR activity.
Role of altered RyR Phosphorylation in Heart Failure
Marx et al. (2000) have proposed that enhanced levels of circulating catecholamines lead to increased phosphorylation of RyR in heart failure. Based on biochemical observations as well as on studying properties of single RyRs incorporated into artificial lipid bilayers, these investigators have hypothesized that
hyperphosphorylation of RyRs contributes to pathogenesis of heart failure
by making the channel excessively leaky due to dissociation of FKBP12.6 from the channel.
We show that the mode of modulation of RyRs by phosphatases does not support this hypothesis as
dephosphorylation caused activation instead of
Interestingly, our results provide the basis for a different possibility in which
dephophosphorylation of RyR rather than its phosphorylation causes depletion of SR Ca(2+) stores by stimulating RyRs in failing hearts.
It has been reported thatPP1 and PP2 activities are increased in heart failure (Huang et al. 1999; Neumann et al. 1997; Neuman, 2002). Furthermore, overexpression of PP1 or ablation of the endogenous PP1 inhibitor, l-1, results in
depressed contractile performance and heart failure (Carr et al. 2002).
Our finding that PP1 causes depletion of SR Ca(2+) stores by activating RyRs could account for, or contribute to, these results.
References
1 DelPrincipe F, Egger M, Pignier C & Niggli E (2001). Enhanced E-C coupling efficiency after beta-stimulation of cardiac myocytes. Biophys J 80, 64a. 2 Gyorke I & Gyorke S (1998). Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites. Biophys J 75, 2801–2810. 3 Gyorke S, Gyorke I, Lukyanenko V, Terentyev D, Viatchenko-Karpinski S & Wiesner TF (2002). Regulation of sarcoplasmic reticulum calcium release by luminal calcium in cardiac muscle. Front Biosci 7, d1454–d1463. 4 Gyorke I, Lukyanenko V & Gyorke S (1997). Dual effects of tetracaine on spontaneous calcium release in rat ventricular myocytes. J Physiol 500, 297–309. 5 MacDougall LK, Jones LR & Cohen P (1991). Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban. Eur J Biochem 196, 725–734. 6 Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N & Marks AR (2000). PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101, 365–376. 7 Rodriguez P, Bhogal MS & Colyer J (2003). Stoichiometric phosphorylation of cardiac ryanodine receptor on serine-2809 by calmodulin-dependent kinase II and protein kinase A. J Biol Chem (in press).
The δC Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure
T Zhang, LS Maier, ND Dalton, S Miyamoto, J Ross, DM Bers, JH Brown. University of California, San Diego, La Jolla, Calif; and Loyola University, Chicago, Ill. Circ Res. 2003;92:912-919. http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5 Recent studies have demonstrated that transgenic (TG) expression of either Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKIIδB, both of which localize to the nucleus, induces cardiac hypertrophy. However,
CaMKIV is not present in heart, and
cardiomyocytes express not only the nuclear CaMKIIδB
but also a cytoplasmic isoform, CaMKII δC.
In the present study, we demonstrate that
expression of the δC isoform of CaMKII is selectively increased and
its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload.
To determine whether enhanced activity of this cytoplasmic δC isoform of CaMKII can lead to phosphorylation of Ca(2+) regulatory proteins and induce hypertrophy, we generated TG mice that expressed the δC isoform of CaMKII. Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca2(2+) handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and
CaMKII is found associated with the RyR from the CaMKII TG mice.
Phosphorylation of phospholamban is increased specifically at the CaMKII but not at the PKA phosphorylation site.
These findings are the first to demonstrate that CaMKIIδC can mediate phosphorylation of Ca(2+) regulatory proteins in vivo and provide evidence for the involvement of CaMKIIδC activation in the pathogenesis of dilated cardiomyopathy and heart failure. Multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM kinases or CaMKs) are transducers of Ca2+ signals that phosphorylate a wide range of substrates and thereby affect Ca(2+)-mediated cellular responses.1 The family includes CaMKI and CaMKIV, monomeric enzymes activated by CaM kinase kinase,2,3 and CaMKII, a multimer of 6 to 12 subunits activated by autophosphorylation.1 The CaMKII subunits α, β, γ, and δ show different tissue distributions,1 with
the δ isoform predominating in the heart.4–7
Splice variants of the δ isoform, characterized by the presence of a second variable domain,4,7 include δB, which contains a nuclear localization signal (NLS), and
δC, which does not. CaMKII composed of δB subunits localizes to the nucleus, whereas CaMKIIδC localizes to the cytoplasm.4,8,9
CaMKII has been implicated in several key aspects of acute cellular Ca(2+) regulation related to cardiac excitation-contraction (E-C) coupling. CaMKII
phosphorylates sarcoplasmic reticulum (SR) proteins including the ryanodine receptors (RyR2) and
phospholamban (PLB).10–14
Phosphorylation of RyR has been suggested to alter the channel open probability,14,15 whereas phosphorylation of PLB has been suggested to regulate SR Ca(2+) uptake.14 It is also likely that CaMKII phosphorylates the L-type Ca(2+) channel complexor an associated regulatory protein and thus
mediates Ca(2+) current (ICa) facilitation.16-18 and
the development of early after-depolarizations and arrhythmias.19
Thus, CaMKII has significant effects on E-C coupling and cellular Ca(2 +) regulation. Nothing is known about the CaMKII isoforms regulating these responses. Contractile dysfunction develops with hypertrophy, characterizes heart failure, and is associated with changes in cardiomyocyte (Ca2+) homeostasis.20 CaMKII expression and activity are altered in the myocardium of rat models of hypertensive cardiac hypertrophy21,22 and heart failure,23 and
in cardiac tissue from patients with dilated cardiomyopathy.24,25
Several transgenic mouse models have confirmed a role for CaMK in the development of cardiac hypertrophy, as originally suggested by studies in isolated neonatal rat ventricular myocytes.9,26–28 Hypertrophy develops in transgenic mice that overexpress CaMKIV,27 but this isoform is not detectable in the heart,4,29 and CaMKIV knockout mice still develop hypertrophy after transverse aortic constriction (TAC).29 Transgenic mice overexpressing calmodulin developed severe cardiac hypertrophy,30 later shown to be associated with an increase in activated CaMKII31; the isoform of CaMKII involved in hypertrophy could not be determined from these studies. We recently reported that transgenic mice that overexpress CaMKIIδB, which is highly concentrated in cardiomyocyte nuclei, develop hypertrophy and dilated cardiomyopathy.32 To determine whether
in vivo expression of the cytoplasmic CaMKIIδC can phosphorylate cytoplasmic Ca(2+) regulatory proteins and
induce hypertrophy or heart failure,
we generated transgenic (TG) mice that expressed the δC isoform of CaMKII under the control of the cardiac specific α-myosin heavy chain (MHC) promoter. Our findings implicate CaMKIIδC in the pathogenesis of dilated cardiomyopathy and heart failure and suggest that
this occurs at least in part via alterations in Ca(2+) handling proteins.33
Ca(2+) and contraction RyR yuan_image3 Ca++ exchange
Results
Expression and Activation of CaMKIIδC Isoform After TAC
To determine whether CaMKII was regulated in pressure overload–induced hypertrophy, CaMKIIδ expression and phosphorylation were examined by Western blot analysis using left ventricular samples obtained at various times after TAC. A selective increase (1.6-fold) in the lower band of CaMKIIδwas observed as early as 1 day and continuously for 4 days (2.3-fold) and 7 days (2-fold) after TAC (Figure 1A). To confirm that CaMKIIδC was increased and determine whether this occurred at the transcriptional level, we performed semiquantitative RT-PCR using primers specific for the CaMKIIδC isoform. These experiments revealed that
mRNA levels for CaMKIIδC were increased 1 to 7 days after TAC (Figure 1B).
In addition to examining CaMKII expression, the activation state of CaMKII was monitored by its autophosphorylation, which confers Ca2-independent activity.
Figure 1. Expression and activation of CaMKII δC isoform after TAC.
see http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5 A, Western blot analysis of total CaMKII in left ventricular (LV) homogenates obtained at indicated times after TAC. Cardiomyocytes transfected with CaMKIIδB and δC (right) served as positive controls and molecular markers. Top band (58 kDa) represents CaMKIIδB plus δ9, and the bottom band (56 kDa) corresponds to CaMKIIδC. *P0.05 vs control. B, Semiquantitative RT-PCR using primers specific for CaMKIIδC isoform (24 cycles) and GAPDH (19 cycles) using total RNA isolated from the same LV samples. C, Western blot analysis of phospho-CaMKII in LV homogenates obtained at various times after TAC. Three bands seen for each sample represent CaMKIIγ subunit (uppermost), CaMKIIδB plus δ9 (58 kDa), and CaMKIIδC (56 kDa). Quantitation is based on the sum of all of the bands. *P0.05 vs control.
Figure 2. Expression and activation of CaMKII in CaMKIIδC transgenic mice.
see http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5 A, Transgene copy number based on Southern blots using genomic DNA isolated from mouse tails (digested with EcoRI). Probe (a 32P-labeled 1.7-kb EcoRI-SalI -MHC fragment) was hybridized to a 2.3-kb endogenous fragment (En) and a 3.9-kb transgenic fragment (TG). Transgene copy number was determined from the ratio of the 3.9-kb/2.3-kb multiplied by 2. B, Immunocytochemical staining of ventricular myocytes isolated from WT and CaMKIIδTG mice. Myocytes were cultured on laminin-coated slides overnight. Transgene was detected by indirect immunofluorescence staining using rabbit anti-HA antibody (1:100 dilution) followed by FITC-conjugated goat antirabbit IgG antibody (1:100 dilution). CaMKIIδB localization to the nucleus in CaMKIIδB TG mice (see Reference 32) is shown here for comparative purpose. C, Quantitation of the fold increase in CaMKIIδprotein expression in TGL and TGM lines. Different amounts of ventricular protein (numbers) from WT control, TG () and their littermates () were immunoblotted with an anti-CaMKIIδ antibody. Standard curve from the WT control was used to calculate fold increases in protein expression in TGL and TGM lines. D, Phosphorylated CaMKII in ventricular homogenates was measured by Western blot analysis (n5 for each group). **P0.01 vs WT.
Generation and Identification of CaMKIIδC Transgenic Mice
TG mice expressing HA-tagged rat wild-type CaMKIIδC under the control of the cardiac-specific α-MHC promoter were generated as described in Materials and Methods. By Southern blot analysis, 3 independent TG founder lines carrying 3, 5, and 15 copies of the transgene were identified. They were designated as TGL (low copy number), TGM (medium copy number), and TGH (high copy number), The founder mice from the TGH line died at 5 weeks of age with marked cardiac enlargement. The other two lines showed germline transmission of the transgene. The transgene was expressed only in the heart. Although CaMKII protein levels in TGL and TGM hearts were increased 12- and 17-fold over wild-type (WT) controls (Figure 2C), the amount of activated CaMKII was only increased 1.7- and 3-fold in TGL and TGM hearts (Figure 2D). The relatively small increase in CaMKII activity in the TG lines probably reflects the fact that the enzyme is not constitutively activated and that the availability of Ca2/CaM, necessary for activation of the overexpressed CaMKII, is limited. Importantly,
the extent of increase in active CaMKII in the TG lines was similar to that elicited by TAC.
Cardiac Overexpression of CaMKIIδC Induces Cardiac Hypertrophy and Dilated Cardiomyopathy
There was significant enlargement of hearts from CaMKIIδC TGM mice by 8 to 10 weeks [see http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5%5D (Figure 3A) and from TGL mice by 12 to 16 weeks. Histological analysis showed ventricular dilation (Figure 3B), cardiomyocyte enlargement (Figure 3C), and mild fibrosis (Figure 3D) in CaMKIIδC TG mice. Quantitative analysis of cardiomyocyte cell volume from 12-week-old TGM mice gave values of 54.7 + 0.1 pL for TGM (n = 96) versus 28.6 + 0.1 pL for WT littermates (n=94; P0.001). Ventricular dilation and cardiac dysfunction developed over time in proportion to the extent of transgene expression. Left ventricular end diastolic diameter (LVEDD) was increased by 35% to 45%, left ventricular posterior wall thickness (LVPW) decreased by 26% to 29% and fractional shortening decreased by 50% to 60% at 8 weeks for TGM and at 16 weeks for TGL. None of these parameters were significantly altered at 4 weeks in TGM or up to 11 weeks in TGL mice, indicating that heart failure had not yet developed. Contractile function was significantly decreased. Figure 6. Dilated cardiomyopathy and dysfunction in CaMKIIδC TG mice at both whole heart and single cell levels. [see Fig 6: http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5] C, Decreased contractile function in ventricular myocytes isolated from 12-week old TGM and WT controls presented as percent change of resting cell length (RCL) stimulated at 0.5 Hz. Representative trace and mean values are shown. *P0.05 vs WT. Figure 7. Phosphorylation of PLB in CaMKIIδC TG mice. [see Fig 7: http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5] Thr17 and Ser16 phosphorylated PLB was measured by Western blots using specific anti-phospho antibodies. Ventricular homogenates were from 12- to 14-week-old WT and TGM mice (A) or 4 to 5-week-old WT and TGM mice (B). Data were normalized to total PLB examined by Western blots (data not shown here). n = 6 to 8 mice per group; *P0.05 vs WT.
Cardiac Overexpression of CaMKIIδC Results in Changes in the Phosphorylation of Ca2 Handling Proteins
To assess the possible involvement of phosphorylation of Ca2cycling proteins in the phenotypic changes observed in the CaMKIIC TG mice, we first compared PLB phosphorylation state in homogenates from 12- to 14-week-old TGM and WT littermates. Western blots using antibodies specific for phosphorylated PLB showed a 2.3-fold increase in phosphorylation of Thr17 (the CaMKII site) in hearts from TGM versus WT (Figure 7A). Phosphorylation of PLB at the CaMKII site was also increased 2-fold in 4- to 5-week-old TGM mice (Figure 7B). Significantly, phosphorylation of the PKA site (Ser16) was unchanged in either the older or the younger TGM mice (Figures 7A and 7B). (see http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5) To demonstrate that the RyR2 phosphorylation changes observed in the CaMKII transgenic mice are not secondary to development of heart failure, we performed biochemical studies examining RyR2 phosphorylation in 4- to 5-week-old TGM mice. At this age, most mice showed no signs of hypertrophy or heart failure (see Figure 6B) and there was no significant increase in myocyte size (21.3 + 1.3 versus 27.7 + 4.6 pL; P0.14). Also, twitch Ca2 transient amplitude was not yet significantly depressed, and mean δ [Ca2+]i (1 Hz) was only 20% lower (192 + 36 versus 156 + 13 nmol/L; P0.47) versus 50% lower in TGM at 13 weeks.33 The in vivo phosphorylation of RyR2, determined by back phosphorylation, was significantly (2.10.3-fold; P0.05) increased in these 4- to 5-week-old TGM animals (Figure 8C), an increase equivalent to that seen in 12- to 14-week-old mice. We also performed the RyR2 back-phosphorylation assay using purified CaMKII rather than PKA. RyR2 phosphorylation at the CaMKII site was also significantly increased (2.2 + 0.3-fold; P0.05) in 4- to 5-week-old TGM mice (Figure 8C). (http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5) The association of CaMKII with the RyR2 is consistent with a physical interaction between this protein kinase and its substrate. The catalytic subunit of PKA and the phosphatases PP1 and PP2A were also present in the RyR2 immunoprecipitates, but not different in WT versus TG mouse hearts (Figure 8D). These data provide further evidence that
the increase in RyR2 phosphorylation, which precedes development of failure in the 4- to 5-week-old CaMKIIδC TG hearts, can be attributed to the increased activity of CaMKII.
Discussion
CaMKII is involved in the dynamic modulation of cellular
Ca2 regulation and has been implicated in the development of cardiac hypertrophy and heart failure.14
Published data from CaMK-expressing TG mice demonstrate that forced expression of CaMK can induce cardiac hypertrophy and lead to heart failure.27,32
However, the CaMK genes expressed in these mice are neither the endogenous isoforms of the enzyme nor the isoforms likely to regulate cytoplasmic Ca(2+) handling, because they localize to the nucleus.
the cytoplasmic cardiac isoform of CaMKII is upregulated at the expression level and is in the active state (based on autophosphorylation) after pressure overload induced by TAC.
two cytoplasmic CaMKII substrates (PLB and RyR) are phosphorylated in vivo when CaMKII is overexpressed and its activity increased to an extent seen under pathophysiological conditions.
CaMKIIδ is found to associate physically with the RyR in the heart.
heart failure can result from activation of the cytoplasmic form of CaMKII and this may be due to altered Ca(2+) handling.
Differential Regulation of CaMKIIδ Isoforms in Cardiac Hypertrophy
The isoform of CaMKII that predominates in the heart is the δ isoform.4–7 Neither the α nor the β isoforms are expressed and there is only a low level of expression of the γ isoforms.39
Both δB and δC splice variants of CaMKIIδ are present in the adult mammalian myocardium36,40 and expressed in distinct cellular compartments.4,8,9
We suggest that the CaMKIIδ isoforms are differentially regulated in pressure-overload–induced hypertrophy, because the expression of CaMKIIδC is selectively increased as early as 1 day after TAC. Studies using RT-PCR confirm that
CaMKIIδC is regulated at the transcriptional level in response to TAC. In addition,
activation of both CaMKIIδB and CaMKIIδC, as indexed by autophosphorylation, increases as early as 2 days after TAC.
Activation of CaMKIIδB by TAC is relevant to our previous work indicating its role in hypertrophy.9,32
The increased expression, as well as activation of the CaMKIIδC isoform, suggests that it could also play a critical role in both the acute and longer responses to pressure overload.
In conclusion, we demonstrate here that CaMKIIδC can phosphorylate RyR2 and PLB when expressed in vivo at levels leading to 2- to 3-fold increases in its activity. Similar increases in CaMKII activity occur with TAC or in heart failure. Data presented in this study and in the accompanying article33 suggest that altered phosphorylation of Ca(2+) cycling proteins is a major component of the observed decrease in contractile function in CaMKIIδC TG mice. The occurrence of increased CaMKII activity after TAC, and of RyR and PLB phosphorylation in the CaMKIIδC TG mice suggest that
CaMKIIδC plays an important role in the pathogenesis of dilated cardiomyopathy and heart failure.
These results have major implications for considering CaMKII and its isoforms in exploring new treatment strategies for heart failure.
Cardiac Electrophysiological Dynamics From the Cellular Level to the Organ Level
Daisuke Sato and Colleen E. Clancy Department of Pharmacology, University of California – Davis, Davis, CA. Biomedical Engineering and Computational Biology 2013:5: 69–75 http://www.la-press.com. http://dx.doi.org/10.4137/BECB.S10960 Abstract: Cardiac alternans describes contraction of the ventricles in a strong-weak-strong-weak sequence at a constant pacing frequency. Clinically, alternans manifests as alternation of the T-wave on the ECG and predisposes individuals to arrhythmia and sudden cardiac death. In this review, we focus on the fundamental dynamical mechanisms of alternans and show how alternans at the cellular level underlies alternans in the tissue and on the ECG. A clear picture of dynamical mechanisms underlying alternans is important to allow development of effective anti-arrhythmic strategies. The cardiac action potential is the single cellular level electrical signal that triggers contraction of the heart.1 Under normal conditions, the originating activation signal comes from a small bundle of tissue in the right atrium called the sinoatrial node (SAN). The action potentials generated by the SAN initiate an excitatory wave that, in healthy tissue, propagates smoothly through a well-defined path and causes excitation and contraction in the ventricles. In disease states, the normal excitation pathway is disrupted and a variety of abnormal rhythms can occur, including cardiac alternans, a well-known precursor to sudden cardiac death. Cardiac alternans was initially documented in 1872 by a German physician, Ludwig Traube.2 He observed contraction of the ventricles in a strong-weak-strong-weak sequence even though the pacing frequency was constant. Clinically, alternans manifests as alternation of the T-wave on the ECG, typically in the microvolt range. It is well established that individuals with microvolt T-wave alternans are at much higher risk for arrhythmia and sudden cardiac death. A clear picture of physiological mechanisms underlying alternans is important to allow development of effective anti-arrhythmic drugs. It is also important to understand dynamical mechanisms because while the cardiac action potential is composed of multiple currents, each of which confers specific properties, revelation of dynamical mechanisms provides a unified fundamental view of the emergent phenomena that holds independently of specific current interactions. The ventricular myocyte is an excitable cell providing the cellular level electrical activity that underlies cardiac contraction. Under resting conditions, the membrane potential is about -80 mV. When the cell is stimulated, sodium (Na) channels open and the membrane potential goes above 0 mV. Then, a few ms later, the inward current L-type calcium (Ca) current activates and maintains depolarization of the membrane potential. During this action potential plateau, several types of outward current potassium (K) channels also activate. Depending on the balance between inward and outward currents, the action potential duration (APD) is determined.The diastolic interval (DI) that follows cellular repolarization describes the duration the cell resides in the resting state until the next excitation. During the DI, channels recover with kinetics determined by intrinsic time constants. APD restitution defines the relationship between the APD and the previous DI (Fig. 1 top panel). In most cases1, the APD becomes longer as the previous DI becomes longer due to recovery of the L-type Ca channel (Fig. 1, bottom panel), and thus the APD restitution curve has a positive slope. Figure 1. (Top): APD and DI. (Bottom): The physiological mechanism of APD alternans involves recovery from inactivation of ICaL. [see http://dx.doi.org/10.4137/BECB.S10960]
Action Potential Duration Restitution
In 1968 Nolasco and Dahlen showed graphically that APD alternans occurs when the slope of the APD restitution curve exceeds unity. Why is the steepness of the slope important? As shown graphically in Figure 2, APD alternans amplitude is multiplied by the slope of the APD restitution curve in each cycle. When the slope is larger than one, then the alternans amplitude will be amplified until the average slope reaches 1 or the cell shows a 2:1 stimulus to response ratio. The one-dimensional mapping between APD and DI fails to explain quasi-periodic oscillation of the APD. Figure 2. APD restitution and dynamical mechanism of APD alternans. [see http://dx.doi.org/10.4137/BECB.S10960]
Calcium Driven Alternans
A strong-weak-strong-weak oscillation in contraction implies that the Ca transient (CaT) is alternating. Until 1999 it was assumed that if the APD is alternating then the CaT alternates because the CaT follows APD changes. However, Chudin et al showed that CaT can alternate even when APD is kept constant during pacing with a periodic AP clamp waveform.14 This implies that the intracellular Ca cycling has intrinsic nonlinear dynamics. A critical component in this process is the sarcoplasmic reticulum (SR), a subcellular organelle that stores Ca inside the cell. When Ca enters a cell through the L-type Ca channel (or reverse mode Na-Ca exchanger (NCX) ryanodine receptors open and large Ca releases occur from the SR (Ca induced Ca release). The amount of Ca release steeply depends on SR Ca load. This steep relation between Ca release and SR Ca load is the key to induce CaT alternans. A one-dimensional map between Ca release and SR calcium load can be constructed to describe the relationship21 similar to the map used in APD restitution.
