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Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Writer and Curator: Larry H Bernstein, MD, FCAP
and
Curator and Content Editor: Aviva Lev-Ari, PhD, RN

This article is Part V in a series of TWELVE articles, listed at the end of this article,  on the

  1. cytoskeleton,
  2. calcium calmodulin kinase signaling,
  3. muscle and nerve transduction, and
  4. calcium,
  5. Na+-K+-ATPase,
  6. 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
  • phosphorylation is 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 cascades have 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+ concentration can be controlled by a buffered solution.

  • Indirect readout of neurotransmitter release using a postsynaptic cell is replaced by
  • direct readout of [3H]NE released 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),

or

  • calmodulin binding inhibitory peptides (22) inhibited Ca2+-activated exocytosis.

However, in other studies, calmodulin-binding peptides and an anti-calmodulin antibody led to the conclusion that

  • calmodulin is only involved in endocytosis,
  • not exocytosis (23).

More recently, it was reported that

  1. Ca2+/ calmodulin signals the completion of docking and
  2. 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

  1. SNAP-25 (37),
  2. synaptotagmin (28),
  3. CAPS (38), and
  4. 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 PKC as 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.
  1. 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.
  2. 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 
  1. does not allow Ca2+-dependent priming events to be assayed, as EGTA is present in the priming reaction.
  2. a different approach revealed the existence of both high and low Ca2+-dependent processes (Fig. 2).
  3. this analysis indicated that late triggering events require high [Ca2+], whereas
  4. 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.
Fig 2A. measurements of range of [Ca2+]total - average [Ca2+]free values._page_004
Fig. 2B. measurements of range of [Ca2+] total - average [Ca2+]free values_edited-1
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+].

FIG. 5 calmodulin action_page_005

FIG. 8. PKC and calmodulin stimulate... the late triggering reaction_page_006
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.
  1. In the case of constitutive fusion, calmodulin may be the predominant Ca2+ sensor.
  2. In the case of slow, non-local exocytosis of large dense core granules, an additional requirement for
  3. 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
    1. hexokinase
    2. MgCl2,
    3. 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 of protein extracts or cracked cells.
Fig. 2B. measurements of range of [Ca2+] total - average [Ca2+]free values_edited-1
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
  1. incubated at 37 °C ,
  2. mixed with fura-2 pentapotassium salt
    (100 mM; Molecular Probes, Eugene, OR), and
  3. 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

  1. filtered through cheesecloth and
  2. loaded overnight onto a column packed with DEAE-Sepharose
    CL-6B beads (Amersham Pharmacia Biotech).

The column was then

  1. washed with
    (20 mM Hepes, pH 7.5, 0.25 mM sucrose, 2 mM EGTA, 1 mM dithiothreitol) and 
  2. 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
  3. dialyzed overnight into PGB, and
  4. 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

  1. collected,
  2. concentrated to about 2 ml using a Centriprep-10 (Amicon), and
  3. 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).

FIG. 3. Gel filtration chromatography reveals two stimulatory_page_004

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.

FIG. 6. Purification of the high molecular weight active factor_page_001

The two polypeptide sequences obtained from the upper band were:

  1. LLNQEEGEYYNVPIXEGD
  2. IRSTLNPRWDESFT.

The only bovine protein that contains both polypeptides is PKCa.
The four polypeptide sequences obtained from the lower band were:

  1. YELTGKFERLIVGLMRPPAY,
  2. LIEILASRTNEQIHQLVAA,
  3. MLVVLLQGTREEDDVVSEDL, and
  4. 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 run was 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). 

Fig. 1 EGTA extract can support NE release_page_003_edited-2

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 EGTA extract can support NE release_page_003_edited-3
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
  1. an early role for the protein, and
  2. 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 enriched in factors that
  • act during triggering stage of NE releaseas
  • 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). 

Fig 2A. measurements of range of [Ca2+]total - average [Ca2+]free values._page_004

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

  1. part of the activity in the membrane EGTA extract binds to and
  2. can be efficiently eluted from an anion exchanger and hydroxyapatite resin,
  3. 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_page_004

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 weight active factor is calmodulin_page_004

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).