Subcellular Alternans
A number of experimental and computational studies have been undertaken to identify molecular mechanisms of CaT alternans by identifying the specific components in the calcium cycling process critical to formation of CaT alternans. These components include SR Ca leak and load, Ca spark frequency and amplitude, and rate of SR refilling. For example, experiments have shown that alternation in diastolic SR Ca is not required for CaT alternans.24 In addition, stochastic openings of ryanodine receptors (RyR) lead to Ca sparks that occur randomly, not in an alternating sequence that would be expected to underlie Ca altern-ans. So, how do local random sparks and constant diastolic SR calcium load lead to global CaT alternans? Mathematical models with detailed representations of subcellular Ca cycling have been developed in order to elucidate the underlying mechanisms. Modeling studies have shown that even when SR Ca load is not changing, RyRs, which are analogous to ICaL in APD alternans, recover gradually from refractoriness. As RyR availability increases (for example during a long diastolic interval) a single Ca spark from a RyR will be larger in amplitude and recruit neighboring Ca release units to generate more sparks. The large resultant CaT causes depletion of the SR and when complete recovery of RyRs does not occur prior to the arrival of the next stimulus, the subsequent CaT will be small. This process results in an alternans of CaT amplitude from beat-to-beat.
Coupling Between the Membrane Potential and Subcellular Calcium Dynamics
Importantly, the membrane voltage and intracellular Ca cycling are coupled via Ca sensitive channels such as the L-type Ca channel and the sodium-calcium exchanger (NCX). The membrane voltage dynamics and the intracellular Ca dynamics are bi-directionally coupled. One direction is from voltage to Ca. As the DI becomes longer, the CaT usually becomes larger since the recovery time for the L-type Ca channel in increased and the SR Ca release becomes larger. The other direction is from Ca to voltage. Here we consider two major currents, NCX and ICaL. As the CaT becomes larger, forward mode NCX becomes larger and prolongs APD. On the other hand, as the CaT becomes larger, ICaL becomes smaller due to Ca-induced inactivation, and thus, larger CaT shortens the APD. Therefore, depending on which current dominates, larger CaT can prolong or shorten APD. If a larger CaT prolongs (shortens) the APD, then the coupling is positive (negative). The coupled dynamics of the membrane voltage and the intracellular Ca cycling can be categorized by the instability of membrane voltage (steep APD restitution), instability of the intracellular Ca cycling (steep relation between Ca release versus SR Ca load), and the coupling (positive or negative). If the coupling is positive, alternans is electromechanically concordant (long-short-long-short APD corresponds to large-small-large-small CaT sequence) regardless of the underlying instability mechanism. On the other hand, if the coupling is negative, alternans is electromechanically concordant in a voltage-driven regime. However, if alternans is Ca driven, alternans becomes electromechanically discordant (long-short-long-short APD corresponds to small-large-small-large CaT sequence). It is also possible to induce quasi- periodic oscillation of APD and CaT when voltage and Ca instabilities contribute equally.
Alternans in Higher Dimensions
Tissue level alternans in APD and CaT also occur and here we describe how the dynamical mechanism of alternans at the single cell level determines the phenomena in tissue. Spatially discordant alternans (SDA) where APDs in different regions of tissue alternate out-of-phase, is more arrhythmogenic since it causes large gradients of refractoriness and wave-break, which can initiate ventricular tachycardia and ventricular fibrillation. How is SDA induced? As the APD is a function of the previous DI, conduction velocity (CV) is also function of the previous DI (CV restitution) since the action potential propagation speed depends on the availability of the sodium channel. As the DI becomes shorter, sodium channels have less time to recover. Therefore, in general, as the DI becomes shorter, the CV becomes slower. When tissue is paced rapidly, action potentials propagate slowly near the stimulus, and thenac-celerate downstream as the DI becomes longer. This causes heterogeneity in APD (APD is shorter near the stimulus). During the following tissue excitation, APD becomes longer and the CV becomes faster at the pacing site then gradually APD becomes shorter and the CV becomes slower. The interaction between steep APD restitution and steep CV restitution creates SDA. This mechanism applies only when the cellular instability is voltage driven. When the cellular instability is Ca driven, the mechanism of SDA formation is different. If the voltage-Ca coupling is negative, SDA can form without steep APD and CV restitution. The mechanism can be understood as follows. First, when cells are uncoupled, alternans of APD and Ca are electromechanically discordant. If two cells are alternating in opposite phases, once these cells are coupled by voltage, due to electrotonic coupling, the membrane voltage of both cells is synchronized and thus APD becomes the same. This synchronization of APD amplifies the difference of CaT between two cells (Fig. 5 in). In other words it desynchronizes CaT. This instability mechanism is also found in subcellular SDA. In the case where the instability is Ca driven and the coupling is positive, there are several interesting distinctive phenomena that can occur. First, the profile of SDA of Ca contains a much steeper gradient at the node (point in space where no alternans occurs–cells downstream of the node are alternating out of phase with those upstream of the node) compared to the case of voltage driven SDA. Thus, the cellular mechanism of instability can be identified by evaluating the steepness of the alternans amplitude gradient in space around the node. When the cellular instability is voltage driven, the steady-state wavelength (separation of nodes in space) depends on electrotonic coupling between cells and the steepness of APD and CV restitution, regardless of the initial conditions. However, if the cellular instability is Ca driven, the location of nodes depends on the pacing history, which includes pacing cycle length and other parameters affected by pacing frequency. In this case, once the node is formed, the location of the node may be fixed, especially when Ca instability is strong. Such an explanation may apply to recent experimental results. Summary In this review, we described how the origin of alternans at the cellular level (voltage driven, Ca drive, coupling between voltage and Ca) affects the formation of spatially discordant alternans at the tissue level. Cardiac alternans is a multi-scale emergent phenomenon. Channel properties determine the instability mechanism at the cellular level. Alternans mechanisms at cellular level determine SDA patterns at the tissue level. In order to understand alternans and develop anti-arrhythmic drug and therapy, multi-scale modeling of the heart is useful, which is increasingly enabled by emerging technologies such as general-purpose computing on graphics processing units (GPGPU) and cloud computing.
The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Author and Curator: Larry H Bernstein, MD, FCAP
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
This is the Part IV of a series on the cytoskeleton and structural shared thematics in cellular movement and cellular dynamics. The last two are specific to the heart, and the third was renal tubular caicium exchange and the effects of Na+ and hormones.
In Part I, Identification of Biomarkers that are Related to the Actin Cytoskeleton
The prior articles discussed common management motifs across cell-types that are essential for cell division, embryogenesis, cancer metastasis, osteogenesis, musculoskeletal function, vascular compliance, and cardiac contractility. This second article concentrates on specific functionalities for cardiac contractility based on Ca++ signaling in excitation-contraction coupling, addressing modifications specific to cardiac muscle and not to skeletal muscle. In Part I there was discussion of the importance of Ca2+ signaling on innate immune system, and the roles of calcium in immunology will be further expanded in a third article of the series.
The Series consists of the following articles:
Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
Observations of Tissues Dependent on Electrical Impulses and Differences in Calcium-Efflux Mechanisms
Voice of Justin Pearlman
Skeletal muscles are named for muscle bundles attached to skeleton elements, including head and neck, thorax, and the long bones of limbs, but the same structural and neuronally controlled muscle type is also in the abdomenal wall and the scalp, face, and eyes (for eye motion), each serving the function of movement on demand. The skeletal element these muscles attach to are tendons (fibrous tissue), often anchored to bone before and after an articulation (joint). There are several features that distinguish skeletal muscle from smooth muscle and from myocardium (heart muscle). Skeletal muscles are striated. They have fast-twitch and slow-twitch fibers in various proportions. They are under voluntary neural control, not autonomic (involuntary).
In distinction, smooth muscles line arterial blood vessels, lymphatics, the urinary bladder, the gastrointestinal tract, the respiratory tract, and also the uterus, the pili of the skin (goose bumps), and are in the eyes to control pupil diameter and lens focus. They are controlled by autonomic innervation.
The myocardium, or heart muscle, is distinct in many ways. The heart muscle has a unique architecture with Z-bands. The heart muscle a syncytium of cardiac muscle made of cardiomyocytes, which means instead of a bundle of separate cells each distinctly bounded by a cell membrane, the entire heart muscle can be viewed as a single multinucleated cell (or merger of cells). Skeletal muscle has multinucleated cells also from the merger of multiple blast cells, but unlike the heart there are distinct cell boundaries between skeletal myocytes, known as myofibers. The heart has fiber layers with different orientations (spiral clockwise and counterclockwise arrangement of muscle fibers) that result in multiple types of motion, but technically all of the heart muscle fibers are part of a single conglomerate cell. The motions of the heart include: translation, tilting, shortening, thickening, narrowing, twisting, rotating, lengthening and widening. The heart cell contracts and has innervation to the AV node and the SA node, with both sympathetic and parasymptathetic innervation.
All three types of muscle apply a basic Motif of proteins that change length in response to a calcium signal. The calcium is stored is sacks called the sarcoplasmic reticulum. The calcium is released into the main fluid of the cell (the cytoplasm), where it controls different functions. Even in skeletal muscle there is a difference between thigh and thorax, and we know from comparative ornithology that the enzymology and energy metabolism of the wings of birds that soar, hawks and eagles, differs from the chicken, or the turkey.
Key features are illustrated below.
Figure 1….. skeletal muscle vs heart calcium channels.
receptors voltage gated Ca(2) channel
We see in Figure 1 that both the skeletal muscle and the cardiomyocyte have a Ryanodyne receptor that is the flow device for carrying the Ca(2+) ions from the sarcoplasm into the cytoplasm. In the skeletal muscle there is a dihydropyridine receptor. The heart muscle is voltage gated. The interaction with calmodulin (not shown) via Calcium/calmodulin-dependent Protein Kinase Type II delta = CaMKI, II – IV. CaMKII has isoforms a, b, c, d – and CaMKIId has two splice variants (cytoplasmic and nuclear). These will be discussed fully in the fifth of the series. Take note of the fact the CaMKII isoform is found only in the heart. So we have here molecules with similar structure, but not completely homologous. Structure and function have made small, requiring significant adaptations.
Figure 2. A cardiomycyte structure with the sarcomere and calcium efflux into the cytoplasn, and with the mitochondrion available for Ca(2+) exchange with the cytoplasm, and with Ca(2+), Na(+) and K(+) channels contiguous with the extracellular space.
RyR
The arterial endothelium is functionally protected by eNOS converting arginine to citrulline. This does not occur with adult form of urea cycle (Krebs Henseleit) disorder, as there is no substrate. iNOS, a nitric oxide isoform present in macrophages that invade through intercellular spaces into the underlying matrix. A large study presented at the European Society of Cardiology (ESC) 2013 Congress has indicated that there is not a relationship of tight control of type 2 diabetes and cardiovascular events, even though we know that there is a relationship between diabetes and
insulin resistance
endothelial activation
inflammatory markers
homocysteine
Adipokines interact in type 2 diabetes with inflammatory cytokines for development of insulin resistance, and these are markers of arterial vascular disease. But the association of diabetes with heart disease, long considered valid, has come into some dispute. Recently, saxagliptin was associated with a significant 27% increased risk of hospitalizations for heart failure in the Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus (SAVOR-TIMI 53) study, a component of the prespecified secondary end point. In the Examination of Cardiovascular Outcomes with Alogliptin versus Standard of Care in Patients with Type 2 Diabetes Mellitus and Acute Coronary Syndrome (EXAMINE) study, there was no increased risk of heart failure with alogliptin. While saxagliptin and alogliptin significantly reduced glycated hemoglobin levels, there was some debate about the role of the drugs, which are dipeptidyl peptidase-4 (DPP-4) inhibitors, in clinical practice. There is some disappointment with respect to the diabetes issue, but that might be remedied by improvement based on the appropriate combination of biomarkers for prediction asnd monitoring at the earliest onset. Dr William White said alogliptin lowers the glycemic index significantly, and such reductions can reduce the risk of microvascular complications. We know from the prior literature that it might take five years-plus before we determine a microvascular benefit. A serious problem in the validity of the results was that statistically, saxagliptin met the primary end point of noninferiority, with the drug no worse than placebo. Glycated hemoglobin levels were reduced with saxagliptin, down from 8.0% at baseline to 7.7% at the end of the trial (p<0.001 vs placebo). In addition, more patients in the saxagliptin arm had glycated hemoglobin levels reduced to less than 7.0%. The relevant question is what the effect was for patients who achieved a glycated Hb of < 7.7%, which makes the p-value meaningless for an 0.3% change overall.
Implications of ca(2+) handling dysfunction
A. if the dysfuction is in smooth muscle – effect on arterial elasticity
B. if the dysfunction is in cardiomyocytes – Ventricular contractility & arrhythmias
We now review the calcium cycling of smooth muscle based on extracted work at MIT and Harvard Medical School, and at the University of Iowa. The work focuses on the disordered Ca(2+) signaling that plays a large role in the development of “arterial stiffness”, not disregarding the competing roles of endothelial nitric oxide and the inflammatory cell mediated oxidative stress related iNOS in the arterial circulation, and the preference for stress points at the junction of arteries. Disordered Ca(2+) in vascular smooth muscle leads to ischemic arterial disease, vascular rigidity from loss of flexibility, which can lead to ischemic myocardial damage.
Calcium Cycling in Synthetic and Contractile Phasic or Tonic Vascular Smooth Muscle Cells
Calcium ions (Ca2+) are present in low concentrations in the cytosol (~100 nM) and in high concentrations (in mM range) in both the extracellular medium and intracellular stores (mainly sarco/endo/plasmic reticulum, SR). This differential allows the calcium ion to be a ubiquitous 2nd messenger that carries information essential for cellular functions as diverse as contraction, metabolism, apoptosis, proliferation and/or hypertrophic growth. The mechanisms responsible for generating a Ca2+ signal greatly differ from one cell type to another. In the different types of vascular smooth muscle cells (VSMC), enormous variations do exist with regard to the mechanisms responsible for generating Ca2+ signal. In each VSMC phenotype (synthetic/proliferating1 and contractile2 [1], tonic or phasic), the Ca2+ signaling system is adapted to its particular function and is due to the specific patterns of expression and regulation of Ca2+ handling molecules (Figure 1).
1Synthetic VSMCs have a fibroblast appearance, proliferate readily, and synthesize increased levels of various extracellular matrix components, particularly fibronectin, collagen types I and III, and tropoelastin [1].
2Contractile VSMCs have a muscle-like or spindle-shaped appearance and well-developed contractile apparatus resulting from the expression and intracellular accumulation of thick and thin muscle filaments [1].
in contractile VSMCs, the initiation of contractile events is driven by membrane depolarization; and the principal entry-point for extracellular Ca2+ is the voltage-operated L-type calcium channel (LTCC). In contrast, in synthetic/proliferating VSMCs, the principal way-in for extracellular Ca2+ is the store-operated calcium (SOC) channel. Whatever the cell type, the calcium signal consists of limited elevations of cytosolic free calcium ions in time and space. The calcium pump, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), has a critical role in determining the frequency of SR Ca2+ release by controlling the velocity of Ca2+ upload into the sarcoplasmic reticulum (SR) and the Ca2+ sensitivity of SR calcium channels, Ryanodin Receptor, RyR and Inositol tri-Phosphate Receptor, IP3R.
Figure 1. Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs.
Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs
Left panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Contractile response is initiated by extracellular Ca2* influx due to activation of Receptor Operated Ca2* channels (through phosphoinositol-coupled receptor) or to activation of L-Type Calcium channels (through an increase in luminal pressure). Small increase of cytosolic due IP3 binding to IP3R (puff) or RyR activation by LTCC or ROC-dependent Ca2* influx leads to large SR Ca2* release due to the activation of IP3R or RyR clusters (“Ca2*-induced Ca2*release” phenomenon). Cytosolic Ca2* is rapidly reduced by SR calcium pumps (both SERCA2a and SERCA2b are expressed in quiescent VSMCs), maintaining high concentration of cytosolic Ca2* and setting the sensitivity of RyR or IP3R for the next spike. Contraction of VSMCs occurs during oscillatory Ca2* transient. Middle panel: schematic representation of atherosclerotic vessel wall. Contractile VSMC are located in the media layer, synthetic VSMC are located in sub-endothelial intima. Right panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Agonist binding to phosphoinositol-coupled receptor leads to the activation of IP3R resulting in large increase in cytosolic Ca2*. Calcium is weakly reduced by SR calcium pumps (only SERCA2b, having low turnover and low affinity to Ca2* is expressed). Store depletion leads to translocation of SR Ca2* sensor STIM1 towards PM, resulting in extracellular Ca2* influx though opening of Store Operated Channel (CRAC). Resulted steady state Ca2* transient is critical for activation of proliferation-related transcription factors ‘NFAT). Abbreviations: PLC – phospholipase C; PM – plasma membrane; PP2B – Ca2*/calmodulin-activated protein phosphatase 2B (calcineurin); ROC- receptor activated channel; IP3 – inositol-1,4,5-trisphosphate, IP3R – inositol-1,4,5-trisphosphate receptor; RyR – ryanodine receptor; NFAT – nuclear factor of activated T-lymphocytes; VSMC – vascular smooth muscle cells; SERCA – sarco(endo)plasmic reticulum Ca2* ATPase; SR – sarcoplasmic reticulum.
General aspects of calcium cycling and signaling in vascular smooth muscle cells
Besides maintaining vascular tone in mature vessels, VSMCs also preserve blood vessel integrity. VSMCs are instrumental for vascular remodeling and repair via proliferation and migration. Interestingly, Ca2* plays a central role in both physiological processes. In VSMCs, calcium signaling involves a cross-regulation of Ca2* influx, sarcolemmal membrane signaling molecules and Ca2* release and uptake from the sarco/endo/plasmic reticulum and mitochondria, which plays a central role in both vascular tone and integrity.
Calcium handling by the plasma membrane’s calcium channels and pumps
Membrane depolarization is believed to be a key process for the activation of calcium events in mature VSMCs. Thus, much attention has been given to uncovering the various mechanisms responsible for triggering this depolarization. Increased intra-vascular pressure of resistance arteries stimulates gradual membrane depolarization in VSMCs, increasing the probability of opening L-type high voltage-gated Ca2* channels (Cav1.2) (LTCC). Alternatively, the calcium-dependent contractile response can be induced through the activation of specific membrane receptors coupled to phospholipase C (PLC) isoforms3. The various isoforms of transient receptor potential (TRP) ion channel family, particularly TRPC3, TRPC6 and TRPC7 possibly activated directly by diacyl glycerol (DAG), can also contribute to initial plasma membrane Ca2* influx and subsequent membrane depolarization.
Among voltage-insensitive calcium influx pathways, the store-operated Ca2* channels (SOC), maintain a long-term cellular Ca2* signal. They are activated upon a decrease of internal store Ca2* concentration resulting from a Ca2* release via the opening of SR Ca2* release channels. SOC has two essential regulatory components, the SR/ER located Ca2* sensor STIM1 (stromal interaction molecule) and the Ca2* channels Orai. Upon decrease of [Ca2*] in the reticulum (<500µM), Ca2* dissociates from STIM1; then STIM1 molecules oligomerize and translocate to specialized cortical reticulum compartments adjacent to the plasma membrane. There, the STIM1 cytosolic activating domains bind to and cluster the Orai proteins into an opened archaic Ca2* channel known as Ca2*-release activated Ca2* channel (CRAC).
All isoforms of PLC, catalyze the hydrolysis of phosphatidylinositol4,5-biphosphate (PIP2) to produce the intracellular messengers IP3 increase and diacylglycerol (DAG); both of which promote cytosolic Ca2* rise through activation of plasma membrane or sarcoplasmic reticulum calcium channels.
The CRAC is responsible for the “2h cytosolic Ca2* increase” required to induce VSMCs proliferation.
The calcium signal is terminated by membrane hyper-polarization and cytosolic Ca2+ removal. First, calcium sparks resulting from the opening of sub-plasmalemmal clusters of RyR activate large-conductance Ca2+ sensitive K+ (BK) channels. Then, the resulting spontaneous transient outward currents (STOC) hyperpolarize the membrane and decrease the open probability of L-type Ca2+ channels. Cytosolic calcium is extruded at the level of plasma membrane by plasma membrane Ca2+ ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX). The principal amount of cytosolic Ca2+ (> 70%) is re-uploaded to the internal store.
Calcium handling by the sarco/endoplasmic reticulum’s calcium channels and pumps
The initial entry of Ca2+ through plasma membrane channels triggers large Ca2+ release from the internal store via the process of Ca2+-induced Ca2+-release (CICR). The mechanism responsible for initiating Ca2+ release depends on Ca2+ sensitive SR calcium channels, the ryanodin receptor (RyR)5 or the IP3 receptor (IP3R). Indeed, IP3R and RyR are highly sensitive to cytosolic Ca2+ concentrations and when cytosolic Ca2+ concentration ranges from nM to µM, they open up. On the contrary, a higher cytosolic Ca2+ concentration (from µM to mM) closes them. In other words, cytosolic Ca2+ increase first exerts a positive feedback and facilitates SR channels opening whereas a further increase has an opposite effect and actually inhibits the SR channels opening. Importantly enough to be mentioned, RyR phosphorylation by the second messenger cyclic ADP ribose (cADPR) and protein kinase A (PKA) enhances Ca2+ sensitivity, the phosphorylation induced by the protein kinase C (PKC) decreases RyR sensitivity to Ca2+.
Sarco/Endoplasmic Ca2+ATPases (SERCA), the only calcium transporters expressed within sarco/endoplasmic reticulum (SR), serve to actively return calcium into this organelle. In mammals, three SERCA genes ATP2A1, ATP2A2 and ATP2A3 coding for SERCA1, SERCA2 and SERCA3 isoforms respectively have been identified [35]. Each gene gives rise to a different SERCA isoform through alternative splicing (Figure 2); they all have discrete tissue distributions and unique regulatory properties, providing a potential focal point within the cell for the integration of diverse stimuli to adjust and fine-tune calcium homeostasis in the SR/ER. In VSMCs, SERCA2a and the ubiquitous SERCA2b isoforms are expressed; besides vascular smooth muscle, SERCA2a is preferentially expressed in cardiac and skeletal muscles. SERCA2b differs from SERCA2a by an extension of 46 amino acids. Diversity of SERCA isoforms in the same cell suggests that each of them could be responsible for controlling unique cell functions.
RyR are structurally and functionally analogous to IP3R, although they are approximately twice as large and have twice the conductance of IP3R [27]; RyR channels are sensitive to store loading and IP3R channels are sensitized by the agonist-dependent formation of IP3.
SERCA2’s activity depends on its interaction with phospholamban and is inhibitory in its de-phosphorylated form. PKA phosphorylation of phospholamban results in its dissociation from SERCA2, thus activating the Ca2+ pumps. Cyclic ADP-ribose was also reported to stimulate SERCA pump activity.
As previously mentioned, SR Ca2+ content controls the sensitivity of SR Ca2+ channels, RyR and IP3R, as well as functioning of SOC-mediated Ca2+ entry, thereby determining the type of intracellular calcium transient. Since SOCs opening depends on Ca2+ content of the store, one may suggest that SERCA participates to its regulation. Consistent with this, SOCs open up when the leak of Ca2+ from intracellular stores is not compensated with SERCA activity; SERCA inhibitors such as thapsigargin which prevent Ca2+ uptake are commonly used to chemically induce SOC currents; several works have established that SERCA can cluster with STIM1 and Orai1 in various cellular types.