FIG. 5A calmodulin action_page 5

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),

FIG. 5B calmodulin action_page_5

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+ requirement for
  • calmodulin-stimulated secretion that we observed.

FIG. 5 calmodulin action_page_005

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 

  •  constitutes about 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_page_006

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

  1. a Ca2+-dependent Na1 current
     (calmodulin N-terminal lobe mutants: E54K, G40E/D50N, and
     V35I/D50N) or
  2. 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

  • defining calmodulin’s binding specificity (46, 47).

Our finding that these mutant calmodulins can stimulate exocytosis suggests that

  • calmodulin-binding domains similar to those of Paramecium Ca2+/calmodulin-dependent
    ion channels, myosin light chain kinase, CaMKII, and CaMKIV,
  • are unlikely to mediate release utilizing the conserved SNARE fusion machinery, as they
  • could be completely abolished by addition of exogenous syntaxin H3 domains (data not shown).
  • the same molecular pathway was not activated, since their effects were additive (data not shown).

 

Acknowledgments
We thank Diana Bautista and Dr. Richard S.Lewis for generous help
with [Ca21]free determination; Dr. Ching Kung for providing the Paramecium calmodulin
mutants, and Dr. Anthony R. Means for providing the chicken calmodulin mutants. We also
thank Dr. Jesse C. Hay for the initial setup of the cracked cell assay, and Dr. Suzie J.
Scales for helpful comments on the manuscript.

REFERENCES

1. Calakos, N., and Scheller, R. H. (1996) Physiol. Rev. 76, 1–29
2. Su¨ dhof, T. C. (1995) Nature 375, 645–653
3. Zucker, R. S. (1996) Neuron 17, 1049–1055
4. Hanson, P. I., Heuser, J. E., and Jahn, R. (1997) Curr. Opin. Neurobiol. 7, 310–315
5. Chen, Y. A., Scales, S. J., Patel, S. M., Doung, Y.-C., and Scheller, R. H. (1999) Cell 97, 165–174
6. Neher, E., and Zucker, R. S. (1993) Neuron 10, 21–30
7. Kamiya, H., and Zucker, R. S. (1994) Nature 371, 603–606
SOURCE

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.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/2013/advanced-medicineprize2013.pdf

Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with
cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-
how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

2. Perspectives on Nitric Oxide in Disease Mechanisms

available on Kindle Store @ Amazon.com

http://www.amazon.com/dp/B00DINFFYC

https://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/
perspectives-on-nitric-oxide-in-disease-mechanisms-v2/

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

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/18/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

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-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/

5.            Seminal Curations by Dr. Aviva Lev-Ari on Genetics and Genomics of Cardiovascular Diseases with a focus on Conduction and Cardiac Contractility

Aviva Lev-Ari, PhD, RN

Aviva Lev-Ari, PhD, RN

Aviva Lev-Ari, PhD, RN and Larry H. Bernstein, MD, FCAP

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

https://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

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:

ION CHANNEL and Cardiovascular Diseases

https://pharmaceuticalintelligence.com/?s=Ion+Channel

Calcium Role in Cardiovascular Diseases

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP
https://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
https://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
https://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
https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-
involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-
post-ischemic-arrhythmia-similarities-and-differen/

Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
and
Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

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
Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-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/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure –
Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-
and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

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
https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-
muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction
(Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-
and-calcium-release-related-contractile-dysfunction-ryanopathy/

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of
vesicles with cell membranes during Neurotransmission
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-
regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Part XI: Sensors and Signaling in Oxidative Stress
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

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

https://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

 

Mitochondria and its Role in Cardiovascular Diseases

Mitochondria and Oxidative Stress Role in Cardiovascular Diseases Reversal of Cardiac Mitochondrial Dysfunction
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

Calcium Signaling, Cardiac Mitochondria and Metabolic Syndrome
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/11/09/calcium-signaling-cardiac-mitochondria-and-metabolic-syndrome/