Mechanisms of cytosolic Ca2+ oscillations in VSMC
Ca2+ oscillations are one of the ways that VSMCs respond to agonists. These Ca2+ oscillations are maintained during receptor occupancy and are driven by an endogenous pacemaker mechanism, called the cellular Ca2+ oscillator. Ca2+ oscillators were classified into two main types, the membrane oscillators and the cytosolic oscillators.
Membrane oscillators are those which generate oscillations at the cell membrane by successive membrane depolarization. In most small resistance arteries, inhibitors of plasma membrane voltage-dependent channels reduce or even abolish the membrane potential oscillations which precede rhythmical contractions. This suggests that rhythmic extracellular Ca2+ influx can be required for calcium oscillatory transient. Besides, membrane oscillators greatly depend on Ca2+ entry in order to provide enough Ca2+ to charge up the intracellular stores for each oscillatory cycle.
Cytosolic oscillators do not depend on the cell membrane to generate oscillations. Instead, they arise from intracellular store membrane instability. The pacemaker mechanism of cytosolic Ca2+ oscillator is based on the velocity of luminal Ca2+ loading and luminal Ca2+ content. The mechanism responsible for initiating Ca2+ release depends either on RyRs or IP3R activation. As soon as stores are sufficiently charged with Ca2+, the SR Ca2+ channels become sensitive to cytosolic Ca2+ and can participate to the process of Ca2+-induced Ca2+-release, which is responsible for orchestrating the regenerative release of Ca2+ from the SR/ER. Importantly, extracellular Ca2+ influx is not required for cytosolic oscillator function. Indeed, the Ca2+ oscillations can be observed in the absence of extracellular Ca2+.
In mature vessels, VSMCs mainly exhibit a tonic or phasic contractile phenotype. In contractile VSMCs extracellular calcium influx predominantly takes place through the voltage-dependent L-type calcium channel, LTCC9 (Figure 3). Extracellular Ca2* influx causes a small increase of cytosolic Ca2* generated by the opening of IP3R clusters, called puff and/or RyR2 clusters, called spark. These local rises of cytosolic Ca2* generate a larger SR Ca2* release through the Ca2*-induced Ca2* release phenomenon. Elevation of free cytosolic calcium triggers VSMC contraction.
In contractile VSMCs, NFAT can be activated by sustained Ca2* influx (persistent Ca2* sparklets) mediated by clusters of L-type Ca2* channels operating in a high open probability mode
Steady state increase in cytosolic Ca2* triggers tonic contraction; oscillatory type of Ca2* transient triggers phasic contraction. It is worth mentioning that accumulating evidence indicate that SR Ca2*ATPase functioning/location within the cell (which greatly influences the velocity of calcium upload) determines the mode of Ca2* transient in VSMCs. Consistent with this, i) “phasic” VSMCs display a greater number of peripherally located SR than “tonic” VSMCs; indeed “tonic” VSMCs exhibit centrally located SR; (rev in [61, 77]); ii) drugs which interfere with the IP3 pathway or intracellular stores abolish spontaneous vaso-motion; iii) blocking SERCA strongly inhibits the Ca2* oscillations, demonstrating that they are induced by SR Ca2* release; this latter argument is further supported by the fact that oscillations are present even in the absence of extracellular Ca2*
SERCA2a has a higher catalytic turnover when compared to SERCA2b due to a higher rate of de-phosphorylation and a lower affinity for Ca2+; ii) SER-CA2a is absent in synthetic VSMCs, which only exhibit tonic contraction, iii) transferring the SERCA2a gene to synthetic cultured VSMCs modifies the agonist-induced calcium transient from steady-state to oscillatory mode. Therefore, one might suggest that the physiological role of SERCA2a in VSMCs consists of controlling the “cytosolic oscillator”, thereby determining phasic vs tonic type of smooth muscle contraction.
SERCA2a as a potential target for treating vascular proliferative diseases
Abundant proliferation of VSMCs is an important component of the chronic inflammatory response associated to atherosclerosis and related vascular occlusive diseases (intra-stent restenosis, transplant vasculopathy, and vessel bypass graft failure). Great efforts have been made to prevent/reduce trans-differentiation and proliferation of synthetic VSMCs. Anti-proliferative therapies including the use of pharmacological agents and gene therapy approaches are, until now, considered as a suitable approach in the treatment of these disorders. Indeed, coronary stenting is the only procedure that has been proven to reduce the incidence of late restenosis after percutaneous transluminal coronary angioplasty. Nevertheless, post-interventional intra-stent restenosis, characterized by the re-narrowing of the arteries caused by VSMC proliferation, occurs in 10 to 20 % of patients. These disorders remain the major limitation of revascularization by percutaneous transluminal angioplasty and artery bypass surgery. The use of drug-eluting stents (stent eluting anti-proliferative drug) significantly reduces restenosis but impairs the re-endothelialization process and subsequently often induces late thrombosis. In human, trans-differentiation of contractile VSMCs towards a synthetic/proliferating inflammatory/migratory phenotype after percutaneous transluminal angioplasty appears to be a fundamental process of vascularproliferative disease.
Concluding remarks
Over the last decade, great progress has been made in the understanding of the various intracellular molecular mechanisms in VSMCs which control calcium cycling and excitation/contraction or excitation/transcription coupling. VSMCs employ a great variety of Ca2+ signaling systems that are adapted to control their different contractile functions. Alterations in the expressions of Ca2+ handling molecules are closely associated with VSMC phenotype modulation. Furthermore, these changes in expression are inter-connected and each acquired or lost Ca2+ signaling molecule represents a component of signaling module functioning as a single unit.
In non-excitable synthetic VSMCs, calcium cycling results from the protein module ROC/IP3R/STIM1/ORAI1 which controls SOC influx. Agonist stimulation of synthetic VSMCs translates into a sustained increase in cytosolic Ca2+. This increase is required for the activation of NFAT downstream cellular signaling pathways inducing proliferation, migration and possibly an inflammatory response. Calcium cycling in excitable contractile VSMCs is governed by the protein module composed of ROC/LTCC/RyR2/SERCA2a and controls the contractile response.
Author details
Larissa Lipskaia
Mount Sinai School of Medicine, Department of Cardiology, New York, NY, USA
Isabelle Limon
Univ Paris 6, UR4 stress inflammation and aging, Paris, France
B. cardiomyocyte or smooth muscle. Let’s look a little further.
CaM kinase and disordering of intracellular calcium homeostasis , molecular link to arrhythmias
Mark E. Anderson, MD, PhD, Professor of Medicine and Pharmacology, University of Iowa, Iowa City, IADr. Anderson has presented a large body of work done at Vanderbilt University and University of Iowa Medical Schools for over a decade. The major hypothesis is that in the aftermath of a heart attack, the structural and electrical remodeling renders the heart prone to arrhythmias . The signaling molecule called calmodulin (CaM) kinase is a key and the work suggests that drugs that block CaM kinase activity might make good anti-arrhythmic medications. CaM kinase is a molecule that is intricately involved in calcium signaling and regulation. CaM kinase regulates calcium entry into the cell and calcium storage and release inside the cell.
Calcium enters heart cells through proteins called L-type calcium channels, donut-like pores in the cell membrane that open and close. If these channels stay open and let too much calcium into the cell, the risk of arrhythmia increases. Studies have shown that CaM kinase activity is increased in animal models and human heart disease. Dr. Anderson poses the question – does CaM kinase — which we know is elevated in heart disease — drive arrhythmias? The question is driven by their findings that the addition of activated CaM kinase allowed more calcium than normal to flow into isolated heart cells. The investigators measured the opening and closing of single calcium channels using a technique called patch-clamp electrophysiology. Then they added an already-activated form of CaM kinase to the preparation. When we added the activated CaM kinase, the calcium channels opened like crazy,” Anderson said. “In fact, they were more likely to open and stay open for long periods of time.
They also showed that cardiac cells with added CaM kinase had electrical changes called early afterdepolarizations (EADs). EADs are believed to be the triggering cause of arrhythmias in cardiomyopathy, hypertrophy, and long QT syndrome. The investigators implanted tiny telemeters into the mice and recorded electrocardiograms (ECGs) , which revealed not only the electrical changes expected in diseased hearts, Anderson said, but also an increased tendency for arrhythmias. Next, they treated the mice with a drug that blocks CaM kinase activity significantly suppressed the arrhythmias. They also found that cardiac cells isolated from the mice and found spontaneous EADs, which disappeared when the cells were treated with the CaM kinase-blocking drug. The evidence all points to CaM kinase driving arrhythmias.
They have demonstrated that CaM kinase is also important for calcium-activated gene expression and that it may be involved in the changes that occur in association with cardiac hypertrophy and heart failure. Anderson suggests that CaM kinase could be the link to explain why calcium channels open more frequently in heart failure, why people in heart failure have arrhythmias. He postulates that it would good to have a target that addresses both phenotypic disorders — the arrhythmia phenotype and the heart failure phenotype — and CaM kinase may be that target. Further, he observes that with the exception of so-called beta blockers, none of the current anti-arrhythmic drugs have been shown to reduce the mortality rate. More recent work in Iowa has identified a new link – a link between the inflammation in heart muscle following a heart attack and the enzyme calcium/calmodulin-dependent protein kinase II or CaM kinase II.
CaM kinase II, a pivotal enzyme that registers changes in calcium levels and oxidative stress and translates these signals into cellular effects, including changes in heart rate, cell proliferation and cell death. CaM kinase II also regulates gene expression — which genes are turned on or off at any given time. We have seen how Inhibition of CaM kinase II in mice protects the animals’ hearts against some of the damaging effects of a heart attack. A study compared a large number of genes that were expressed in the protected mice compared to the non-protected control mice. A particularly interesting finding was that a cluster of inflammatory genes was differently expressed depending on whether CaM kinase II was active or inhibited. Specifically, the research showed that heart attack triggered increased expression of a set of pro-inflammatory genes, and inhibition of CaM kinase II substantially reduced this effect.
The main research themes pursued by the Anderson laboratory are
Oxidative activation of CaMKII;
CaMKII signaling to ion channels;
The role of CaMKII in inflammation;
The role of CaMKII in cardiac pacemaker cells;
The role of CaMKII in cell survival.
Keywords: Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium, Calcium-Calmodulin-Dependent Protein Kinases, Calcium Channels, L-Type, Calmodulin, Arrhythmia, Ion channel, Hypertrophy, Cell Signaling, Signal Transduction
Regulation of cardiac ATP-sensitive potassium channel surface expression by calcium/calmodulin-dependent protein kinase II.
Ana Sierra; Asipu Sivaprasadarao; Peter M Snyder; Ekaterina Subbotina; Michel Vivaudou; Zhiyong Zhu; Leonid V Zingman; et al.
Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits.
Grueter, CE, Abiria, SA, Wu, Y, Anderson, ME, Colbran, RJ.
Biochemistry, 47(6), 1760-7, 2008.
Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies.
Werdich, AA, Lima, EA, Dzhura, I, Singh, MV, Li, J, Anderson, ME, Baudenbacher, FJ.
Am J Physiol Heart Circ Physiol, 294(5), H2352-62, 2008.
Conserved Regulation of Cardiac Calcium Uptake by Peptides Encoded in Small Open Reading Frames
Emile G. Magny1, Jose Ignacio Pueyo1, Frances M.G. Pearl1,2, MA Cespedes1, et al.
1 School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex, UK.
2 Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK Science http:/dx.doi.org/10.1126/science.1238802
Small Open Reading Frames (smORFs) are short DNA sequences able to encode small peptides of less than 100 amino acids. Study of these elements has been neglected despite thousands existing in our genomes. We and others showed previously that peptides as short as 11 amino acids are translated and provide essential functions during insect development. Here, we describe two peptides of less than 30 amino acids regulating calcium transport in the Drosophila heart influencing regular muscle contraction. These peptides seem conserved for more than 550 million years in a range of species from flies to humans, where they have been implicated in cardiac pathologies. Such conservation suggests that the mechanisms for heart regulation are ancient and that smORFs may be a fundamental genome component that should be studied systematically.
Excitation-contraction coupling in the heart: the state of the question.
Recent developments have led to great progress toward determining the mechanism by which calcium is released from the sarcoplasmic reticulum in the heart. The data support the notion of calcium-induced calcium release via a calcium-sensitive release channel. Calcium release channels have been isolated and cloned. This situation creates a paradox, as it has also been found that calcium release is smoothly graded and closely responsive to sarcolemmal membrane potential, properties that would not be expected of calcium-induced calcium release, which has intrinsic positive feedback. There is, therefore, no quantitative understanding of how the properties of the calcium release channel can lead to the macroscopic physiology of the whole cell. This problem could, in principle, be solved by various schemes involving heterogeneity at the ultrastructural level. The simplest of these require only that the sarcolemmal calcium channel be located in close proximity to one or more sarcoplasmic reticulum release channels. Theoretical modeling shows that such arrangements can, in fact, resolve the positive feedback paradox. An agenda is proposed for future studies required in order to reach a specific, quantitative understanding of the functioning of calcium-induced calcium release.
The role of protein kinases and protein phosphatases in the regulation of cardiac sarcoplasmic reticulum function
Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3,5-monophosphate (cAMP)-dependent and by calcium calmodulin-dependent protein kinases on a 27 000 proteolipid, called phospholamban. Both types of phosphorylation are associated with an increase in the initial rates of Ca(2+) transport by SR vesicles which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence. The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which can dephosphorylate both the CAMP-dependent and the calcium calmodulin-dependent sites on phospholamban. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases.
Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites
The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca(2+) was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca(2+) (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca(2+) on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low [trans Ca(2+)] (20 microM), increasing [cis Ca(2+)] from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating [cis Ca(2+)] above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca(2+)] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca(2+) enhanced the sensitivity of the channel to activating cytosolic Ca(2+), and it essentially reversed the inhibition of the channel by high cytosolic Ca(2+). Potentiation of Po by increased luminal Ca(2+) occurred irrespective of whether the electrochemical gradient for Ca(2+) supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca(2+) through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca(2+) acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca(2+)-sensitive site(s) at the luminal face of the channel or associated protein.
Contemporary Definitions and Classification of the Cardiomyopathies
AHA Scientific Statement: Council on Clin. Cardiol.; HF and Transplant. Committee; Quality of Care and Outcomes Res. and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention
BJ Maron, Chair; JA Towbin; G Thiene; C Antzelevitch; D Corrado; D Arnett; AJ Moss; et al.
Circulation. 2006; 113: 1807-1816 http://dx.doi.org/10.1161/CIRCULATIONAHA.106.174287
Classifications of heart muscle diseases have proved to be exceedingly complex and in many respects contradictory. Indeed, the precise language used to describe these diseases is profoundly important. A new contemporary and rigorous classification of cardiomyopathies (with definitions) is proposed here. This reference document affords an important framework and measure of clarity to this heterogeneous group of diseases. Of particular note, the present classification scheme recognizes the rapid evolution of molecular genetics in cardiology, as well as the introduction of several recently described diseases, and is unique in that it incorporates ion channelopathies as a primary cardiomyopathy.
Ryanopathy: causes and manifestations of RyR2 dysfunction in heart failure
Belevych AE, Radwański PB, Carnes CA, Györke S.
College of Medicine, The Ohio State University, Columbus, OH.
Cardiovasc Res. 2013; 98(2):240-7. http://dx.doi.org/10.1093/cvr/cvt024.
Epub 2013 Feb 12. PMID: 23408344 PMCID: PMC3633158 [Available on 2014/5/1]
The cardiac ryanodine receptor (RyR2), a Ca(2+) release channel on the membrane of the sarcoplasmic reticulum (SR), plays a key role in determining the strength of the heartbeat by supplying Ca(2+) required for contractile activation. Abnormal RyR2 function is recognized as an important part of the pathophysiology of heart failure (HF). While in the normal heart, the balance between the cytosolic and intra-SR Ca(2+) regulation of RyR2 function maintains the contraction-relaxation cycle, in HF, this behaviour is compromised by excessive post-translational modifications of the RyR2. Such modification of the Ca(2+) release channel impairs the ability of the RyR2 to properly deactivate leading to a spectrum of Ca(2+)-dependent pathologies that include cardiac systolic and diastolic dysfunction, arrhythmias, and structural remodeling. In this article, we present an overview of recent advances in our understanding of the underlying causes and pathological consequences of abnormal RyR2 function in the failing heart. We also discuss the implications of these findings for HF therapy.
Up-regulation of Sarcoplasmic Reticulum Ca(2+) Uptake Leads to Cardiac Hypertrophy, Contractile Dysfunction and Early Mortality in mice deficient in CASQ2
Kalyanasundaram A, Lacombe VA, Belevych AE, Brunello L, Carnes CA, Janssen PM, … Gyørke S.
Department of Physiology and Cell Biology, College of Medicine, Ohio State University, Columbus, OH.
Cardiovasc Res. May 2013; 98(2):297-306. http://dx.doi.org/10.1093/cvr/cvs334. Epub 2012 Nov 6.
Aberrant Ca(2+) release (i.e. Ca(2+) ‘leak’) from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptors (RyR2) is linked to heart failure (HF). Does SR-derived Ca(2+) can actually cause HF? We ask whether and by what mechanism combining dysregulated RyR2 function with facilitated Ca(2+) uptake into SR exacerbates abnormal SR Ca(2+) release and induces HF.
We crossbred mice deficient in expression of cardiac calsequestrin (CASQ2) with mice overexpressing the skeletal muscle isoform of SR Ca(2+)ATPase (SERCA1a). The new double-mutant strains displayed early mortality, congestive HF with left ventricular dilated hypertrophy, and decreased ejection fraction. Intact right ventricular muscle preparations from double-mutant mice preserved normal systolic contractile force but were susceptible to spontaneous contractions. Double-mutant cardiomyocytes while preserving normal amplitude of systolic Ca(2+) transients displayed marked disturbances in diastolic Ca(2+) handling in the form of multiple, periodic Ca(2+) waves and wavelets. Dysregulated myocyte Ca(2+) handling and structural and functional cardiac pathology in double-mutant mice were associated with increased rate of apoptotic cell death. Qualitatively similar results were obtained in a hybrid strain created by crossing CASQ2 knockout mice with mice deficient in phospholamban.
We demonstrate that enhanced SR Ca(2+) uptake combined with dysregulated RyR2s results in sustained diastolic Ca(2+) release causing apoptosis, dilated cardiomyopathy, and early mortality. Further, up-regulation of SERCA activity must be advocated with caution as a therapy for HF in the context of abnormal RyR2 function.
Comment in
Mind the store: modulating Ca(2+) reuptake with a leaky sarcoplasmic reticulum. [Cardiovasc Res. 2013] PMID: 23135969 [PubMed – in process] PMCID: PMC3633154 [Available on 2014/5/1]
Myocardial Delivery of Stromal Cell-Derived Factor 1 in Patients With Ischemic Heart Disease: Safe and Promising Circ. Res.. 2013;112:746-747
Circulation Research Thematic Synopsis: Cardiovascular Genetics Circ. Res.2013;112:e34-e50,
Ryanodine Receptor Phosphorylation and Heart Failure: Phasing Out S2808 and ³Criminalizing² S2814 ,
By the time the heart reaches the pathological state clinically recognized as heart failure (HF), it has undergone profound and often irreversible alterations in structure and function at the molecular, cellular and organ level. Although the etiologies of HF are diverse:
hypertension,
myocardial infarction,
atherosclerosis,
valvular insufficiency,
mutations in genes encoding sarcomeric proteins
Some alterations are commonly found in most forms of HF, and they may account for the maladaptive structural remodeling and systolic dysfunction that characterize this syndrome.
At the cellular level, there are well documented changes in
ionic channel density and function (electrical remodeling),
increased ROS production,
mitochondrial dysfunction,
imbalanced energy intake and consumption,
genetic reprogramming,
altered excitation-contraction coupling,
and in general, dysregulation of a multitude of other processes and pathways that are essential for proper cardiac function. Combined, this myriad of alterations leads to
loss in contractility and
loss ejection fraction,
ventricular wall remodeling,
increased vascular resistance, and
dysregulated fluid homeostasis.
In this issue of Circulation Research, Respress et al.2 report that preventing phosphorylation of cardiac ryanodine receptors (RyR2) at a single residue, S2814, is sufficient to avert many of these alterations and improve cardiac function in HF. The results presented here follow a string of papers that touch on the delicate and controversial subject of ryanodine receptor phosphorylation and HF. They offer a new twist to a contentious story and attempt to reconcile many apparently contradicting results, but key issues remain.
Calcium “Leak” in HF
It appears that suppressing the dysfunction of a select group of biological and molecular signaling pathways may substantially improve or even reverse the cardiac deterioration observed in HF. For example, correcting the characteristically depressed sarcoplasmic reticulum (SR) calcium content of failing cardiomyocytes is a target of HF gene therapy. SR calcium “leak”, an operational term that indicates increased and untimely calcium release by RyR2s, also appears common to several models of HF. Therefore, stemming off calcium “leak” may prevent the progression of cardiac malfunction in HF patients. However, a rationalized therapy towards this aim must be founded on the precise knowledge of the mechanisms leading to calcium leak. Marks group, in a landmark publication in 2000 (ref. 6) and later in multiple other high-impact factor papers (many of them co-authored by Wehrens 7-10) postulated that RyR2 “hyperphosphorylation” at S2808 by PKA was the primary mechanism leading to increased calcium “leak” in HF. This idea was initially appealing and fueled intensive research in the subject, but many groups failed to reproduce central tenets of this hypothesis. (11 and 12) The controversies surrounding the Marks-Wehrens hypothesis of increased calcium leak by hyperphosphorylation of RyR2-S2808 have been recently and comprehensibly reviewed by Bers.13 Here I will focus on the modifications to this hypothesis as derived from the new findings of Respress et al.2 Emerging points from these new findings will be the demotion of S2808, to intervene not as universal player in HF but only in selective forms of this syndrome, and the role of S2814 as pre-eminent generator of calcium leak that leads to arrhythmias and exacerbates other forms of HF. The “criminalization” of S2814 has begun in earnest.
CaMKII Effect on Calcium Leak and the Role of S2808 and S2814
Many studies have provided evidence that persistent CaMKII activity can lead to cardiac arrhythmias and promote HF.14-16 Animals and patients with congestive HF display increased levels of CaMKII,17,18 and overexpression of AC3-I, a peptide inhibitor of CaMKII, delays the onset of HF in mice.19 There is also good agreement4,20 (although not universal21) that CaMKII, and not PKA, increases calcium leak, and therefore, it is likely that the arrhythmogenic and deleterious activity of CaMKII in HF may be associated with this effect. Obviously, if PKA does not cause calcium leak directly, this by itself imposes insurmountable constraints on the Marks-Wehrens hypothesis that posits that PKA phosphorylation of RyR2-S2808 is responsible for the high calcium leak of HF. With the focus now on CaMKII, the obligated question is then, by what mechanisms CaMKII increases calcium leak from the SR? To increase calcium leak, the cell must either increase SR calcium content, and/or increase the activity of the RyR2 (albeit the latter alone would have only transient effects due to autoregulatory mechanisms22). Since both PKA and CaMKII increase SR calcium load by phosphorylating phospholamban (but at different residues) and relieving the inhibition it exerts on SERCA2a, the differential effect of these kinases must result from the regulation they exert on RyR2s. Wehrens group offers here2 at least a partial explanation of this complex mechanism and, along with previous papers co- authored with Marks, these groups set specific roles for S2808 and S2814 on regulation of RyR2 activity and their protective effect (or lack thereof) in HF. In their view, PKA exclusively phosphorylates S2808 and dissociates FKBP12.6, which destabilizes the closed state of the channel and increases RyR2 activity, whereas CaMKII (almost) exclusively phosphorylates S2814, has no effect on FKBP12.6 binding, and equally activates RyR2s. In this issue, Respress et al.2 report that preventing phosphorylation of S2814 (by genetic substitution of Ser by Ala, S2814A) protects against non-ischemic (pressure overload) HF but has no effect on ischemic HF; conversely, and against other data by the same groups, S2808 phosphorylation was not significantly different in non-ischemic HF, implying that it is relevant only in ischemic HF. This clean targeting of RyR2 phospho-epitopes by PKA and CaMKII and their nice “division of labor” for pathogenicity in distinct forms of HF would really simplify phosphorylation schemes and reconcile apparent contradictions. However, as is generally the case, the proposal appears oversimplified and almost too good to be true. Let’s discuss each of the premises on which the Respress et al.2 results have been interpreted and the problems associated with these premises.
One kinase = one site = one effect. Is it really that simple?
The RyR2 is a huge protein. It is assembled as a tetrameric complex of ~2 million Da, with each subunit composed of ~5,000 amino acids.
Using canonical phosphorylation consensus and high confidence values, the RyR2 may be phosphorylated in silico at more than 100 sites by the combined action of PKA,
CaMKII,
PKG, and
PKC, to name a few.11
Granted, a “potential” phosphorylation site is very different than a demonstrated, physiologically-relevant phosphorylation site and it is possible that many of the predicted residues are not phosphorylated in vivo. Even then, several groups have demonstrated that CaMKII phosphorylates RyR2 with stoichiometry of at least 3 or 4 to 1 with respect to PKA.23-26 This fact is by itself compelling evidence that there are multiple phosphorylation sites in RyR2. Now, let’s make the optimistic assumption that all the PKA sites have already been mapped, and that S2808 and S2030 (ref. 27) are the only PKA sites. Taking into account the CaMKII:PKA phosphorylation ratio (3:1 or 4:1), this would then yield a minimum of ~6 – 8 CaMKII phosphorylation sites (per channel subunit!). In this perspective, it is almost disingenuous to label S2808 as “the” PKA site, and we may purposely deceive ourselves when we label S2814 “the” CaMKII site. Against this sense of pessimism and intractability, let’s not forget that S2808 was actually discovered as a CaMKII site.24 It is possible then that the number of CaMKII sites is smaller if only S2030 remains as a bona fide PKA site. Still, neither scheme supports one CaMKII site per channel subunit.
But let’s go along for a moment with the possibility, however unlikely, that PKA phosphorylates S2808 only, and CaMKII phosphorylates S2814 only. When calling these sites by their distinctive numbers, it is easy to forget that these phospho-sites are only 6 residues apart, that is, a minuscule proportion (~0.000003%) in the context of the whole channel protein. How can the same reaction (phosphorylation) that occurs at sites so close to one another be differentially transmitted to the very distant gating domains of the channel? If these residues were lining the pore of the channel, where critical differences emerge by substituting one residue but not the neighboring one, then it would be easier to understand how S2808 and S2814 could transmit distinct signals. But both are part of a “phosphorylation hot spot”, a cytoplasmic loop that contains additional potential phospho-sites11 and that has been mapped to the external surface of the channel.28 Marks and Wehrens groups have shown that phosphorylation of S2808A by CaMKII or of S2814A by PKA fully activate the channel.7,9 At face value, this means that knocking out one phospho-residue does not cripple this “hot spot” and that phosphorylation of at least one residue in this external loop enables it to transmit conformational changes to the gating domains of the channel. Seen in this structural context in which the “hot spot” works in unison upon phosphorylation of at least one residue, it is very difficult (but not impossible) to accommodate the notion that phosphorylation of S2808 or S2814 alone dictates the differential response of the RyR2 to PKA and CaMKII.
An Alternative Model to explain Differential PKA and CaMKII Effects
An alternative model to explain the differential effect of PKA and CaMKII to elicit calcium leak from RyR2 that takes into account other phospho-sites is needed. Before formulating it, let’s consider some important points. First, it is not difficult to assume that the role of the “phosphorylation hot spot” is to readily pick up signals from different kinases. The multi-valence of this “hot spot” is demonstrated so far by the fact that S2808 may be phosphorylated by CaMKII24,25,26 and by PKA,6,25,26 and its eagerness to undergo phosphorylation by the fact that S2808 is at least ~50% phosphorylated even at basal state25-27,29,30 and phospho-signals from these sites may be readily detected upon β-adrenergic stimulation of the heart.30,31Second, if we accept the Shannon and Bers results that CaMKII, and not PKA, elicits calcium leak from the SR,4,20 this obligatorily means that PKA phosphorylation of S2808 is not responsible for eliciting calcium leak (in direct conflict with the Marks-Wehrens hypothesis). In support of this notion, studies by the Houser and Valdivia groups have provided evidence that preventing S2808 phosphorylation has negligible impact on the β-adrenergic response of the heart and on the progression of non-ischemic and ischemic HF.30-32 Third, another PKA site, S2030, largely ignored in the Marks-Wehrens scheme, has been mapped and shown to activate channel openings27 and although its place in the larger context of RyR2 phosphorylation has not been determined yet, I think it is illogical to assume that its existence is futile and that it contributes nothing to regulation of the channel. Thus, according to the preceding discussion, it is almost unsustainable to postulate that the differential effects of CaMKII and PKA to elicit calcium leak stems from their effects on the RyR2 “phosphorylation hot spot” alone. Instead, I would like to posit an alternative model that integrates findings by many of the above-referenced groups (Fig. 1). In this model, the surface domain of the RyR2 comprising residues 2804-2814 (mouse nomenclature) is an eager target for phosphorylation by PKA, CaMKII and probably other kinases (4 Ser/Thr).11,24-26,29 Phosphorylation of this “hot spot” by either PKA or CaMKII (or both) “primes” the RyR2 for subsequent signals and is probably responsible for the coordinated openings in response to fast calcium stimuli detected in single channel recordings33 and in cellular settings34 (but this has yet to be demonstrated). The differential effect of PKA and CaMKII on RyR2 activity would then depend on the integrated response of the phosphorylated “hot spot” and of additional phosphorylation sites. For example, phosphorylation of S2808 and S2030 by PKA could coordinate channel openings in response to fast calcium stimuli, and phosphorylation of S2814 and other CaMKII site(s) could open RyR2s at diastolic [Ca2+], which would translate in calcium leak. Examples of proteins acting as molecular switchboards in response to various degrees of phosphorylation are not unprecedented.35 In fact, RyR2s are activated by phosphorylation and dephosphorylation as well36,37 and their relative degree of phosphorylation determines a final functional output.38 It is therefore conceivable that the complex response of RyR2s to any type of phosphorylation and the variable results obtained by investigators apparently using the same experimental conditions may be due to the variable degree of phosphorylation in which the RyR2s were found. Of course, until the 3D structure of the RyR2 is solved and we understand the mechanism by which the “phosphorylation hot spot” and other phospho-sites “talk” to the channel’s gating domains this structurally-based model will remain speculative, but it at least takes into consideration compelling evidence on the existence of various phosphorylation sites and departs substantially from the simplified notion of one kinase = one site = one effect.
Fig. 1 Models of RyR2 modulation by phosphorylation
Models of RyR2 modulation by phosphorylation. In the Marks-Wehrens model (A), S2808 is the only site phosphorylated by PKA, and S2814 by CaMKII. PKA phosphorylation of S2808 dissociates FKBP12.6, which destabilizes the closed state of the channel and induces subconductance states, eliciting calcium leak. Calcium leak from the SR then causes deleterious effects such as arrhythmias and worsening of (ischemic) HF. CaMKII phosphorylation of S2814 does not dissociate FKBP12.6 but also causes calcium leak. This leak is also arrhythmogenic but is not relevant in ischemic HF, only in nonischemic HF. In the multiphosphorylation site model (B), S2808 and S2814 are part of a “phosphorylation hot spot” that is located in a protruding part of the channel, is targeted by several kinases, and may contain other phospho-epitopes not yet characterized. Phosphorylation of individual residues within this “hot spot” may be undistinguishable by the channel’s gating domains; instead, the differential regulation of PKA and CaMKII on channel gating may come about by the combined effect of each kinase on phospho-residues of the “hot spot” and other phosphorylation sites.
In situations of stress the heart beats faster and stronger. According to Marks and colleagues, this response is, to a large extent, the consequence of facilitated Ca2+ release from intracellular Ca2+ stores via ryanodine receptor 2 (RyR2), thought to be due to catecholamine-induced increases in RyR2 phosphorylation at serine 2808 (S2808). If catecholamine stimulation is sustained (for example, as occurs in heart failure), RyR2 becomes hyperphosphorylated and “leaky,” leading to arrhythmias and other pathology. This “leaky RyR2 hypothesis” is highly controversial. In this issue of the JCI, Marks and colleagues report on two new mouse lines with mutations in S2808 that provide strong evidence supporting their theory.
In the signalling scheme outlined in Figure1 of this commentary, which prevailed until the end of the last century, the two major determinants of intracellular Ca2+ transients and thereby the contractile force of the heart were (a) the size of the Ca2+ current entering via the LTCC (well exemplified by the negative inotropic effects of LTCC blockers) and (b) the activity of SERCA and thus the Ca2+ load of the SR. The critical role of the latter was convincingly demonstrated by the fact that Plb–/– mice, which have maximal SERCA activity, exhibit higher basal force and reduced inotropic response to isoprenaline (1).
In the Marks-Wehrens model, S2808 is the only site phosphorylated by PKA, and S2814 by CaMKII. PKA phosphorylation of S2808 dissociates FKBP12.6, which destabilizes the closed state of the channel and induces subconductance states, eliciting calcium leak. Calcium leak from the SR then causes deleterious effects such as arrhythmias and worsening of (ischemic) HF. CaMKII phosphorylation of S2814 does not dissociate FKBP12.6 but also causes calcium leak. This leak is also arrhythmogenic, but is not relevant in ischemic HF, only in non-ischemic HF. In the multi-phosphorylation site model, S2808 and S2814 are part of a “phosphorylation hot spot” that is located in a protruding part of the channel, is targeted by several kinases, and may contain other phospho-epitopes not yet characterized. Phosphorylation of individual residues within this “hot spot” may be undistinguishable by the channel’s gating domains; instead, the differential regulation of PKA and CaMKII on channel gating may come about by the combined effect of each kinase on phospho-residues of the “hot spot” and other phosphorylation sites.
Appealing as Marks’ theory is, the concept has been challenged and remains controversial (Tables1 and 2). On the one hand, some theoretical considerations argue against it. For example, it seems counterintuitive that phosphorylation at a single residue in a protein of more than 5,000 amino acids could profoundly affect channel open probability. Second, S2808, the proposed site of phosphorylation by PKA, is located in an area distant from the FKBP12.6/RyR2 interaction site (3), making it somewhat unlikely that phosphorylation affects FKPB12.6 binding. Third, it seems unlikely and to contradict experimental results (4) that an isolated increase in RyR2 open probability has more than a transient consequence on Ca2+ handling, because an isolated increase in Ca2+release from the RyR2 will automatically lead to reduced Ca2+ load in the SR and therefore fast normalization of Ca2+ transients (autoregulation).
More concerning than theoretical considerations are numerous reports that failed to reproduce important aspects of the data that support the leaky RyR2 hypothesis and the critical importance of S2808 (Tables (Tables11and and2).2). (a) Phosphorylation of RyR2 at S2808 has been found by others to be either not altered in heart failure at all or to be only moderately increased (5–8). Others have reported that 75% of the available RyR2 S2808 sites are phosphorylated under normal conditions, making a 9-fold change in chronic heart failure somewhat unlikely (9). (b) Whereas general consensus exists that β-adrenergic stimulation increases spontaneous Ca2+ release (the “Ca2+ leak”) from the SR, the role of RyR2 phosphorylation and FKBP12.6 dissociation remains controversial. Importantly, PKA had no effect on Ca2+release in permeabilized Plb–/– mouse myocytes, i.e., cells in which the SR is maximally loaded with Ca2+ and one would have expected a particularly strong effect of increasing RyR2 open probability.
Now, let’s go back to the results of Respress et al.2 and consider them in this light. They found that preventing phosphorylation of S2814 alone mitigates non-ischemic HF induced by transverse aortic constriction (TAC) in mice. This implies that other CaMKII sites are not necessary to mitigate the CaMKII-induced calcium leak that they propose is responsible for the deleterious effect in WT mice subjected to TAC. If phosphorylation of the “hot spot” is compulsory to prime the RyR2 to process and discriminate other phosphorylation signals, then other residues in that “hot spot” must have been phosphorylated to fulfill this need. Surprisingly, S2808 was not significantly phosphorylated in this setting. This leaves a very difficult conundrum: if S2808 was not phosphorylated significantly and the other CaMKII sites are not necessary to stop calcium leak, how then can we explain the results of Respress et al.2? Of course there are always alternatives, and we would be inconsistent if we rigidly adhere to one model and fell into the dogmatism we are criticizing. The conclusions of Respress et al.2 are in line with their findings, but at this point the numbers do not add up and it’s obvious that the great complexity of this process (RyR2 phosphorylation) precludes simplified and neatly organized schemes. As a clear example of this, in the landmark paper by Marks group,6 S2808 was found substantially hyperphosphorylated in tachypacing-induced failing dogs, also a non-ischemic model of HF. This does not fit well in the current scheme of Wehrens where S2808A protects against ischemic HF, but has no prominent role in non-ischemic HF.
In summary, CaMKII and PKA may have specific roles in calcium leak and, since they both increase SR calcium load, their differential effect likely resides on their effect on RyR2s. However, the effect of PKA- or CaMKII-phosphorylation of RyR2s does not appear solved yet. Starting in 2000 and up to the present day, Marks and Wehrens have provided high-quality data in prominent journals aggressively pursuing the notion that PKA phosphorylates S2808 only, that CaMKII phosphorylates S2814 only, and that these sites alone integrate multiple signals to open RyR2s. Many key aspects of their general hypothesis including dissociation of FKBP12.6 by PKA phosphorylation of S2808, subconductance states as hallmarks of phosphorylation, and the prominent role of S2808 as promoter of arrhythmias and HF have not been confirmed by several groups. The present paper by the Wehrens group modifies slightly the original claim that S2808 was involved in ischemic and non-ischemic forms of HF and continues to shift the lion’s share of pathogenicity to S2814. However, as discussed above, the Marks-Wehrens model largely ignores compelling data on the presence of multiple phosphorylation sites and the complexity they add to the finely graded response of RyR2s to phosphorylation.
2. Respress JL, van Oort RJ, Li N, Rolim N, Dixit S, Dealmeida A, Voigt N, Lawrence WS, Skapura DG, Skårdal K, Wisloff U, Wieland T, Ai X, Pogwizd SM, Dobrev D, Wehrens XH. Role of RyR2 Phosphorylation at S2814 During Heart Failure Progression. Circ Res. 2012;xx:xx–xx. [in the issue; printer, please update] [PMC free article] [PubMed]
6. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000;101(4):365–376. [PubMed]
7. Wehrens XH, Lehnart SE, Reiken S, Vest JA, Wronska A, Marks AR. Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression. Proc Natl Acad Sci U S A. 2006;103:511–518. [PMC free article] [PubMed]
36. Lokuta AJ, Rogers TB, Lederer WJ, Valdivia HH. Modulation of cardiac ryanodine receptors of swine and rabbit by a phosphorylation-dephosphorylation mechanism. J Physiol. 1995;487:609–622. [PMC free article] [PubMed]
37. Terentyev D, Viatchenko-Karpinski S, Gyorke I, Terentyeva R, Gyorke S. Protein phosphatases decrease sarcoplasmic reticulum calcium content by stimulating calcium release in cardiac myocytes. J Physiol. 2003;552(Pt 1):109–118. [PMC free article] [PubMed]
38. Carter S, Colyer J, Sitsapesan R. Maximum phosphorylation of the cardiac ryanodine receptor at Ser-2809 by protein kinase A produces unique modifications to channel gating and conductance not observed at lower levels of phosphorylation. Circ Res. 2006; 98:1506–1513. [PubMed]
The Cardiac Ryanodine Receptor (calcium release channel) – Emerging role in Heart Failure and Arrhythmia Pathogenesis
The cardiac sarcoplasmic reticulum calcium release channel, commonly referred to as the ryanodine receptor, is a key component in cardiac excitation–contraction coupling, where it is responsible for the release of calcium from the sarcoplasmic reticulum. As our knowledge of the ryanodine receptor has advanced an appreciation that this key E–C coupling component may have a role in the pathogenesis of human cardiac disease has emerged. Heart failure and arrhythmia generation are both pathophysiological states that can result from deranged excitation–contraction coupling. Evidence is now emerging that hyperphosphorylation of the cardiac ryanodine receptor is an important event in chronic heart failure, contributing to impaired contraction and the generation of triggered ventricular arrhythmias.
Furthermore the therapeutic benefits of β blockers in heart failure appear to be partly explained through a reversal of this phenomenon. Two rare inherited arrhythmogenic conditions, which can cause sudden death in children, have also been shown to result from mutations in the cardiac ryanodine receptor. These conditions,
catecholaminergic polymorphic ventricular tachycardia and
arrhythmogenic right ventricular cardiomyopathy (subtype 2),
further implicate the ryanodine receptor as a potentially arrhythmogenic substrate and suggest this channel may offer a new therapeutic target in the treatment of both cardiac arrhythmias and heart failure.
Protein phosphatases decrease sarcoplasmic reticulum calcium content by stimulating calcium release in cardiac myocytes
D Terentyev, S Viatchenko-Karpinski, I Gyorke, R Terentyeva and S Gyorke
Texas Tech University Health Sciences Center, Lubbock, TX
J Physiol 2003; 552(1), pp. 109–118. http:/dx.doi.org/10.1113/jphysiol.2003.046367
Phosphorylation/dephosphorylation of Ca2+ transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation–contraction coupling; furthermore, abnormal protein kinase and phosphatase activities have been implicated in heart failure. However, the precise mechanisms of action of these enzymes on intracellular Ca2+ handling in normal and diseased hearts remains poorly understood. We have investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca(2+) sparks and SR Ca(2+) load in myocytes permeabilized with saponin. Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca(2+) sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca(2+) stores, as determined by application of caffeine. These changes in Ca(2+) release and SR Ca(2+) load could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1-medited RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti-RyR antibody. Our results suggest that increased intracellular phosphatase activity stimulates RyR mediated SR Ca(2+) release leading to depleted SR Ca(2+) stores in cardiac myocytes.
In heart muscle cells, the process of excitation–contraction (EC) coupling is mediated by Ca(2+) influx through sarcolemmal L-type Ca(2+) channels activating Ca(2+) release channels (ryanodine receptors, RyRs) in the sarcoplasmicreticulum (SR). Once activated, the RyR channels allow Ca(2+) to be released from the SR into the cytosol to induce contraction. This mechanism is known as Ca(2+)-induced calcium release (CICR) (Fabiato, 1985; Bers, 2002).
During relaxation, most of the Ca(2+) is resequestered into the SR by the Ca(2+)-ATPase. The amount of Ca(2+) released and the force of contraction depend on the magnitude of the Ca(2+) trigger signal, the functional state of the RyRs and the amount of Ca(2+) stored in the SR. Reversible phosphorylation of proteins composing the EC coupling machinery plays an important role in regulation of cardiac contractility (Bers, 2002). Thus, during stimulation of the b-adrenergic pathway, phosphorylation of several target proteins, including the L-type Ca(2+) channels, RyRs and phospholamban, by protein kinase A (PKA) leads to an overall increase in SR Ca2+ release and contractile force in heart cells (Callewaert et al. 1988, Spurgeon et al. 1990; Hussain & Orchard, 1997; Zhou et al. 1999; Song et al. 2001; Viatchenko-Karpinski & Gyorke, 2001). PKA-dependent phosphorylation of the L-type Ca(2+) channels increases the Ca2+ current (ICa), increasing both the Ca2+ trigger for SR Ca2+ release and the SR Ca(2+) content (Callewaert et al. 1988; Hussain & Orchard, 1997; Del Principe et al. 2001). Phosphorylation of phospholamban (PLB) relieves the tonic inhibition dephosphorylated PLB exerts on the SR Ca(2+)-ATPase (SERCA) resulting in enhanced SR Ca(2+) accumulation and enlarged Ca(2+) release (Kranias et al. 1985; Simmermann & Jones, 1998). With regard to the RyR, despite clear demonstration of phosphorylation of the channel in biochemical studies (Takasago et al. 1989; Yoshida et al. 1992), the consequences of this reaction to channel function have not been clearly defined. RyR phosphorylation by PKA and Ca(2+)–calmodulin dependent protein kinase (CaMKII) has been reported to increase RyR activity in lipid bilayers (Hain et al. 1995; Marx et al. 2000; Uehara et al. 2002). Moreover, it has been reported that in heart failure (HF), hyperphosphorylation of RyR causes the release of FK-506 binding protein (FKBP12.6) from the RyR, rendering the channel excessively leaky for Ca(2+) (Marx et al. 2000). However, other studies have reported no functional effects (Li et al. 2002) or even found phosphorylation to reduce RyR channel steady-state open probability (Valdivia et al. 1995; Lokuta et al. 1995).
The Action of Protein Kinases is Opposed by Dephosphorylating Phosphatases.
Three types of protein: phosphatases (PPs), referred to as
PP1,
PP2A and
PP2B (calcineurin),
have been shown to influence cardiac performance (Neumann et al. 1993; Rusnak & Mertz, 2000). Overall, according to most studies phosphatases appear to downregulate SR Ca(2+) release and contractile performance (Neumann et al. 1993; duBell et al. 1996, 2002; Carr et al. 2002; Santana et al. 2002). Furthermore, PP1 and PP2A activities appear to be increased in heart failure (Neumann, 2002; Carr et al. 2002). However, again the precise mode of action of these enzymes on intracellular Ca(2+) handling in normal and diseased hearts remains poorly understood. In the present study, we have investigated the effects of protein phosphatases PP1 and PP2A on local Ca(2+) release events, Ca(2+) sparks, in cardiac cells. Our results show that phosphatases activate RyR mediated SR Ca(2+) release leading to depletion of SR Ca(2+) stores. These results provide novel insights into the mechanisms and potential role of protein phosphorylation/dephosphorylation in regulation of Ca(2+) signaling in normal and diseased hearts.