Mitochondrial Dysfunction and Cardiac Disorders
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

Mitochondrial Metabolism and Cardiac Function
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing
Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix Identified
Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/02/03/mit-scientists-on-proteomics-all-the-proteins-
in-the-mitochondrial-matrix-identified/

Mitochondrial Dynamics and Cardiovascular Diseases
Ritu Saxena, Ph.D.
https://pharmaceuticalintelligence.com/2012/11/14/mitochondrial-dynamics-and-cardiovascular-diseases/

Mitochondrial Damage and Repair under Oxidative Stress
Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Nitric Oxide has a Ubiquitous Role in the Regulation of Glycolysis -with a Concomitant Influence on Mitochondrial Function
Larry H. Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-
glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Mitochondrial Mechanisms of Disease in Diabetes Mellitus
Aviva Lev-Ari, PhD, RN
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Mitochondria Dysfunction and Cardiovascular Disease – Mitochondria: More than just the “Powerhouse of the Cell”
Ritu Saxena, PhD
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Read Full Post »


Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation

Author: Larry H. Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

 

Introduction

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:

  1. 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.
  2. Estalished a critical role of ox-CaMKII in promoting AF
  3. 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.
  4. The protection from AF in MsrA TG mice appeared to be independent of pressor effects that are critical for the proarrhythmic actions.
  5. These findings suggest that NADPH oxidase dependent ROS and elevated ox-CaMKII drive Ang II  -pacing-induced AF and that
  6. targeted antioxidant therapy, by MsrA over-expression, can reduce or prevent AF in Ang -II-infused mice.  
  7. 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
  8. 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
  9. 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.
  10. Enhanced CaMKII-mediated phosphorylation of serine 2814 on RyR2 is associated with an increased susceptibility to acquired arrhythmias, including AF
  11. Proarrhythmic actions of ox-CaMKII require access to RyR2 serine 2814.
  12. Mutant S2814A knock-in mice (lacking serine 2814) were highly resistant to Ang II mediated AF
  13. 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
  14. 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.
  15. selectively targeted antioxidant therapies could be effective in preventing or reducing AF 
  16. half of patients enrolled in the Mode Selection Trial (MOST) with sinus node dysfunction had a history of AF
  17. Ang II and diabetes-induced CaMKII oxidation caused sinus node dysfunction by increased pacemaker cell death and fibrosis
  18.  ox-CaMKII increases susceptibility for AF via increased diastolic sarcoplasmic reticulum Ca2+ release
  19. 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:

Contributions to cardiomyocyte interactions and signaling
Author and Curator: Larry H Bernstein, MD, FCAP  and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/10/21/contributions-to-cardiomyocyte-interactions-and-signaling/

Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Editor: Justin Pearlman, MD, PhD, FACC, Author and Curator: Larry H Bernstein, MD, FCAP, and Article Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part I. Identification of Biomarkers that are Related to the Actin Cytoskeleton
Curator and Writer: Larry H Bernstein, MD, FCAP
https://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
https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

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
https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/

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
Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-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/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

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
https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

Oxidized CaMKII Triggers Atrial Fibrillation

Running title: Purohit et al.; oxCaMKII and AF

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
Circulation Sept 12, 2013;

http://circ.ahajournals.org/content/early/2013/09/12/CIRCULATIONAHA.113.003313
http://circ.ahajournals.org/content/suppl/2013/09/12/CIRCULATIONAHA.113.003313.DC1.html

http://dx.doi.org/10.1161/CIRCULATIONAHA.113.003313

Journal Subject Codes: Basic science research:[132] Arrhythmias – basic studies, Etiology:[5] Arrhythmias, clinical electrophysiology, drugs

 Abstract

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 of methionines (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 AF provides 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 dysfunction evoked 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.

Selected References

1. Benjamin EJ, Wolf PA, D’Agostino RB, Silbershatz H, Kannel WB, Levy D. Impact of atrial fibrillation on the risk of death: the Framingham Heart Study. Circulation. 1998;98:946-952.
2. Khatib R, Joseph P, Briel M, Yusuf S, Healey J. Blockade of the renin-angiotensinaldosterone system (RAAS) for primary prevention of non-valvular atrial fibrillation: A systematic review and meta analysis of randomized controlled trials. Int J Cardiol. 2013;165:17-24.

4. Shimano M, Shibata R, Inden Y, Yoshida N, Uchikawa T, Tsuji Y, Murohara T. Reactive oxidative metabolites are associated with atrial conduction disturbance in patients with atrial
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 6. Erickson JR, Joiner M-LA, Guan X, Kutschke W, Yang J, Oddis CV, Bartlett RK, Lowe JS, O’Donnell SE, Aykin-Burns N, Zimmerman MC, Zimmerman K, Ham A-JL, Weiss RM, Spitz DR, Shea MA, Colbran RJ, Mohler PJ, Anderson ME. A dynamic pathway for calciumin-dependent activation of CaMKII by methionine oxidation. Cell. 2008;133:462-474.

7. Swaminathan PD, Purohit A, Soni S, Voigt N, Singh MV, Glukhov AV, Gao Z, He BJ, Luczak ED, Joiner M-LA, Kutschke W, Yang J, Donahue JK, Weiss RM, Grumbach IM, Ogawa M, Chen P-S, Efimov I, Dobrev D, Mohler PJ, Hund TJ, Anderson ME. Oxidized CaMKII
causes cardiac sinus node dysfunction in mice. J Clin Invest. 2011;121:3277-3288.

8. Erickson JR, He BJ, Grumbach IM, Anderson ME. CaMKII in the cardiovascular system: sensing redox states. Physiol Rev. 2011;91:889-915.
9. Chelu MG, Sarma S, Sood S, Wang S, van Oort RJ, Skapura DG, Li N, Santonastasi M, Müller FU, Schmitz W, Schotten U, Anderson ME, Valderrábano M, Dobrev D, Wehrens XHT. Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice. J Clin Invest. 2009;119:1940-1951.
15. Moskovitz J, Bar-Noy S, Williams WM, Requena J, Berlett BS, Stadtman ER. Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc Natl Acad Sci USA. 2001;98:12920-12925.
16. He BJ, Joiner M-LA, Singh MV, Luczak ED, Swaminathan PD, Koval OM, Kutschke W, Allamargot C, Yang J, Guan X, Zimmerman K, Grumbach IM, Weiss RM, Spitz DR, Sigmund CD, Blankesteijn WM, Heymans S, Mohler PJ, Anderson ME. Oxidation of CaMKII determines the cardiotoxic effects of aldosterone. Nat Med. 2011;17:1610-1618.
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.

http://circ.ahajournals.org/content/early/2013/09/12/CIRCULATIONAHA.113.003313
http://circ.ahajournals.org/content/suppl/2013/09/12/CIRCULATIONAHA.113.003313.DC1.html

http://dx.doi.org/10.1161/CIRCULATIONAHA.113.003313

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

Author and Curator: Larry H Bernstein, MD, FCAP and  Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/10/21/contributions-to-cardiomyocyte-interactions-and-signaling/
This is a review of left ventricular cardiac hypertrophy and interaction with heparin-binding EGF,  based on work in the laboratory of Richard Lee, at Brigham and Women Hospital, Harvard Medical School, and MIT, titled…

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.

Xun Ai, JW Curran, TR Shannon, DM Bers and SM Pogwizd.   
Circ Res. 2005;97:1314-1322  http://dx.doi.org/10.1161/01.RES.0000194329.41863.89
http://circres.ahajournals.org/content/97/12/1314

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.)

Key Words: ryanodine receptor -CaMKII -phosphorylation -heart failure -arrhythmia

III.  The Fire From Within: The Biggest Ca2+ Channel Erupts and Dribbles  – Mark E. Anderson

Circ Res. 2005;97:1213-1215  http://dx.doi.org/10.1161/01.RES.0000196744.62327.36
http://circres.ahajournals.org/content/97/12/1213

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).

 

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