RESULTS
Effects of PP1 and PP2A on Ca2+ Sparks and SR Ca(2+) Content.
PP1 caused an early transient potentiation of Ca2+ spark frequency followed by a delayed inhibition of event occurrence.
PP1 produced similar biphasic effects on the magnitude and spatio-temporal characteristics of Ca(2+) sparks
Specifically, during the potentiatory phase (1 min after addition of the enzyme), PP1 significantly increased the amplitude, rise-time, duration and width of Ca(2+) sparks; during the inhibitory phase (5 min after addition of the enzyme), all these parameters were significantly suppressed by PP1.
The SR Ca(2+) content decreased by 35 % or 69 % following the exposure of myocytes to either 0.5 or 2Uml_1 PP1, respectively (Fig. 1C).
Qualitatively similar results were obtained with phosphatase PP2A. Similar to the effects of PP1, PP2A (5Uml_1) produced a transient increase in Ca(2+) spark frequency (~4-fold) followed by a depression of event occurrence and decreased SR Ca(2+) content (by 82 % and 65 %, respectively). Also similar to the action of PP1, PP2A increased the amplitude and spatio-temporal spread (i.e. rise-time, duration and width) of Ca(2+) sparks at 1 min and suppressed the same parameters at 5 min of exposure to the enzyme (Table 1). Together, these results suggest that phosphatases enhance spark-mediated SR Ca2+ release, leading to decreased SR Ca(2+) content.
Preventive effects of calyculin A and okadaic acid
Preventive effects of ryanodine
PP1-mediated RyR dephosphorylation
The cardiac RyR is phosphorylated at Ser-2809 (in the rabbit sequence) by both PKA and CAMKII (Witcher et al. 1991; Marx et al. 2000). Although additional phosphorylation sites may exist on the RyR (Rodriguez et al. 2003), Ser-2809 is believed to be the only site that is phosphorylated by PKA, and RyR hyperphosphorylation at this site has been reported in heart failure (Marx et al. 2000). To test whether indeed phosphatases dephosphorylated the RyR in our permeabilized myocyte experiments we performed quantitative immunoblotting using an antibody that specifically recognizes the phosphorylated form of the RyR at Ser-2809 (Rodriguez et al. 2003). Myocytes exhibited a significant level of phosphorylation under baseline conditions. Maximal phosphorylation was 201 % of control. When exposed to 2Uml_1 PP1, RyR phosphorylation was 58 % of the control basal condition. Exposing to a higher PP1 concentration (10Uml_1) further reduced RyR phosphorylation to 22% of control. Thus, consistent with the results of our functional measurements, PP1 decreased RyR phosphorylation in cardiac myocytes.
A, spontaneous Ca(2+) spark images recorded under reference conditions, and 1 or 5 min after exposure of the cell to 2Uml_1 PP1. Traces below the images are Ca(2+) transients induced by application of 10 mM caffeine immediately following the acquisition of sparks before (3 min) and after (5 min) application of PP1 in the same cell. The Ca(2+) transients were elicited by a whole bath application of 10 mM caffeine. B, averaged spark frequency at early (1 min) and late (5 min) times following the addition of either 0.5 or 2Uml_1 of PP1 to the bathing solution. C, averaged SR Ca(2+) content for 0.5 or 2Uml_1 of PP1 measured before and 5 min after exposure to the enzyme. Data are presented as means ± S.E.M. of 6 experiments in different cells.
A, spontaneous Ca(2+) spark images recorded under reference conditions, and 1 or 5 min after exposure of the cell to 5Uml_1 PP2A. Traces below the images are Ca(2+) transients induced by application of 10 mM caffeine immediately following the acquisition of sparks before (3 min) and after (5 min) application of PP2A in the same cell. B and C, averaged spark frequency (B) and SR Ca(2+) content (C) for the same conditions as in A. Data are presented as means ± S.E.M. of 6 experiments in different cells.
DISCUSSION
In the present study, we have investigated the impact of physiologically relevant exogenous protein phosphatases PP1 and PP2A on RyR-mediated SR Ca(2+) release (measured as Ca(2+) sparks) in permeabilized heart cells. Our principal finding is that phosphatases stimulated RyR channels leading to depleted SR Ca(2+) stores. These results have important ramifications for understanding the mechanisms and role of protein phosphorylation/dephosphorylation in modulation of Ca(2+) handling in normal and diseased heart.
Modulation of SR Ca2+ release by Protein Phosphorylation/Dephophorylation
Since protein dephosphorylation clearly resulted in increased functional activity of the Ca(+)release channel, our results imply that a reverse, phosphorylation reaction should reduce RyR activity. If indeed such effects take place, why do they not manifest in inhibition of Ca(+)sparks? One possibility is that enhanced Ca(+) uptake by SERCA masks or overcomes the effects phosphorylation may have on RyRs. In
addition, the potential inhibitory influence of protein phosphorylation on RyR activity in myocytes could be countered by feedback mechanisms involving changes in luminal Ca(+)(Trafford et al. 2002; Gyorke et al. 2002). In particular, reduced open probability of RyRs would be expected to lead to increased Ca2+ accumulation in the SR; increased intra-SR [Ca(2+)] in turn would increase activity of RyRs at their luminal Ca(2+) regulatory sites as demonstrated for the RyR channel inhibitor tetracaine (Gyorke et al. 1997; Overend et al. 1997). Thus potentiation of SERCA combined with the intrinsic capacity of the release mechanism to self-regulate could explain at least in part why PKA-mediated protein phoshorylation results in maintained potentiation of Ca(2+) sparks despite a potential initial decrease in RyR activity.
Role of altered RyR Phosphorylation in Heart Failure
Marx et al. (2000) have proposed that enhanced levels of circulating catecholamines lead to increased phosphorylation of RyR in heart failure. Based on biochemical observations as well as on studying properties of single RyRs incorporated into artificial lipid bilayers, these investigators have hypothesized that hyperphosphorylation of RyRs contributes to pathogenesis of heart failure by making the channel excessively leaky due to dissociation of FKBP12.6 from the channel. We show that the mode of modulation of RyRs by phosphatases does not support this hypothesis as dephosphorylation caused activation instead of inhibition of activity of RyR channels in a relatively intact setting. Interestingly, our results provide the basis for a different possibility in which dephophosphorylation of RyR rather than its phosphorylation causes depletion of SR Ca(2+) stores by stimulating RyRs in failing hearts. It has been reported that PP1 and PP2 activities are increased in heart failure (Huang et al. 1999; Neumann et al. 1997; Neuman, 2002). Furthermore, overexpression of PP1 or ablation of the endogenous PP1 inhibitor, l-1, results in depressed contractile performance and heart failure (Carr et al. 2002). Our finding that PP1 causes depletion of SR Ca(2+) stores by activating RyRs could account for, or contribute to, these results.
DelPrincipe F, Egger M, Pignier C & Niggli E (2001). Enhanced E-C coupling efficiency after beta-stimulation of cardiac myocytes. Biophys J 80, 64a.
Gyorke I & Gyorke S (1998). Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites. Biophys J 75, 2801–2810.
Gyorke S, Gyorke I, Lukyanenko V, Terentyev D, Viatchenko-Karpinski S & Wiesner TF (2002). Regulation of sarcoplasmic reticulum calcium release by luminal calcium in cardiac muscle. Front Biosci 7, d1454–d1463.
Gyorke I, Lukyanenko V & Gyorke S (1997). Dual effects of tetracaine on spontaneous calcium release in rat ventricular myocytes. J Physiol 500, 297–309.
MacDougall LK, Jones LR & Cohen P (1991). Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban. Eur J Biochem 196, 725–734.
Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N & Marks AR (2000). PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101, 365–376.
Rodriguez P, Bhogal MS & Colyer J (2003). Stoichiometric phosphorylation of cardiac ryanodine receptor on serine-2809 by calmodulin-dependent kinase II and protein kinase A. J Biol Chem (in press).
Proc Natl Acad Sci U S A. 2010 August 3; 107(31): E124.
The ryanodine receptor/calcium-release channel (RyR2) on the sarcoplasmic reticulum (SR) is the source of Ca2+ required for myocardial excitation–contraction (EC) coupling. During stress (i.e., exercise), contractility of the cardiac muscle is increased largely because of phosphorylation and activation of key proteins that regulate SR Ca2+ release. These include the voltage-gated calcium channel (Cav1.2) on the plasma membrane through which Ca2+ enters the cardiomyocyte, the sarco/endoplasmic reticulum calcium ATPase (SERCA2a)/phospholamban complex that pumps Ca2+ into the SR, and the RyR2 channel that releases Ca2+ from the SR, all of which are activated by phosphorylation.
For the past 10 y, Eisner et al. (1) have advanced the idea that activation of the RyR2 channel (e.g., by phosphorylation) cannot play a role in regulating systolic Ca2+ release and cardiac contractility. They base their position on an experiment in which they used caffeine to activate the RyR2 channel and showed that Ca2+ release was increased but after a few beats, returned to baseline (1). However, their experiment is not a good model for the physiological response to stress in which the three key regulators of EC coupling are all activated by the same signal (i.e., phosphorylation) such that there is increased Ca2+ influx, increased SR Ca2+ uptake, and increased SR Ca2+ release.
In the Eisner caffeine experiment, RyR2 was activated, but the Cav1.2 and SERCA2a were not. Selective activation of RyR2 is not physiological, and the outcome of their experiment was predictable. Caffeine-induced activation of RyR2 resulted in a transient increase in SR Ca2+ release, but because there was no concomitant increase in Ca2+ influx or SR Ca2+ uptake, the increase in SR Ca2+ release could not be sustained. However, on the basis of this experiment, Eisner et al. (1) concluded that activation of RyR2 plays no role in stress-induced increased cardiac contractility.
We have shown that, during stress, the increased heart rate results in a rate-dependent activation of CaMKII that phosphorylates and activates RyR2. We showed the essential role of this rate-dependent activation of RyR2 by CaMKII by showing that genetically engineered mice, lacking the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), exhibit blunted increases in systolic Ca2+-transient amplitudes and contractile responses as heart rate increases (2). We also showed that a reduction in the amount of CaMKII in the RyR2 complex in failing hearts results in defective regulation of the channel, which could explain the loss of the rate-dependent increase in contractility in heart failure.
Eisner et al. (3) challenge all of our findings based on their caffeine experiment. However, our experiments have been conducted under physiological conditions in which all three components involved in Ca2+signaling during muscle contraction are activated, not just one. The only perturbation that we have introduced is to ablate the CaMKII phosphorylation site on RyR2 using a single amino acid substitution. This results in a blunted contractile response, leading us to conclude that CaMKII phosphorylation of RyR2 does indeed play a key role in enhancing contractility as the heart rate increases.
Cardiac Ryanodine Receptor Function and Regulation in Heart Disease
Cardiac Engineering: From Genes and Cells to Structure and Function 2004; 1015(1), pp 144–159
The cardiac ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR) controls intracellular Ca2+ release and muscle contraction in the heart. Ca2+ release via RyR2 is regulated by several physiological mediators. Protein kinase (PKA) phosphorylation dissociates the stabilizing FKBP12.6 subunit (calstabin2) from the RyR2 complex, resulting in increased contractility and cardiac output. Congestive heart failure is associated with
elevated plasma catecholamine levels, and
chronic stimulation of β-adrenergic receptors
leads to PKA hyperphosphorylation of RyR2 in failing hearts.
PKA hyperphosphorylation results in calstabin2-depleted RyR2 that displays altered channel gating and
may cause aberrant SR Ca2+ release,
depletion of SR Ca2+ stores, and
reduced myocardial contractility in heart failure.
Calstabin2-depleted RyR2 may also trigger cardiac arrhythmias that cause sudden cardiac death. In patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), RyR2 missense mutations cause reduced calstabin2 binding to RyR2. Increased RyR2 phosphorylation and pathologically increased calstabin2 dissociation during exercise results in aberrant diastolic calcium release, which may trigger ventricular arrhythmias and sudden cardiac death. In conclusion, heart failure and exercise-induced sudden cardiac death have been linked to defects in RyR2-calstabin2 regulation, and this may represent a novel target for the prevention and treatment of these forms of heart disease
The δC Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure
T Zhang, LS Maier, ND Dalton, S Miyamoto, J Ross, DM Bers, JH Brown
University of California, San Diego, La Jolla, Calif; and Loyola University, Chicago, Ill.
Circ Res. 2003;92:912-919. http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5
Recent studies have demonstrated that transgenic (TG) expression of either Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKIIδB, both of which localize to the nucleus, induces cardiac hypertrophy. However, CaMKIV is not present in heart, and cardiomyocytes express not only the nuclear CaMKIIδB but also a cytoplasmic isoform, CaMKII δC. In the present study, we demonstrate that expression of the δC isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload. To determine whether enhanced activity of this cytoplasmic δC isoform of CaMKII can lead to phosphorylation of Ca(2+) regulatory proteins and induce hypertrophy, we generated TG mice that expressed the δC isoform of CaMKII. Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca2(2+) handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and CaMKII is found associated with the RyR in immunoprecipitates from the CaMKII TG mice. Phosphorylation of phospholamban is also increased specifically at the CaMKII but not at the PKA phosphorylation site. These findings are the first to demonstrate that CaMKIIδC can mediate phosphorylation of Ca(2+) regulatory proteins in vivo and provide evidence for the involvement of CaMKIIδC activation in the pathogenesis of dilated cardiomyopathy and heart failure.
Multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM kinases or CaMKs) are transducers of Ca2+ signals that phosphorylate a wide range of substrates and thereby affect Ca(2+)-mediated cellular responses.1 The family includes CaMKI and CaMKIV, monomeric enzymes activated by CaM kinase kinase,2,3 and CaMKII, a multimer of 6 to 12 subunits activated by autophosphorylation.1 The CaMKII subunits α, β, γ, and δ show different tissue distributions,1 with the δisoform predominating in the heart.4–7 Splice variants of the δisoform, characterized by the presence of a second variable domain,4,7 include δB, which contains a nuclear localization signal (NLS), and δC, which does not. CaMKII composed of δB subunits localizes to the nucleus, whereas CaMKIIδC localizes to the cytoplasm.4,8,9
CaMKII has been implicated in several key aspects of acute cellular Ca(2+) regulation related to cardiac excitation-contraction (E-C) coupling. CaMKII phosphorylates sarcoplasmic reticulum (SR) proteins including the ryanodine receptors (RyR2) and phospholamban (PLB).10–14 Phosphorylation of RyR has been suggested to alter the channel open probability,14,15 whereas phosphorylation of PLB has been suggested to regulate SR Ca(2+) uptake.14 It is also likely that CaMKII phosphorylates the L-type Ca2 channel complex or an associated regulatory protein and thus mediates Ca2 current (ICa) facilitation.16-18 and the development of early after-depolarizations and arrhythmias.19 Thus, CaMKII has significant effects on E-C coupling and cellular Ca2 regulation. Nothing is known about the CaMKII isoforms regulating these responses.
Contractile dysfunction develops with hypertrophy, characterizes heart failure, and is associated with changes in cardiomyocyte Ca2homeostasis.20 CaMKII expression and activity are altered in the myocardium of rat models of hypertensive cardiac hypertrophy21,22 and heart failure,23 and in cardiac tissue from patients with dilated cardiomyopathy.24,25
Several transgenic mouse models have confirmed a role for CaMK in the development of cardiac hypertrophy, as originally suggested by studies in isolated neonatal rat ventricular myocytes.9,26–28 Hypertrophy develops in transgenic mice that overexpress CaMKIV,27 but this isoform is not detectable in the heart,4,29 and CaMKIV knockout mice still develop hypertrophy after transverse aortic constriction (TAC).29
Transgenic mice overexpressing calmodulin developed severe cardiac hypertrophy,30 later shown to be associated with an increase in activated CaMKII31; the isoform of CaMKII involved in hypertrophy could not be determined from these studies. We recently reported that transgenic mice that overexpress CaMKIIδB, which is highly concentrated in cardiomyocyte nuclei, develop hypertrophy and dilated cardiomyopathy.32 To determine whether in vivo expression of the cytoplasmic CaMKIIδC can phosphorylate cytoplasmic Ca2regulatory proteins and induce hypertrophy or heart failure, we generated transgenic (TG) mice that expressed the δC isoform of CaMKII under the control of the cardiac specific α-myosin heavy chain (MHC) promoter. Our findings implicate CaMKIIδC in the pathogenesis of dilated cardiomyopathy and heart failure and suggest that this occurs at least in part via alterations in Ca2handling proteins.33
Results
Expression and Activation of CaMKIIδC Isoform After TAC
To determine whether CaMKII was regulated in pressureoverload–induced hypertrophy, CaMKIIδ expression and phosphorylation were examined by Western blot analysis using left ventricular samples obtained at various times after TAC. A selective increase (1.6-fold) in the lower band of CaMKIIδwas observed as early as 1 day and continuously for 4 days (2.3-fold) and 7 days (2-fold) after TAC (Figure 1A). To confirm that CaMKIIδC was increased and determine whether this occurred at the transcriptional level, we performed semiquantitative RT-PCR using primers specific for the CaMKIIδC isoform. These experiments revealed that mRNA levels for CaMKIIδC were increased 1 to 7 days after TAC (Figure 1B). In addition to examining CaMKII expression, the activation state of CaMKII was monitored by its autophosphorylation, which confers Ca2-independent activity.
A, Western blot analysis of total CaMKII in left ventricular (LV) homogenates obtained at indicated times after TAC. Cardiomyocytes transfected with CaMKIIδB and δC (right) served as positive controls and molecular markers. Top band (58 kDa) represents CaMKIIδB plus δ9, and the bottom band (56 kDa) corresponds to CaMKIIδC. *P0.05 vs control. B, Semiquantitative RT-PCR using primers specific for CaMKIIδC isoform (24 cycles) and GAPDH (19 cycles) using total RNA isolated from the same LV samples. C, Western blot analysis of phospho-CaMKII in LV homogenates obtained at various times after TAC. Three bands seen for each sample represent CaMKIIγ subunit (uppermost), CaMKIIδB plus δ9 (58 kDa), and CaMKIIδC (56 kDa). Quantitation is based on the sum of all of the bands. *P0.05 vs control.
A, Transgene copy number based on Southern blots using genomic DNA isolated from mouse tails (digested with EcoRI). Probe (a 32P-labeled 1.7-kb EcoRI-SalI -MHC fragment) was hybridized to a 2.3-kb endogenous fragment (En) and a 3.9-kb transgenic fragment (TG). Transgene copy number was determined from the ratio of the 3.9-kb/2.3-kb multiplied by 2. B, Immunocytochemical staining of ventricular myocytes isolated from WT and CaMKIIδTG mice. Myocytes were cultured on laminin-coated slides overnight. Transgene was detected by indirect immunofluorescence staining using rabbit anti-HA antibody (1:100 dilution) followed by FITC-conjugated goat antirabbit IgG antibody (1:100 dilution). CaMKIIδB localization to the nucleus in CaMKIIδB TG mice (see Reference 32) is shown here for comparative purpose. C, Quantitation of the fold increase in CaMKIIδprotein expression in TGL and TGM lines. Different amounts of ventricular protein (numbers) from WT control, TG () and their littermates () were immunoblotted with an anti-CaMKIIδ antibody. Standard curve from the WT control was used to calculate fold increases in protein expression in TGL and TGM lines. D, Phosphorylated CaMKII in ventricular homogenates was measured by Western blot analysis (n5 for each group). **P0.01 vs WT.
Generation and Identification of CaMKIIδC Transgenic Mice
TG mice expressing HA-tagged rat wild-type CaMKIIδC under the control of the cardiac-specific α-MHC promoter were generated as described in Materials and Methods. By Southern blot analysis, 3 independent TG founder lines carrying 3, 5, and 15 copies of the transgene were identified. They were designated as TGL (low copy number), TGM (medium copy number), and TGH (high copy number),
The founder mice from the TGH line died at 5 weeks of age with marked cardiac enlargement. The other two lines showed germline transmission of the transgene. The transgene was expressed only in the heart.
Although CaMKII protein levels in TGL and TGM hearts were increased 12- and 17-fold over wild-type (WT) controls
(Figure 2C), the amount of activated CaMKII was only increased 1.7- and 3-fold in TGL and TGM hearts (Figure 2D). The relatively small increase in CaMKII activity in the TG lines probably reflects the fact that the enzyme is not constitutively activated and that the availability of Ca2/CaM, necessary for activation of the overexpressed CaMKII, is limited. Importantly, the extent of increase in active CaMKII in the TG lines was similar to that elicited by TAC.
Cardiac Overexpression of CaMKIIδC Induces Cardiac Hypertrophy and Dilated Cardiomyopathy
There was significant enlargement of hearts from CaMKIIδC TGM mice by 8 to 10 weeks (Figure 3A) and from TGL mice by 12 to 16 weeks. Histological analysis showed ventricular dilation (Figure 3B), cardiomyocyte enlargement (Figure 3C), and mild fibrosis (Figure 3D) in CaMKIIδC TG mice. Quantitative analysis of cardiomyocyte cell volume from 12-week-old TGM mice gave values of 54.7 + 0.1 pL for TGM (n = 96) versus 28.6 + 0.1 pL for WT littermates (n=94; P0.001).
Ventricular dilation and cardiac dysfunction developed over time in proportion to the extent of transgene expression. Left ventricular end diastolic diameter (LVEDD) was increased by 35% to 45%, left ventricular posterior wall thickness (LVPW) decreased by 26% to 29% and fractional shortening decreased by 50% to 60% at 8 weeks for TGM and at 16 weeks for TGL. None of these parameters were significantly altered at 4 weeks in TGM or up to 11 weeks in TGL mice, indicating that heart failure had not yet developed. Contractile function was significantly decreased.
C, Decreased contractile function in ventricular myocytes isolated from 12-week old TGM and WT controls presented as percent change of resting cell length (RCL) stimulated at 0.5 Hz. Representative trace and mean values are shown. *P0.05 vs WT.
Figure 7. Phosphorylation of PLB in CaMKIIδC TG mice.
Thr17 and Ser16 phosphorylated PLB was measured by Western blots using specific anti-phospho antibodies. Ventricular homogenates were from 12- to 14-week-old WT and TGM mice (A) or 4 to 5-week-old WT and TGM mice (B). Data were normalized to total PLB examined by Western blots (data not shown here). n = 6 to 8 mice per group; *P0.05 vs WT.
Cardiac Overexpression of CaMKIIδC Results in Changes in the Phosphorylation of Ca2 Handling Proteins
To assess the possible involvement of phosphorylation of Ca2cycling proteins in the phenotypic changes observed in the CaMKIIC TG mice, we first compared PLB phosphorylation state in homogenates from 12- to 14-week-old TGM and WT littermates. Western blots using antibodies specific for phosphorylated PLB showed a 2.3-fold increase in phosphorylation of Thr17 (the CaMKII site) in hearts from TGM versus WT (Figure 7A). Phosphorylation of PLB at the CaMKII site was also increased 2-fold in 4- to 5-week-old TGM mice (Figure 7B). Significantly, phosphorylation of the PKA site (Ser16) was unchanged in either the older or the younger TGM mice (Figures 7A and 7B).
To demonstrate that the RyR2 phosphorylation changes observed in the CaMKII transgenic mice are not secondary to development of heart failure, we performed biochemical studies examining RyR2 phosphorylation in 4- to 5-week-old TGM mice. At this age, most mice showed no signs of hypertrophy or heart failure (see Figure 6B) and there was no significant increase in myocyte size (21.3 + 1.3 versus 27.7 + 4.6 pL; P0.14). Also, twitch Ca2 transient amplitude was not yet significantly depressed, and mean δ [Ca2+]i (1 Hz) was only 20% lower (192 + 36 versus 156 + 13 nmol/L; P0.47) versus 50% lower in TGM at 13 weeks.33
The in vivo phosphorylation of RyR2, determined by back phosphorylation, was significantly (2.10.3-fold; P0.05) increased in these 4- to 5-week-old TGM animals (Figure 8C), an increase equivalent to that seen in 12- to 14-week-old mice. We also performed the RyR2 back-phosphorylation assay using purified CaMKII rather than PKA. RyR2 phosphorylation at the CaMKII site was also significantly increased (2.2 + 0.3-fold; P0.05) in 4- to 5-week-old TGM mice (Figure 8C).
The association of CaMKII with the RyR2 is consistent with a physical interaction between this protein kinase and its substrate. The catalytic subunit of PKA and the phosphatases PP1 and PP2A were also present in the RyR2 immunoprecipitates, but not different in WT versus TG mouse hearts (Figure 8D). These data provide further evidence that the increase in RyR2 phosphorylation, which precedes development of failure in the 4- to 5-week-old CaMKIIδC TG hearts, can be attributed to the increased activity of CaMKII.
Discussion
CaMKII is involved in the dynamic modulation of cellular Ca2 regulation and has been implicated in the development of cardiac hypertrophy and heart failure.14 Published data from CaMK-expressing TG mice demonstrate that forced expression of CaMK can induce cardiac hypertrophy and lead to heart failure.27,32 However, the CaMK genes expressed in these mice are neither the endogenous isoforms of the enzyme nor the isoforms likely to regulate cytoplasmic Ca(2+) handling, because they localize to the nucleus.
First, we demonstrate that the cytoplasmic cardiac isoform of CaMKII is upregulated at the expression level and is in the active state (based on autophosphorylation) after pressure overload induced by TAC. Second, we demonstrate that two cytoplasmic CaMKII substrates (PLB and RyR) are phosphorylated in vivo when CaMKII is overexpressed and its activity increased to an extent seen under pathophysiological conditions. Moreover, CaMKIIδis found to associate physically with the RyR in the heart. Finally, our data indicate that heart failure can result from activation of the cytoplasmic form of CaMKII and this may be due to altered Ca(2+) handling.
Differential Regulation of CaMKIIδ Isoforms in Cardiac Hypertrophy
The isoform of CaMKII that predominates in the heart is the δ isoform.4–7 Neither the α nor the β isoforms are expressed and there is only a low level of expression of the γ isoforms.39 Both δB and δC splice variants of CaMKIIδ are present in the adult mammalian myocardium36,40 and expressed in distinct cellular compartments.4,8,9
We suggest that the CaMKIIδisoforms are differentially regulated in pressure-overload–induced hypertrophy, because the expression of CaMKIIδC is selectively increased as early as 1 day after TAC. Studies using RT-PCR confirm that CaMKIIδC is regulated at the transcriptional level in response to
TAC. In addition, activation of both CaMKIIδB and CaMKIIδC, as indexed by autophosphorylation, increases as early as 2 days after TAC. Activation of CaMKIIδB by TAC is relevant to our previous work indicating its role in hypertrophy.9,32 The increased expression, as well as activation of the CaMKIIδC isoform, suggests that it could also play a critical role in both the acute and longer responses to pressure overload.
In conclusion, we demonstrate here that CaMKIIδC can phosphorylate RyR2 and PLB when expressed in vivo at levels leading to 2- to 3-fold increases in its activity. Similar increases in CaMKII activity occur with TAC or in heart failure. Data presented in this study and in the accompanying article33 suggest that altered phosphorylation of Ca(2+) cycling proteins is a major component of the observed decrease in contractile function in CaMKIIδC TG mice. The early occurrence of increased CaMKII activity after TAC, and of RyR and PLB phosphorylation in the CaMKIIδC TG mice suggest that CaMKIIδC plays an important role in the pathogenesis of dilated cardiomyopathy and heart failure. These results have major implications for considering CaMKII and its isoforms in exploring new treatment strategies for heart failure.
Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity.
DR Witcher, RJ Kovacs, H Schulman, DC Cefali, LR Jones
Krannert Institute of Cardiology and the Indiana University School of Medicine, Indianapolis,
Stanford University School of Medicine, Stanford.
Journal of Biological Chemistry 07/1991; 266(17):11144-52. · 4.77 Impact http://www.jbc.org/content/266/17/11144.full.pdf
Ryanodine receptors have recently been shown to be the Ca2+ release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites are postulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac [3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylation at this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca(2+) release channel by CaM kinase may provide a unique mechanism for regulating intracellular (Ca2+) release.
The Ca(2+) release from the SR causes an increase in Ca(2+) concentration which leads to muscle contraction (1). Recently, the sites of Ca(2+) release have been identified and purified from both cardiac (2-4) and skeletal muscle SR (5- 7) and shown to be the same as the ryanodine receptors or high molecular weight proteins. The structures attach the transverse tubules to the junctional SR both in intact tissues and isolated membrane fractions (1, 8-10). Although the Ca(2+) release channels from cardiac and skeletal muscle show many similarities such as nearly identical
myoplasmic 3- EGTA,
Ca2+ conductances (2-7),
protease sensitivities (11, E ) ,
calmodulin-binding capabilities (ll), and
modulation by allosteric regulators such as Ca2+, Mg2+, ATP, and calmodulin (13-15),
they also exhibit several differences in protein structure and function. Quantitative differences have been noted on the effects of modulators on ryanodine binding to the two proteins (16-18), as well as on Ca(2+) channel kinetics. In addition, the cardiac ryanodine smaller apparent molecular weight than the skeletal muscle receptor on SDS-PAGE (ll), and monoclonal antibodies can be made which react with the cardiac receptor but not the skeletal receptor (16).
Recent work on characterization receptors has culminated in elucidation of structures of the proteins by sequencing of their cDNAs (19-21). Consistent with the differences between the two protein iso- forms noted above, the cardiac and skeletal muscle receptors have been found to be the products of different genes, with overall amino acid identities of 66% (21). Both protein isoforms are very large, containing approximately 5,000 amino acids and exhibiting predicted molecular weights of 564,711 for the cardiac protein (21) and 565,223 (19) or 563,584 (20) for the skeletal muscle protein. In the native state, ryanodine receptors are arranged as tetramers (1-7). In an earlier study (22), we demonstrated that the canine cardiac high molecular weight protein (or ryanodine receptor; Ref. 3) was an excellent substrate CaM kinase (23,24) endogenous to junctional SR membranes. In the work described here, we show that phosphorylation of the cardiac receptor by CaM kinase occurs at a single site, which is not substantially phosphorylated in the skeletal muscle receptor, and that phosphorylation ryanodine receptor at this site activates the Ca2+ channel.
Our data are the first to support the hypothesis (21), that the modulator-binding sites of the cardiac ryanodine receptor are contained within residues 2619-3016. (13, 14). The ryanodine receptor is compared with the primary structure for the multifunctional of the cardiac model of Otsu et al. (21).
Preferential Phosphorylation Receptor-(Fig. 1, arrowheads) is phosphorylated in junctional vesicles by an endogenous calmodulin-requiring proteinase and this phosphorylation is stimulated several fold when exogenous CaM kinase is added. In contrast, the ryanodine receptor in canine fast and vesicles, which migrates with weight on SDS-PAGE (2, 11, 16), is not significantly phosphorylated by either endogenous or exogenous protein kinase (Fig. 1, small arrows).
Similar results were obtained with rabbit skeletal muscle SR vesicles. The identity of the skeletal muscle ryanodine receptor in these studies (Fig. 1, small arrow) was confirmed by immunoblotting with a skeletal muscle isoform-specific antibody (supplied by K. Campbell, University of Iowa). We did detect a low level of phosphorylation of a protein in slow skeletal muscle samples migrating slightly faster than the cardiac receptor, but this protein did not cross-react with skeletal muscle (or cardiac, see below) antibodies, suggesting that it is unrelated to the ryanodine receptor. CaM kinase-catalyzed phosphorylation of the cardiac ryanodine receptor was always at least 10-fold greater than skeletal receptor phosphorylation. These results demonstrate that the skeletal muscle ryanodine receptor phosphorylation is insignificant compared to cardiac protein phosphorylation. Consistent with our results, Otsu et al. (21) have recently shown that, the cardiac isoform receptor is absent from fast and slow skeletal muscle. Phosphorylation of the cardiac ryanodine receptor by cAMP kinase also occurs, but phosphorylation by added cAMP kinase is no greater than that achieved with endogenous CaM kinase. (Fig. 2). In contrast, the amount of exogenous CaM kinase increases receptor phosphorylation 4-fold, to a maximal level of 26 pmol of P/mg of SR protein (Fig. 2). We observed no significant phosphorylation of canine fast and slow or rabbit skeletal muscle ryanodine. Maximal ryanodine binding (3) in these preparations ranged between 5 and 6 pmol/mg of protein, a value nearly identical to the level of receptor phosphorylation achieved with exogenous cAMP kinase (see CaM kinase), but one-fourth the value achieved with added CaM kinase. Since the functional unit release channel contains only one high affinity ryanodine- binding site/tetramer (4), our results suggest that the endogenous CaM kinase is capable of phosphorylating only one-fourth of the available sites, whereas the exogenous kinase can fully phosphorylate the receptor (below) of the Cardiac Ryanodine. The canine Slow skeletal muscle SR receptor of the ryanodine it was recently reported is phosphorylated 1/20th by the of the CaM kinase.
TABLE 1
Immunoprecipitation of Ryanodine receptors from CHAPS-solubilized canine SR membranes. Values are expressed for aliquots of the following fractions: S, solubilized receptors after treatment of membranes with 2% CHAPS; B, bound fraction, containing ryanodine receptors immunoprecipitated from CHAPS superna- tant; F, free fraction, containing ryanodine receptors not immunoprecipitated. Total binding was measured using 20 nM [3H]ryanodine. For nonspecific binding, 10 PM cold ryanodine was added. FIG. 7.
Effect of ATP and calmodulin on the cardiac Ca(2+) release channel. Holding potential was 0 mV, with upward current deflections representing movement of Ba(2+) from the trans to the cis chamber. Gaussian distributions were fit to the peaks of activity in the histograms. Signals were filtered at 300 Hz (low pass Bessel) and digitized at 1 KHz (Axotape, Axon Instruments) for * off-line analysis. In the control (A), p(open) was 0.26. Addition of 1 mM ATP (B) produced prolonged openings of the channel, increasing p(0pen) to 0.81. Subsequent addition of calmodulin (C) decreased p(open) to 0.12, producing long closures and brief aborted openings.
Sequencing of the Cardiac Phosphorylation Site. In order to sequence the phosphorylation site of the cardiac ryanodine receptor, we phosphorylated junctional SR membranes on large scale with added CaM kinase and purified the phosphorylated denatured ryanodine receptor to homogeneity in one step using SDS-gel filtration chromatography (Fig. 3). The purified cardiac ryanodine receptor was digested with trypsin, and the radioactive peptides recovered using Fe(3+) affinity chromatography (30,37). 90% of the loaded radioactivity was recovered in the pH 8.6 and 10 eluates from the Fe column (Fig. 4). These fractions were then combined and subjected to reverse-phase chromatography, yielding a single major radioactive peptide peak eluting at approximately 24% acetonitrile (Fig. 4, inset).
Gas-phase sequencing of the radioactive tryptic peptide gave a single sequence of 18 consecutive residues, which corresponded exactly to residues 2807-2824 reported for the rabbit cardiac ryanodine receptor from cDNA cloning (Fig. 5) (21). When CNBr and endoproteinase Lys-C were used to cleave the receptor, another “P-labeled peptide was isolated and sequenced, which matched with residues 2800-2811 of the rabbit cardiac ryanodine receptor (Fig. 5).
Serine 2809 within the phosphorylated tryptic peptide is situated on the carboxyl-terminal side of 2 arginine residues. The fact that R-R-X-S and R-X-X-S/T are minimal consensus phosphorylation sequences (38,39) for CAMP kinase and CaM kinase, respectively, makes this residue the likely phosphorylation site. Consistent with this, the ratio threitol-serine to phenylthiohydantoin-serine recovered dur- ing cycle 3 of sequencing of this peptide was 10 times greater than that recovered during cycles 6 and 9. It is known that dithiothreitol-serine is the predominant breakdown product of phosphoserine (40, 41). Phosphoamino acid analysis revealed that this peptide contained only phosphoserine; more- over, >90% of the 3’Pi was released from the peptide by cycle 10 (40, 42), demonstrating that no serine residue downstream of this region was significantly labeled.
Based on these results, we conclude that serine 2809 is the amino acid phosphorylated by CaM kinase. When only endogenous CaM kinase was used to phosphorylate the cardiac ryanodine receptor, the same labeled tryptic peptide was recovered and sequenced in four separate runs. Thus, although exogenously added kinase gives a 4-fold stimulation of receptor phosphorylation (Fig. 2), no new sites are phosphorylated. The reason for the low level of phosphorylation obtained with endogenous CaM kinase remains undefined.
Cardiac Electrophysiological Dynamics From the Cellular Level to the Organ Level
Daisuke Sato and Colleen E. Clancy
Department of Pharmacology, University of California – Davis, Davis, CA.
Biomedical Engineering and Computational Biology 2013:5: 69–75
Figure 1. (Top): APD and DI. (Bottom): The physiological mechanism of APD alternans involves recovery from inactivation of ICaL. [see http://dx.doi.org/10.4137/BECB.S10960]
Figure 2. APD restitution and dynamical mechanism of APD alternans. [see http://dx.doi.org/10.4137/BECB.S10960] Review Series. Genetic Causes of Human Heart Failure
Hiroyuki Morita, Jonathan Seidman and Christine E. Seidman
Harvard Medical School, Brigham and Women’s Hospital, Howard Hughes Medical Institute, Boston, MA
J Clin Invest. 2005;115(3):518–526. http://dx.doi.org/10.1172/JCI24351.
Correspondence to: Christine E. Seidman, Department of Genetics, Harvard Medical School, Boston, MA. Ph: (617) 432-7871; E-mail: cseidman@genetics.med.harvard.edu
Factors that render patients with cardiovascular disease at high risk for heart failure remain incompletely defined. Recent insights into molecular genetic causes of myocardial diseases have highlighted the importance of single-gene defects in the pathogenesis of heart failure. Through analyses of the mechanisms by which a mutation selectively perturbs one component of cardiac physiology and triggers cell and molecular responses, studies of human gene mutations provide a window into the complex processes of cardiac remodeling and heart failure. Knowledge gleaned from these studies shows promise for defining novel therapeutic targets for genetic and acquired causes of heart failure.
Introduction
Heart failure currently affects 4.8 million Americans, and each year over 500,000 new cases are diagnosed. In 2003 heart failure contributed to over 280,000 deaths and accounted for 17.8 billion health care dollars (1).
Heart failure almost universally arises in the context of antecedent cardiovascular disease:
atherosclerosis,
cardiomyopathy,
myocarditis,
congenital malformations, or
valvular disease.
The study of single-gene mutations that trigger heart failure provides an opportunity for defining important molecules involved in these processes. Although these monogenic disorders account for only a small subset of overall heart failure cases, insights into the responses triggered by gene mutations are likely to also be relevant to more common etiologies of heart failure.
Early Manifestation – Heart Failure – Ventricular Remodeling.
One of 2 distinct morphologies occurs: left ventricular hypertrophy (increased wall thickness without chamber expansion) or dilation (normal or thinned walls with enlarged chamber volumes).
Each is associated with specific hemodynamic changes. Systolic function is normal, but diastolic relaxation is impaired in hypertrophic remodeling; diminished systolic function characterizes dilated remodeling. Clinical recognition of these cardiac findings usually prompts diagnosis of hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). There is now considerable evidence that many different gene mutations can cause these pathologies (Figure 1), and with these discoveries has come recognition of distinct histopathologic features that further delineate several subtypes of remodeling. The current compendia of genes that remodel the heart already suggest a multiplicity of pathways by which the human heart can fail.
To facilitate a discussion, we have grouped known cardiomyopathy genes according to the probable functional consequences of mutations on
force generation and transmission,
metabolism,
calcium homeostasis, or
transcriptional control.
Gene mutations in one functional category inevitably have an impact on multiple myocyte processes, and, the eventual delineation of signals between functional groups may be critical to understanding cardiac decompensation and heart failure development.
Human gene mutations can cause cardiac hypertrophy (blue), dilation (yellow), or both (green). In addition to these two patterns of remodeling, particular gene defects produce hypertrophic remodeling with glycogen accumulation (pink) or dilated remodeling with fibrofatty degeneration of the myocardium (orange). Sarcomere proteins denote β-myosin heavy chain, cardiac troponin T, cardiac troponin I, α-tropomyosin, cardiac actin, and titin. Metabolic/storage proteins denote AMP-activated protein kinase γ subunit, LAMP2, lysosomal acid α 1,4–glucosidase, and lysosomal hydrolase α-galactosidase A. Z-disc proteins denote MLP and telethonin. Dystrophin-complex proteins denote δ-sarcoglycan, β-sarcoglycan, and dystrophin. Ca2+ cycling proteins denote PLN and RyR2. Desmosome proteins denote plakoglobin, desmoplakin, and plakophilin-2.
Force generation and propagation. Generation of contractile force by the sarcomere and its transmission to the extracellular matrix are the fundamental functions of heart cells. Inadequate performance in either component prompts cardiac remodeling (hypertrophy or dilation), produces symptoms, and leads to heart failure. Given the importance of these processes for normal heart function and overt clinical manifestations of deficits in either force generation or transmission, it is not surprising that more single-gene mutations have been identified in molecules involved in these critical processes than in those of other functional classes.
Human mutations affecting contractile and Z-disc proteins. The schematic depicts one sarcomere,
the fundamental unit of contraction encompassing the protein segment between flanking Z discs. Sarcomere thin filament proteins are composed of actin and troponins C, T, and I. Sarcomere thick filament proteins include myosin heavy chain, myosin essential and regulatory light chains, myosin-binding protein-C and titin. The sarcomere is anchored through titin and actin interactions with Z disc proteins α-actinin, calsarcin-1, MLP, telethonin (T-cap), and ZASP. Human mutations (orange text) in contractile proteins and Z-disc proteins can cause HCM or DCM.
Sarcomere protein mutations. Human mutations in the genes encoding protein components of the sarcomere cause either HCM or DCM. While progression to heart failure occurs with both patterns of remodeling, the histopathology, hemodynamic profiles, and biophysical consequences of HCM or DCM mutations suggest that distinct molecular processes are involved.
Over 300 dominant mutations in genes encoding β-cardiac myosin heavy chain (MYH7), cardiac myosin-binding protein-C (MYBPC3), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), essential myosin light chain (MYL3), regulatory myosin light chain (MYL2), α-tropomyosin (TPM1), cardiac actin (ACTC), and titin (TTN) have been reported to cause HCM (Figure 2) (2, 3). Recent reports of comprehensive sequencing of sarcomere protein genes in diverse patient populations indicate that MYBPC3 and MYH7 mutations are most frequent (4, 5). Sarcomere gene mutations that cause HCM produce a shared histopathology with enlarged myocytes that are disorganized and die prematurely, which results in increased cardiac fibrosis.
The severity and pattern of ventricular hypertrophy,
age at onset of clinical manifestations, and
progression to heart failure
are, in part, dependent on the precise sarcomere protein gene mutation. For example, TNNT2 mutations are generally associated with a high incidence of sudden death despite only mild left ventricular hypertrophy (6, 7). While only a small subset (10–15%) of HCM patients develop heart failure, this end-stage phenotype has a markedly poor prognosis and often necessitates cardiac transplantation. Accelerated clinical deterioration has been observed with MYH7 Arg719Trp, TNNT2 Lys273Glu, TNNI3 Lys183del, and TPM1 Glu180Val mutations (8–11).
Most HCM mutations encode defective polypeptides containing missense residues or small deletions; these are likely to be stably incorporated into cardiac myofilaments and to produce hypertrophy because normal sarcomere function is disturbed. Many HCM mutations in MYBPC3 fall within carboxyl domains that interact with titin and myosin; however, the exact biophysical properties altered by these defects remain unknown (Figure 2). HCM mutations in myosin are found in virtually every functional domain, which suggests that the biophysical consequences of these defects may vary. Genetic engineering of some human myosin mutations into mice has indicated more consistent sequelae. Isolated single-mutant myosin molecules containing different HCM mutations
had increased actin-activated ATPase activity and
showed greater force production and
faster actin-filament sliding,
biophysical properties that may account for hyperdynamic contractile performance observed in HCM hearts and that suggest a mechanism for premature myocyte death in HCM (12–14). Uncoordinated contraction due to
heterogeneity of mutant and normal sarcomere proteins,
increased energy consumption, and
changes in Ca2+ homeostasis
could diminish myocyte survival and trigger replacement fibrosis. With insidious myocyte loss and increased fibrosis, the HCM heart transitions from hypertrophy to failure.
Mice that are engineered to carry a sarcomere mutation replicate the genetics of human disease; heterozygous mutations cause HCM. One exception is a deletion of proximal myosin-binding protein-C sequences; heterozygous mutant mice exhibited normal heart structure while homozygous mutant mice developed hypertrophy (15). Remarkably, while most heterozygous mouse models with a mutation in myosin heavy chain, myosin-binding protein-C, or troponin T developed HCM (16–18), homozygous mutant mice (19, 20) developed DCM with fulminant heart failure and, in some cases, premature death. These mouse studies might indicate that HCM, DCM, and heart failure reflect gradations of a single molecular pathway. Alternatively, significant myocyte death caused by homozygous sarcomere mutations may result in heart failure. Human data suggest a more complicated scenario. The clinical phenotype of rare individuals who carry homozygous sarcomere mutations in either MYH7 (21) or in TNNT2 (22) is severe hypertrophy, not DCM. Furthermore, individuals with compound heterozygous sarcomere mutations exhibit HCM, not DCM. The absence of ventricular dilation in human hearts with 2 copies of mutant sarcomere proteins is consistent with distinct cellular signaling programs that remodel the heart into hypertrophic or dilated morphologies.
DCM sarcomere protein gene mutations affect distinct amino acids from HCM-causing mutations, although the proximity of altered residues is remarkable. The histopathology of sarcomere DCM mutations is quite different from those causing HCM, and is remarkably nonspecific. Degenerating myocytes with increased interstitial fibrosis are present, but myocyte disarray is notably absent. There are 2 mechanisms by which sarcomere mutations may cause DCM and heart failure: deficits of force production and deficits of force transmission. Diminished force may occur in myosin mutations (e.g., MYH7 Ser532Pro) that alter actin-binding residues involved in initiating the power stroke of contraction. Impaired contractile force may also occur in DCM troponin mutations (TNNT2 ΔLys210, ref. 23; and TNNI3 Ala2Val, ref. 24) that alter residues implicated in tight binary troponin interactions. Because troponin molecules modulate calcium-stimulated actomyosin ATPase activity, these defects may cause inefficient ATP hydrolysis and therein decrease contractile power.
Other DCM sarcomere mutations are more likely to impair force transmission (Figure 2). For example, a myosin mutation (at residue 764) located within the flexible fulcrum that transmits movement from the head of myosin to the thick filament is likely to render ineffectual the force generated by actomyosin interactions (23). DCM TPM1 mutations (25) are predicted to destabilize actin interactions and compromise force transmission to neighboring sarcomere. Likewise, ACTC mutations (26) that impair binding of actin to Z-disc may compromise force propagation. TTN mutations provide quintessential evidence that deficits in force transmission cause DCM and heart failure. By spanning the sarcomere from Z-disc to M-line, this giant muscle protein assembles contractile filaments and provides elasticity through serial spring elements. Titin interacts with α-actinin and telethonin (T-cap) at the Z-disc, with calpain3 and obscurin at the I-band (the extensible thin filament regions flanking Z-discs), and with myosin-binding protein-C, calmodulin, and calpain3 at the M-line region. Human mutations identified in
the Z-disc–I-band transition zone (27),
in the telethonin and α-actinin–binding domain, and
in the cardiac-specific N2B domain (an I-band subregion; ref. 28) each cause DCM and heart failure.
Intermediate filaments and dystrophin-associated glycoprotein mutations. Intermediate filaments function as cytoskeletal proteins linking the Z-disc to the sarcolemma. Desmin is a type III intermediate filament protein, which, when mutated, causes skeletal and cardiac muscle disease (Figure 3). The hearts of mice deficient in desmin (29) are more susceptible to mechanical stress, which is consistent with the function of intermediate proteins in force transmission.
Figure 3
Human mutations (orange text) in components of myocyte cytoarchitecture cause DCM and heart failure. Force produced by sarcomeric actin-myosin interactions is propagated through the actin cytoskeleton and dystrophin to the dystrophin-associated glycoprotein complex (composed of α- and β-dystroglycans, α-, β-, γ- and δ-sarcoglycans, caveolin-3, syntrophin, and dystrobrevin). Desmosome proteins plakoglobin, desmoplakin, and plakophilin-2, provide functional and structural contacts between adjacent cells and are linked through intermediate filament proteins, including desmin, to the nuclear membrane, where lamin A/C is localized. (Adapted from ref. 96.)
Through dystrophin and actin interactions, the dystrophin-associated glycoprotein complex (composed of α- and β-dystroglycans, α-, β-, γ- and δ-sarcoglycans, caveolin-3, syntrophin, and dystrobrevin) provides stability to the sarcomere and transmits force to the extracellular matrix. Human mutations in these proteins cause muscular dystrophy with associated DCM and heart failure (Figure 3). Skeletal muscle manifestations can be minimal in female carriers of X-linked dystrophin defects, and some individuals present primarily with heart failure (30). In the mouse experiment, coxsackievirus B3–encoded protease2A, which can cleave dystrophin, was shown to produce sarcolemmal disruption and cause DCM, which suggests that dystrophin is also involved in the pathologic mechanism of DCM and heart failure that follow viral myocarditis (31).
While deficiencies of proteins that link the sarcomere to the extracellular matrix are likely to impair force transmission, recent studies of mice engineered to carry mutations in these molecules indicate other mechanisms for heart failure. A model of desmin-related cardiomyopathies (32) uncovered striking intracellular aggresomes, electron dense accumulations of heat shock and chaperone protein, α-B-crystalline, desmin, and amyloid in association with sarcomeres. While particularly abundant in the amyloid heart, aggresomes were also found in some DCM and HCM specimens, which suggests that excessive degenerative processing induced by myocyte stress or gene mutation may be toxic to sarcomere function.
Analyses of δ-sarcoglycan null mice (33) also yielded unexpected disease mechanisms, primary coronary vasospasm and myocardial ischemia. Selective restoration of δ-sarcoglycan to the cardiac myocytes extinguished this pathology, thereby implicating chronic ischemia as a contributing factor to heart failure development in patients with sarcoglycan mutations.
Mutations in intercalated and Z-disc proteins. To generate contraction, one end of each actin thin filament must be immobilized. The Z-disc defines the lateral boundary of the sarcomere, where actin filaments, titin, and nebulette filaments are anchored. Metavinculin provides attachment of thin filaments to the plasma membrane and plays a key role in productive force transmission. Two metavinculin gene mutations cause DCM by disruption of disc structure and actin-filament organization (34).
Other Z-disc protein constituents may also function as mechano-stretch receptors (35). Critical components include α-actinin, which aligns actin and titin from neighboring sarcomeres and interacts with muscle LIM protein (MLP encoded by CSRP3), telethonin (encoded by TCAP), which interacts with titin and MLP to subserve overall sarcomere function, and Cypher/Z-band alternatively spliced PDZ-motif protein (Cypher/ZASP), a striated muscle-restricted protein that interacts with α-actinin–2 through a PDZ domain and couples to PKC-mediated signaling via its LIM domains (Figure 2). Mutations in these molecules cause either DCM (35, 36) or HCM (37, 38) and predispose the affected individuals to heart failure. Genetically engineered mice with MLP deficiency (39) help to model the mechanism by which mutations in distinct proteins cause disease. Without MLP, telethonin is destabilized and gradually lost from the Z-disc; as a consequence, MLP-deficient cardiac papillary muscle shows an impairment in tension generation following the delivery of a 10% increase in passive stretch of the muscle and a loss of stretch-dependent induction of molecular markers (e.g., brain natriuretic peptide), which suggests that an MLP-telethonin–titin complex is an essential component of the cardiac muscle mechanical stretch sensor machinery. An important question is how signaling proteins (e.g., Cyper/ZASP) within the Z-disc translate mechanosensing into activation of survival or cell death pathways.
Lamin A/C mutations. The inner nuclear-membrane protein complex contains emerin and lamin A/C. Defects in emerin cause X-linked Emery-Dreifuss muscular dystrophy, joint contractures, conduction system disease, and DCM. Dominant lamin A/C mutations exhibit a more cardiac-restricted phenotype with fibrofatty degeneration of the myocardium and conducting cells, although subclinical involvement of skeletal muscles and contractures are sometimes apparent. The remarkable electrophysiologic deficits (progressive atrioventricular block and atrial arrhythmias) observed in mutations of lamin A/C and emerin indicate the particular importance of these proteins in electrophysiologic cells. A recent study of lamin A/C mutant mice showed evidence of marked nuclear deformation, fragmentation of heterochromatin, and defects in mechanotransduction (40, 41), all of which likely contribute to reduced myocyte viability. The similarities of cardiac histopathology (fibrofatty degeneration) observed in mutations of the nuclear envelope and desmosomes raise the possibility that these structures may both function as important mechanosensors in myocytes (Figure 3).
Desmosome protein mutations. Arrhythmogenic right ventricular cardiomyopathy (ARVD) identifies an unusual group of cardiomyopathies characterized by progressive fibrofatty degeneration of the myocardium, electrical instability, and sudden death (42). While right ventricular dysplasia predominates, involvement of the left ventricle also occurs. Progressive myocardial dysfunction is seen late in the course of disease, often with right-sided heart failure. ARVD occurs in isolation or in the context of Naxos syndrome, an inherited syndrome characterized by prominent skin (palmar-plantar keratosis), hair, and cardiac manifestations. Mutations in protein components of the desmosomes (Figure 3) (plakoglobin, ref. 43; desmoplakin, refs. 44, 45; and plakophilin-2, ref. 46) and in the cardiac ryanodine receptor (RyR2) (ref. 47; discussed below) cause syndromic and nonsydromic ARVD. Desmosomes are organized cell membrane structures that provide functional and structural contacts between adjacent cells and that may be involved in signaling processes. Whether mutations in the desmosomal proteins render cells of the heart (and skin) inappropriately sensitive to normal mechanical stress or cause dysplasia via another mechanism is unknown.
Energy production and regulation
Mitochondrial mutations. Five critical multiprotein complexes, located within the mitochondria, synthesize ATP by oxidative phosphorylation. While many of the protein components of these complexes are encoded by the nuclear genome, 13 are encoded by the mitochondrial genome. Unlike nuclear gene mutations, mitochondrial gene mutations exhibit matrilineal inheritance. In addition, the mitochondrial genome is present in multiple copies, and mutations are often heteroplasmic, affecting some but not all copies. These complexities, coupled with the dependence of virtually all tissues on mitochondrial-derived energy supplies, account for the considerable clinical diversity of mitochondrial gene mutations (Figure 4). While most defects cause either dilated or hypertrophic cardiac remodeling in the context of mitochondrial syndromes such as Kearns-Sayre syndrome, ocular myopathy, mitochondrial encephalomyopathy with lactic-acidosis and stroke-like episodes (MELAS), and myoclonus epilepsy with ragged-red fibers (MERFF) (48), there is some evidence that particular mitochondrial mutations can produce predominant or exclusive cardiac disease (49, 50). An association between heteroplasmic mitochondrial mutations and DCM has been recognized (51).
Figure 4
Human gene mutations affecting cardiac energetics and metabolism. Energy substrate utilization is directed by critical metabolic sensors in myocytes, including AMP-activated protein kinase (AMPK), which, in response to increased AMP/ATP levels, phosphorylates target proteins and thereby regulates glycogen and fatty acid metabolism, critical energy sources for the heart. Glycogen metabolism involves a large number of proteins including α-galactosidase A (mutated in Fabry disease) and LAMP2 (mutated in Danon disease). Glycogen and fatty acids are substrates for multiprotein complexes located within the mitochondria for the synthesis of ATP. KATP channels composed of an enzyme complex and a potassium pore participate in decoding metabolic signals to maximize cellular functions during stress adaptation. Human mutations (orange text) that cause cardiomyopathies have been identified in the regulatory SUR2A subunit of KATP, the γ2 subunit of AMPK, mitochondrial proteins, α-galactosidase A, and LAMP2.
Nuclear-encoded metabolic mutations. Nuclear gene mutations affecting key regulators of cardiac metabolism are emerging as recognized causes of hypertrophic cardiac remodeling and heart failure (Figure 4). Mutations in genes encoding the γ2 subunit of AMP-activated protein kinase (PRKAG2), α-galactosidase A (GLA), and lysosome-associated membrane protein-2 (LAMP2) can cause profound myocardial hypertrophy in association with electrophysiologic defects (52). AMP-activated protein kinase functions as a metabolic-stress sensor in all cells. This heterotrimeric enzyme complex becomes activated during energy-deficiency states (low ATP, high ADP) and modulates (by phosphorylation) a large number of proteins involved in cell metabolism and energy (53). Most GLA mutations can cause multisystem classic Fabry disease (angiokeratoma, corneal dystrophy, renal insufficiency, acroparesthesia, and cardiac hypertrophy), but some defects produce primarily cardiomyopathy. LAMP2 mutations can also produce either multisystem Danon disease (with skeletal muscle, neurologic, and hepatic manifestations) or a more restricted cardiac phenotype.
Cardiac histopathology reveals that, unlike sarcomere gene mutations, which cause hypertrophic remodeling, the mutations in PRKAG2, LAMP2, and GLA accumulate glycogen in complexes with protein and/or lipids, thereby defining these pathologies as storage cardiomyopathies. Progression from hypertrophy to heart failure is particularly common and occurs earlier with LAMP2 mutations than with other gene mutations that cause metabolic cardiomyopathies. Since both GLA and LAMP2 are encoded on chromosome X, disease expression is more severe in men, but heterozygous mutations in women are not entirely benign, perhaps due to X-inactivation that equally extinguishes a normal or mutant allele. The cellular and molecular pathways that produce either profound hypertrophy or progression to heart failure from PRKAG2, GLA, or LAMP2 mutations are incompletely understood. While accumulated byproducts are likely to produce toxicity, animal models indicate that mutant proteins cause far more profound consequences by changing cardiac metabolism and altering cell signaling. This is particularly evident in PRKAG2 mutations that increase glucose uptake by stimulating translocation of the glucose transporter GLUT-4 to the plasma membrane, increase hexokinase activity, and alter expression of signaling cascades (54).
The cooccurrence of electrophysiologic defects in metabolic mutations raises the possibility that pathologic cardiac conduction and arrhythmias contribute to cardiac remodeling and heart failure in these gene mutations. One mechanism for electrophysiologic defects appears to be the direct consequence of storage: transgenic mice that express a human PRKAG2 mutation (55) developed ventricular pre-excitation due to pathologic atrioventricular connections by glycogen-filled myocytes that ruptured the annulus fibrosis (the normal anatomic insulator which separates atrial and ventricular myocytes). A second and unknown mechanism may be that these gene defects are particularly deleterious to specialized cells of the conduction system. Little is known about the metabolism of these cells, although historical histopathologic data indicate glycogen to be particularly more abundant in the conduction system than in the working myocardium (56–58).
Ca2+ Cycling
Considerable evidence indicates the presence of abnormalities in myocyte calcium homeostasis to be a prevalent and important mechanism for heart failure. Protein and RNA levels of key calcium modulators are altered in acquired and inherited forms of heart failure, and human mutations in molecules directly involved in calcium cycling have been found in several cardiomyopathies (Figure 5).
Figure 5
Human mutations affecting Ca2+ cycling proteins. Intracellular Ca2+ handling is the central coordinator of cardiac contraction and relaxation. Ca2+ entering through L-type channels (LTCC) triggers Ca2+ release (CICR) from the SR via the RyR2, and sarcomere contraction is initiated. Relaxation occurs with SR Ca2+ reuptake through the SERCA2a. Calstabin2 coordinates excitation and contraction by modulating RyR2 release of Ca2+. PLN, an SR transmembrane inhibitor of SERCA2a modulates Ca2+ reuptake. Dynamic regulation of these molecules is effected by PKA-mediated phosphorylation. Ca2+ may further function as a universal signaling molecule, stimulating Ca2+-calmodulin and other molecular cascades. Human mutations (orange text) in molecules involved in calcium cycling cause cardiac remodeling and heart failure. NCX, sodium/calcium exchanger.
Calcium enters the myocyte through voltage-gated L-type Ca2+ channels; this triggers release of calcium from the sarcoplasmic reticulum (SR) via the RyR2. Emerging data define FK506-binding protein (FKBP12.6; calstabin2) as a critical stabilizer of RyR2 function (59), preventing aberrant calcium release during the relaxation phase of the cardiac cycle (Figure 5). Stimuli that phosphorylate RyR2 (such as exercise) by protein kinase A (PKA) dissociate calstabin2 from the receptor, thereby increasing calcium release and enhancing contractility. At low concentrations of intracellular calcium, troponin I and actin interactions block actomyosin ATPase activity; increasing levels foster calcium binding to troponin C, which releases troponin I inhibition and stimulates contraction. Cardiac relaxation occurs when calcium dissociates from troponin C, and intracellular concentrations decline as calcium reuptake into the SR occurs through the cardiac sarcoplasmic reticulum Ca2+-ATPase pump (SERCA2a). Calcium reuptake into SR is regulated by phospholamban (PLN), an inhibitor of SERCA2a activity that when phosphorylated dissociates from SERCA2a and accelerates ventricular relaxation.
RyR2 mutations.While some mutations in the RyR2 are reported to cause ARVD (47) (see discussion of desmosome mutations), defects in this calcium channel are more often associated with catecholaminergic polymorphic ventricular tachycardia (60, 61), a rare inherited arrhythmic disorder characterized by normal heart structure and sudden cardiac death during physical or emotional stress. Mutations in calsequestrin2, an SR calcium-binding protein that interacts with RyR2, also cause catecholaminergic polymorphic ventricular tachycardia (62, 63). Whether the effect of calsequestrin2 mutations directly or indirectly alters RyR2 function is unknown (Figure 5).
While RyR2 mutations affect residues in multiple functional domains of the calcium channel, those affecting residues involved in calstabin2-binding provide mechanistic insights into the substantial arrhythmias found in affected individuals. Mutations that impair calstabin2-binding may foster calcium leak from the SR and trigger depolarization. Diastolic calcium leak can also affect excitation-contraction coupling and impair systolic contractility.
Studies of mice deficient in FKBP12.6 (64) confirmed the relevance of SR calcium leak from RyR2 to clinically important arrhythmias. RyR2 channel activity in FKBP12.6-null mice was significantly increased compared with that of wild-type mice, consistent with a diastolic Ca2+ leak. Mutant myocytes demonstrated delayed after-depolarizations, and exercise-induced syncope, ventricular arrhythmias, and sudden death were observed in FKBP12.6-null mice.
Calcium dysregulation is also a component of hypertrophic remodeling that occurs in sarcomere gene mutations. Calcium cycling is abnormal early in the pathogenesis of murine HCM (65, 66): SR calcium stores are decreased and calcium-binding proteins and RyR2 levels are diminished. Whether calcium changes contribute to ventricular arrhythmias in mouse and human HCM remains an intriguing question.
Related mechanisms may contribute to ventricular dysfunction and arrhythmias in acquired forms of heart failure, in which chronic phosphorylation of RyR2 reduces calstabin2 levels in the channel macromolecular complex and increases calcium loss from SR stores. These data indicate the potential benefit of therapeutics that improve calstabin2-mediated stabilization of RyR2 (67, 68); such agents may both improve ventricular contractility and suppress arrhythmias in heart failure.
PLN mutations. Rare human PLN mutations cause familial DCM and heart failure (69, 70). The pathogenetic mechanism of one mutation (PLN Arg9Cys) was elucidated through biochemical studies, which indicated unusual PKA interactions that inhibited phosphorylation of mutant and wild-type PLN. The functional consequence of the mutation was predicted to be constitutive inhibition of SERCA2a, a result confirmed in transgenic mice expressing mutant, but not wild-type, PLN protein. In mutant transgenic mice, calcium transients were markedly prolonged, myocyte relaxation was delayed, and these abnormalities were unresponsive to β-adrenergic stimulation. Profound biventricular cardiac dilation and heart failure developed in mutant mice, providing clear evidence of the detrimental effects of protracted SERCA2a inhibition due to excess PLN activity.
The biophysical consequences accounting for DCM in humans who are homozygous for a PLN null mutation (Leu39stop; ref. 70) are less clear. PLN-deficient mice show increased calcium reuptake into the SR and enhanced basal contractility (71). Indeed, these effects on calcium cycling appear to account for the mechanism by which PLN ablation rescues DCM in MLP-null mice (72). However, normal responsiveness to β-adrenergic stimulation is blunted in PLN-deficient myocytes, and cells are less able to recover from acidosis that accompanies vigorous contraction or pathologic states, such as ischemia (73). The collective lesson from human PLN mutations appears to be that too little or too much PLN activity is bad for long-term heart function.
Acquired causes of heart failure are also characterized by a relative decrease in SERCA2a function due to excessive PLN inhibition. Downregulation of β-adrenergic responsiveness attenuates PLN phosphorylation, which compromises calcium reuptake and depletes SR calcium levels, which may impair contractile force and enhance arrhythmias. Heterozygote SERCA2 null mice are a good model of this phenotype and exhibit impaired restoration of SR calcium with deficits in systolic and diastolic function (74).
Cardiac ATP-sensitive potassium channel mutations. In response to stress such as hypoxia and ischemia, myocardial cells undergo considerable changes in metabolism and membrane excitability. Cardiac ATP-sensitive potassium channels (KATP channels) contain a potassium pore and an enzyme complex that participate in decoding metabolic signals to maximize cellular functions during stress adaptation (Figure 4) (75). KATP channels are multimeric proteins containing the inwardly rectifying potassium channel pore (Kir6.2) and the regulatory SUR2A subunit, an ATPase-harboring, ATP-binding cassette protein. Recently, human mutations in the regulatory SUR2A subunit (encoded by ABCC9) were identified as a cause of DCM and heart failure (76). These mutations reduced ATP hydrolytic activities, rendered the channels insensitive to ADP-induced conformations, and affected channel opening and closure. Since KATP-null mouse hearts have impaired response to stress and are susceptible to calcium overload (75), some of the pathophysiology of human KATP mutations (DCM and arrhythmias) may reflect calcium increases triggered by myocyte stress.
Transcriptional Regulators
Investigation of the molecular controls of cardiac gene transcription has led to the identification of many key molecules that orchestrate physiologic expression of proteins involved in force production and transmission, metabolism, and calcium cycling. Given that mutation in the structural proteins involved in these complex processes is sufficient to cause cardiac remodeling, it is surprising that defects in transcriptional regulation of these same proteins have not also been identified as primary causes of heart failure. Several possible explanations may account for this. Transcription factor gene mutations may be lethal or may at least substantially impair reproductive fitness so as to be rapidly lost. The consequences of transcription factor gene mutations may be so pleiotropic that these cause systemic rather than single-organ disease. Changes in protein function (produced by a structural protein mutation) may be more potent for remodeling than changes in levels of structural protein (produced by transcription factor mutation). While many other explanations may be relevant, the few human defects discovered in transcriptional regulators that cause heart failure provide an important opportunity to understand molecular mechanisms for heart failure.
Nkx2.5 mutations. The homeodomain-containing transcription factor Nkx2.5, a vertebrate homolog of the Drosophila homeobox gene tinman, is one of the earliest markers of mesoderm. When Nkx2.5 is deleted in the fly, cardiac development is lost (77). Targeted disruption of Nkx2.5 in mice (Nkx2.5–/–) causes embryonic lethality due to the arrested looping morphogenesis of the heart tube and growth retardation (78, 79). Multiple human dominant Nkx2.5 mutations have been identified as causing primarily structural malformations (atrial and ventricular septation defects) accompanied by atrioventricular conduction delay, although cardiac hypertrophic remodeling has also been observed (80). Although the mechanism for ventricular hypertrophy in humans with Nkx2.5 mutations is not fully understood, the pathology is unlike that found in HCM, which perhaps indicates that cardiac hypertrophy is a compensatory event. Several human Nkx2.5 mutations have been shown to abrogate DNA binding (81), which suggests that the level of functional transcription factor is the principle determinant of structural phenotypes. Heterozygous Nkx2.5+/– mice exhibit only congenital malformations with atrioventricular conduction delay (82, 83). Remarkably, however, transgenic mice expressing Nkx2.5 mutations develop profound cardiac conduction disease and heart failure (84) and exhibit increased sensitivity to doxorubicin-induced apoptosis (85), which suggests that this transcription factor plays an important role in postnatal heart function and stress response.
Insights into transcriptional regulation from mouse genetics. Dissection of the combinatorial mechanisms that activate or repress cardiac gene transcription has led to the identification of several key molecules that directly or indirectly lead to cardiac remodeling. While human mutations in these genes have not been identified, these molecules are excellent candidates for triggering cell responses to structural protein gene mutations.
Hypertrophic remodeling is associated with reexpression of cardiac fetal genes. Molecules that activate this program may also regulate genes that directly cause hypertrophy. Activation of calcineurin (Ca2+/calmodulin-dependent serine/threonine phosphatase) results in dephosphorylation and nuclear translocation of nuclear factor of activated T cells 3 (NFAT3), which, in association with the zinc finger transcription factor GATA4, induces cardiac fetal gene expression. Transgenic mice that express activated calcineurin or NFAT3 in the heart develop profound hypertrophy and progressive decompensation to heart failure (86), responses that were prevented by pharmacologic inhibition of calcineurin. Although these data implicated NFAT signaling in hypertrophic heart failure, pharmacologic inhibition of this pathway fails to prevent hypertrophy caused by sarcomere gene mutations in mice and even accelerates disease progression to heart failure (65). Mice lacking calsarcin-1, which is localized with calcineurin to the Z-disc, showed an increase in Z-disc width, marked activation of the fetal gene program, and exaggerated hypertrophy in response to calcineurin activation or mechanical stress, which suggests that calsarcin-1 plays a critical role in linking mechanical stretch sensor machinery to the calcineurin-dependent hypertrophic pathway (87).
Histone deacetylases (HDACs) are emerging as important regulators of cardiac gene transcription. Class II HDACs (4/5/7/9) bind to the cardiac gene transcription factor MEF2 and inhibit MEF2-target gene expression. Stress-responsive HDAC kinases continue to be identified but may include an important calcium-responsive cardiac protein, calmodulin kinase. Kinase-induced phosphorylation of class II HDACs causes nuclear exit, thereby releasing MEF2 for association with histone acetyltransferase proteins (p300/CBP) and activation of hypertrophic genes. Mice deficient in HDAC9 are sensitized to hypertrophic signals and exhibit stress-dependent cardiac hypertrophy. The discovery that HDAC kinase is stimulated by calcineurin (88) implicates crosstalk between these hypertrophic signaling pathways.
Recent attention has also been focused on Hop, an atypical homeodomain-only protein that lacks DNA-binding activity. Hop is expressed in the developing heart, downstream of Nkx2-5. While its functions are not fully elucidated, Hop can repress serum response factor–mediated (SRF-mediated) transcription. Mice with Hop gene ablation have complex phenotypes. Approximately half of Hop-null embryos succumb during mid-gestation with poorly developed myocardium; some have myocardial rupture and pericardial effusion. Other Hop-null embryos survive to adulthood with apparently normal heart structure and function. Cardiac transgenic overexpression of epitope-tagged Hop causes hypertrophy, possibly by recruitment of class I HDACs that may inhibit anti-hypertrophic gene expression (89–92).
PPARα plays important roles in transcriptional control of metabolic genes, particularly those involved in cardiac fatty acid uptake and oxidation. Mice with cardiac-restricted overexpression of PPARα replicate the phenotype of diabetic cardiomyopathy: hypertrophy, fetal gene activation, and systolic ventricular dysfunction (93). Heterozygous PPARγ-deficient mice, when subjected to pressure overload, developed greater hypertrophic remodeling than wild-type controls, implicating the PPARγ-pathway as a protective mechanism for hypertrophy and heart failure (94).
Retinoid X receptor α (RXRα) is a retinoid-dependent transcriptional regulator that binds DNA as an RXR/retinoic acid receptor (RXR/RAR) heterodimer. RXRα-null mice die during embryogenesis with hypoplasia of the ventricular myocardium. In contrast, overexpression of RXRα in the heart does not rescue myocardial hypoplasia but causes DCM (95).
Integrating Functional and Molecular Signals
Study of human gene mutations that cause HCM and DCM provides information about functional triggers of cardiac remodeling. In parallel with evolving information about molecular-signaling cascades that influence cardiac gene expression, there is considerable opportunity to define precise pathways that cause the heart to fail. To understand the integration of functional triggers with molecular responses, a comprehensive data set of the transcriptional and proteomic profiles associated with precise gene mutations is needed. Despite the plethora of information associated with such studies, bioinformatic assembly of data and deduction of pathways should be feasible and productive for defining shared or distinct responses to signals that cause cardiac remodeling and heart failure. Accrual of this data set in humans is a desirable goal, although confounding clinical variables and tissue acquisition pose considerable difficulties that can be more readily addressed by study of animal models with heart disease. With more knowledge about the pathways involved in HCM and DCM, strategies may emerge to attenuate hypertrophy, reduce myocyte death, and diminish myocardial fibrosis, processes that ultimately cause the heart to fail.
CardioGenomics. Genomics of Cardiovascular Development, Adaptation, and Remodeling.
Morita, H, et al. Molecular epidemiology of hypertrophic cardiomyopathy. Cold Spring Harb. Symp. Quant. Biol. 2002. 67:383-388.
Richard, P, et al. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation. 2003. 107:2227-2232
Palmer, BM, et al. Effect of cardiac myosin binding protein-C on mechanoenergetics in mouse myocardium. Circ. Res. 2004. 94:1615-1622.
Harris, SP, et al. Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice. Circ. Res. 2002. 90:594-601.
Kamisago, M, et al. Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N. Engl. J. Med. 2000. 343:1688-1696.
Itoh-Satoh, M, et al. Titin mutations as the molecular basis for dilated cardiomyopathy. Biochem. Biophys. Res. Commun. 2002. 291:385-393.
Gerull, B, et al. Mutations in the desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy. Nat. Genet. 2004. 36:1162-1164.
Tiso, N, et al. Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2). Hum. Mol. Genet. 2001. 10:189-194.
Anan, R, et al. Cardiac involvement in mitochondrial diseases. A study on 17 patients with documented mitochondrial DNA defects. Circulation. 1995. 91:955-961.
Cardiovascular Autonomic Dysfunction and Predicting Outcomes in Diabetes
Autonomic Dysfunction and Risk of a CV Event In patients with CAD and type 2 diabetes, autonomic dysfunction is common, but its prognostic value is unknown.
A.
data a substudy of patients enrolled in the ARTEMIS trial
,530 patients with CAD and diabetes matched with 530 patients with CAD without diabetes. The patients had a mean age of 67, and 69% were males
patients performed a test on an exercise bicycle, which allowed the researchers to determine their heart-rate recovery, defined as the drop in heart rate from the rate at maximal exercise to the rate one minute after stopping the exercise In univariate analysis, among patients with CAD and type 2 diabetes, those who had a blunted heart-rate recovery after exercise–defined as a drop in heart rate of less than 21 beats per minute–had a 1.69-fold greater risk having a cardiovascular event than their peers. Similarly, those with blunted heart-rate turbulence (<3.4 ms/R-R interval) had a 2.08-fold increased risk of an event, and those with low heart-rate variability (<110 ms) had a 1.96-fold greater risk of having a cardiovascular event. After multivariate analysis, C-reactive protein (CRP), but none of the three measures of autonomic function, still predicted an increased risk of having a cardiovascular event during this short follow-up.
During a two-year follow-up, 127 patients (13%) reached the composite end point of a cardiovascular event, which included
cardiovascular death (2%),
acute coronary event (8%),
stroke (3%), or
hospitalization for heart failure (2%).
B. Autonomic Dysfunction and Risk of Severe Hypoglycemia
Dr Seung-Hyun Ko (Catholic University of Korea, Gyeonggi-do, South Korea
data 894 consecutive patients with type 2 diabetes, aged 25 to 75
heart-rate variability measured at three times: during a Valsalva maneuver, deep breathing, and going from lying down to standing. During close to 10 years of follow-up, 77 episodes of severe hypoglycemia occurred among 62 patients (9.9%). About 16% of patients were diagnosed with early autonomic dysfunction and another 15% were diagnosed with definite autonomic dysfunction. Patients with type 2 diabetes and definite autonomic dysfunction were more than twice as likely to have an episode of severe hypoglycemia as those with normal autonomic function (HR 2.43).
patient education concerning hypoglycemia is essential for patients with definite [cardiovascular autonomic neuropathy] to prevent [severe hypoglycemia] and related mortality
Current Risk-Stratification Role for Heart-Rate Turbulence Monitoring Defined
Measurement of heart-rate turbulence (HRT), an ECG phenomenon that reflects hemodynamic responses to premature ventricular contractions (PVCs), can risk-stratify patients in the post-MI setting and may be similarly useful in heart failure or other heart disease, according to a state-of-the-art review in the October 21, 2008 issue of the Journal of the American College of Cardiology [1]. “Several large-scale retrospective and prospective studies have established beyond any doubt that HRT is one of the strongest independent risk predictors after MI. It thus appears that the stage has now been reached when HRT might be used in large prospective intervention studies,” according to the authors, led by Dr Axel Bauer (Deutsches Herzzentrum, Munich, Germany). The group had been asked to write the review by the International Society for Holter and Noninvasive Electrophysiology (ISHNE), it states. HRT, first published as a potential CV risk stratifier in 1999 [2], and other measures of autonomic function aren’t as well established or even studied as much as some other prognostic markers based on electrocardiography, such as T-wave alternans. As the authors note, it’s usually measured from an average of multiple PVCs on 24-hour Holter monitoring.
The strongest support for the parameter’s risk-stratification role comes from “six large-scale studies and from two prospective studies, both of which have been specifically designed to validate the prognostic value of HRT in post-MI patients receiving state-of-the-art treatment,” the report states.
Other evidence suggests a role for HRT evaluation after PCI to assess the strength of perfusion from the treated coronary artery. “Persistent impairment of HRT after PCI in patients with incomplete reperfusion implies prolonged baroreflex impairment and is consistent with poor prognosis,” write Bauer et al. “Thus, early assessment of HRT may be detecting pathological loss of reflex autonomic response due to incomplete reperfusion or severe microvascular dysfunction after PCI. In heart failure, according to the authors, patients “are known to have significantly impaired baroreflex sensitivity as well as reduced heart-rate variability. . . . This may suggest the possibility of guiding pharmacological therapy [according to HRT responses] in heart-failure patients.” They also note that the prognostic power of HRT in heart failure appears limited to patients with ischemic cardiomyopathy.
Bauer A, Malik M, Schmidt G, et al. Heart rate turbulence: Standards of measurement, physiological interpretation, and clinical use. International Society for Holter and Noninvasive Electrophysiology consensus. J Am Coll Cardiol 2008; 52:1353–1365. http://dx.doi.org/10.1016/j.jacc.2008.07.041
Schmidt G, Malik M, Barthel P, et al. Heart-rate turbulence after ventricular premature beats as a predictor of mortality after acute myocardial infarction. Lancet 1999; 353:1390–1396. Abstract http://www.medscape.com/viewarticle/582091
Integration of expression data in genome-scale metabolic network reconstructions Anna S. Blazier and Jason A. Papin*
Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA
Front. Physiol., 06 August 2012 | http://dx.doi.org/10.3389/fphys.2012.00299
http://
The other side of cardiac Ca2+ signaling: transcriptional control Alejandro Domínguez-Rodríguez1, Gema Ruiz-Hurtado2, Jean-Pierre Benitah1 and Ana M. Gómez1*
Ca2+ is probably the most versatile signal transduction element used by all cell types. In the heart, it is essential to activate cellular contraction in each heartbeat. Nevertheless Ca2+ is not only a key element in excitation-contraction coupling (EC coupling), but it is also
a pivotal second messenger in cardiac signal transduction, being able to control processes such as
excitability, metabolism, and transcriptional regulation.
Regarding the latter, Ca2+ activates Ca2+-dependent transcription factors by a process called excitation-transcription coupling (ET coupling). ET coupling is an integrated process by which
the common signaling pathways that regulate EC coupling
activate transcription factors.
In studies on the development of cardiac hypertrophy, two Ca2+-dependent enzymes are key actors:
both of which are activated by the complex Ca2+/Calmodulin.
The question now is how ET coupling occurs in cardiomyocytes, where intracellular Ca2+ is continuously oscillating. We draw attention to location of Ca2+ signaling:
intranuclear ([Ca2+]n) or cytoplasmic ([Ca2+]c), and
the specific ionic channels involved in the activation of cardiac ET coupling.
We highlight the role of the 1,4,5 inositol triphosphate receptors (IP3Rs) in the elevation of [Ca2+]n levels, which are important to
locally activate CaMKII, and
the role of transient receptor potential channels canonical (TRPCs) in [Ca2+]c,
needed to activatecalcineurin (Cn).
Keywords: heart, calcium, excitation-transcription coupling, TRPC, nuclear calcium
Citation: Domínguez-Rodríguez A, Ruiz-Hurtado G, Benitah J-P and Gómez AM (2012) The other side of cardiac Ca2+ signaling: transcriptional control.
Front. Physio. 3:452. http://dx.doi.org/10.3389/fphys.2012.00452 Published online: 28 November 2012.
Edited by:Eric A. Sobie, Mount Sinai School of Medicine, USA; Reviewed by: Jeffrey Varner, Cornell University, USA; Ravi Radhakrishnan, University of Pennsylvania, USA
Integration of expression data in genome-scale metabolic network reconstructions Anna S. Blazier and Jason A. Papin*
Front. Physiol., 06 August 2012 | doi: 10.3389/fphys.2012.00299
With the advent of high-throughput technologies, the field of systems biology has amassed an abundance of “omics” data,
quantifying thousands of cellular components across a variety of scales,
ranging from mRNA transcript levels to metabolite quantities.
Methods are needed to not only
integrate this omics data but to also
use this data to heighten the predictive capabilities of computational models.
Several recent studies have successfully demonstrated how flux balance analysis (FBA), a constraint-based modeling approach, can be used
to integrate transcriptomic data into genome-scale metabolic network reconstructions
to generate predictive computational models.
We summarize such FBA-based methods for integrating expression data into genome-scale metabolic network reconstructions, highlighting their advantages as well as their limitations.
Introduction
Genomics provides data on a cell’s DNA sequence,
transcriptomics on the mRNA expression of cells,
proteomics on a cell’s protein composition, and
metabolomics on a cell’s metabolite abundance.
Computational methods are needed to reduce this dimensionality across the wide spectrum of omics data to improve understanding of the underlying biological processes (Cakir et al., 2006; Pfau et al., 2011).
Metabolic network reconstructions are an advantageous platform for the integration of omics data (Palsson, 2002). Assembled in part from
annotated genomes as well as
biochemical, genetic, and cell phenotype data,
a metabolic network reconstruction is a manually-curated, computational framework that
enables the description of gene-protein-reaction relationships (Chavali et al., 2012).
After applying constraints, the solution space of possible phenotypes narrows, allowing for more accurate characterization of the reconstructed metabolic network,
Omics data can be used to further constrain the possible solution space and
Given the wealth of transcriptomic data, efforts to integrate mRNA expression data with metabolic network reconstructions, have, in particular, made significant progress when using FBA as an analytical platform (Covert and Palsson, 2002; Akesson et al., 2004; Covert et al., 2004). However, despite this abundance of data, the integration of expression data faces unique challenges such as
experimental and inherent biological noise,
variation among experimental platforms,
detection bias, and the
unclear relationship between gene expression and reaction flux
The past few years have witnessed several advances in the integration of transcriptomic data with genome-scale metabolic network reconstructions. Specifically, numerous FBA-driven algorithms have been introduced that use experimentally derived mRNA transcript levels to modify the network’s reactions either by
inactivating them entirely or
by constraining their activity levels.
Such algorithms have demonstrated their applicability by, for example,
classifying tissue-specific metabolic activity in the human network and
by identifying novel drug targets in Mycobacterium tuberculosis
We summarize various FBA-driven methods for integrating expression data into genome-scale metabolic network reconstructions.
We survey the limitations of these algorithms as well as look to the future of
multi-omics data integration using genome-scale metabolic network reconstructions as the scaffold.
Flux balance analysis
FBA is a constraint-based modeling approach that characterizes and predicts aspects of an organism’s metabolism (Gianchandani et al., 2009) To use FBA, the user supplies a metabolic network reconstruction in the form of a stoichiometric matrix, S, where
the rows in S correspond to the metabolites of the reconstruction and
the columns in S represent reactions in the reconstruction.
a stoichiometric coefficient sij conveys the molecularity of a certain metabolite in a particular reaction, with
sij ≥ 1 indicating that the metabolite is a product of the reaction,
sij ≤ −1 a reactant, and
sij = 0 signifies that the metabolite is not involved.
A system of linear equations is established by multiplying the S matrix by a column vector, v, which contains the unknown fluxes through each of the reactions of the S matrix. Under the assumption that the system operates at steady-state, that is to say there is no net production or consumption of mass within the system, the product of this matrix multiplication must equal zero, S · v = 0 (Gianchandani et al., 2009). Because the resulting system is underdetermined (i.e., too few equations, too many unknowns), linear programming (LP) is used to optimize for a particular flux,Z, the objective function, subject to underlying constraints. The objective function typically takes on the form of: Z = c ⋅ v
where c is a row vector of weights for each of the fluxes in column vector v, indicating how much each reaction in v contributes to the objective function,Z (Lee et al., 2006; Orth et al., 2010). Examples of objective functions include maximizing biomass, ATP production, and the production of a metabolite of interest (Lewis et al., 2012).
(1)
subject to
S ⋅ v = 0
(2)
lb ≤ v ≤ ub
(3)
(1) outlines the objective function to be optimized,
(2) the steady state assumption, and
(3) describes the upper and lower bounds, ub and lb, of each of the fluxes in v according to such constraints as
Through this application of constraints, the solution space of physiologically feasible flux distributions for v shrinks. Thus, the task of FBA is to find a solution to v that lies within the bounded solution space and that optimizes the objective function at the same time.
Several recently developed algorithms have demonstrated how expression data can be incorporated into FBA models to further constrain the flux distribution solution space in genome-scale metabolic network reconstructions . Summary of the algorithms for the integration of expression data. Table 1 image URL http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3429070/table/T1/?report=thumb
List of Methods:
GIMME guarantees to both produce a functioning metabolic model based on gene expression levels and quantify the agreement between the model and the data is called the Gene Inactivity Moderated by Metabolism and Expression (GIMME) algorithm (Becker and Palsson, 2008).
iMAT Similar to GIMME, the Integrative Metabolic Analysis Tool (iMAT) results in a functioning model in which the fluxes of reactions correlated with high mRNA levels are maximized and the fluxes of reactions associated with low mRNA levels are minimized (Shlomi et al., 2008; Zur et al., 2010). A key difference is that iMAT does not require a priori knowledge of a defined metabolic functionality. Briefly, this method establishes a tri-valued gene-to-reaction mapping for each reaction in the model according to the level of gene expression in the data. iMAT requires that reactions catalyzed by the products of highly expressed genes are able to carry a minimum flux. By removing this need for user-specified objective functions, iMAT bypasses assumptions about metabolic functionalities of a particular network, which proves advantageous for models where there is no clear objective function, as in models of mammalian cells.
MADE While both GIMME and iMAT rely on user-specified threshold values to determine which reactions are highly expressed and which reactions are lowly expressed, Metabolic Adjustment by Differential Expression (MADE) uses statistically significant changes in gene expression measurements to determine sequences of highly and lowly expressed reactions (Jensen and Papin, 2011). The lack of correlation between mRNA levels and protein levels makes it difficult to accurately determine when genes are “turned on,” and when they are “turned off.” Therefore, in eliminating this need for thresholding, MADE removes significant user-bias from the system.
E-Flux Whereas GIMME, iMAT, and MADE incorporate gene expression data into their models by reducing gene expression levels to binary states, the method E-Flux attempts to more directly incorporate gene expression data into FBA optimization problems by constraining the maximum possible flux through the reactions (Colijn et al., 2009). Rather than setting the upper bounds of a reaction to some large constant or 0, mirroring the implementation of binary-based algorithms, E-Flux constrains the upper bound of a reaction according to its respective gene expression level relative to a particular threshold. In cases where the gene expression data is below a certain threshold, tight constraints are placed on the flux through the corresponding reactions in the reconstruction; conversely, in cases where the gene expression is above a certain threshold, loose constraints are placed on the flux through the corresponding reactions.
PROM In contrast to the other methods discussed, which focused solely on integrating gene expression data into genome-scale metabolic network reconstructions, Probabilistic Regulation of Metabolism (PROM) aims to fuse together metabolic networks and transcription regulatory networks with expression data (Chandrasekaran and Price, 2010). To run PROM, the user supplies a genome-scale metabolic network reconstruction, a regulatory network structure describing transcription factors and their targets, and a range of expression data from various environmental and genetic perturbations. Given this expression data, PROM binarizes the genes with respect to a user-supplied threshold to evaluate the likelihood of the expression of a target gene given the expression of that gene’s transcription factor.
Challenges facing the integration of expression data
Each of the methods discussed hinges on the assumption that mRNA transcript levels are a strong indicator for the level of protein activity. For instance, GIMME and iMAT assume that mRNA levels below a certain threshold suggest that the corresponding reactions are inactive. MADE follows a similar logic, turning reactions on and off depending on the changes in mRNA transcript levels. E-Flux and PROM assume that transcript levels indicate the degree to which reactions are active, evident in the constraining of the upper bounds in the FBA optimization problems associated with these methods.
Rather than requiring that the reconstruction mirror the expression data exactly, the methods allow for deviations in the FBA flux solution space in order to generate a functioning model that adheres to the specified constraints. In the case of GIMME, highly expressed reactions are prioritized relative to lowly expressed reactions; however, in the event that an optimal, functioning solution cannot be found, the assumption can be violated and lowly expressed reactions can be added back into the reconstruction. Thus, this assumption that mRNA transcript levels correlate to protein levels serves as a cue rather than a mandate.
Conclusion
The above methods have been used to not only integrate expression data from a variety of sources but to also make progress toward overcoming key challenges in the field of systems biology. For instance, iMAT, highlighting its applicability in multi-cellular organisms, was used to curate the human metabolic network reconstruction and predict tissue-specific gene activity levels in ten human tissues (Duarte et al., 2007; Shlomi et al., 2008). Additionally, both E-Flux and PROM have been used to discover novel drug targets in Mycobacterium tuberculosis (Colijn et al., 2009; Chandrasekaran and Price, 2010).
Given the recent success with using genome-scale metabolic network reconstructions as a platform for integrating expression data, efforts should focus on multi-omics data integration. A handful of methods have already been introduced that integrate two or more types of omics data into genome-scale metabolic network reconstructions. For example, despite the current dearth of quantitative metabolomics data, a method has been developed that demonstrates how semi-quantitative metabolomics data can be used with transcriptomic data to curate genome-scale metabolic network reconstructions and identify key reactions involved in the production of certain metabolites (Cakir et al., 2006). Another algorithm, called Integrative Omics-Metabolic Analysis (IOMA), integrates metabolomics data and proteomics data into a genome-scale metabolic network reconstruction by evaluating kinetic rate equations subject to quantitative omics measurements (Yizhak et al., 2010). Furthermore, Mass Action Stoichiometric Simulation (MASS) uses metabolomic, fluxomic, and proteomic data to transform a static stoichiometric reconstruction of an organism into a large-scale dynamic network model (Jamshidi and Palsson, 2010). And finally, building off of iMAT, the Model-Building Algorithm (MBA) utilizes literature-based knowledge, transcriptomic, proteomic, metabolomic, and phenotypic data to curate the human metabolic network reconstruction to derive a more complete picture of tissue-specific metabolism (Jerby et al., 2010). Such algorithms show promise in their ability to easily integrate high-throughput data into genome-scale metabolic network reconstructions to generate phenotypically accurate and predictive computational models.
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