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:
- Ang II infusion increased the susceptibility of mice to AF induction by rapid right atrial pacing and established a framework for us to test the hypothesized role of ox-CaMKII in promoting AF. ox-CaMKII is critical for AF.
- Estalished a critical role of ox-CaMKII in promoting AF
- Ang II induced increases in ROS production seen in WT atria were absent in atria from MsrA TG mice suggesting that MsrA sensitive targets represent an important component of Ang II mediated atrial oxidation.
- The protection from AF in MsrA TG mice appeared to be independent of pressor effects that are critical for the proarrhythmic actions.
- These findings suggest that NADPH oxidase dependent ROS and elevated ox-CaMKII drive Ang II -pacing-induced AF and that
- targeted antioxidant therapy, by MsrA over-expression, can reduce or prevent AF in Ang -II-infused mice.
- Atrial myocytes from Ang II treated WT mice showed a significant (p<0.05) increase in spontaneous Ca2+ sparks compared to atrial myocytes from saline treated control mice
- In contrast to findings in WT mice, the atrial myocytes isolated from Ang II treated MM-VV mice did not show an increase in Ca2+ sparks compared to saline treated MM-VV mice
- These data to suggest that in ox–the proarrhythmic effects of Ang I I infusion depend upon an increaseCaMKII, sarcoplasmic reticulum Ca2+ leak and DADs.
- Enhanced CaMKII-mediated phosphorylation of serine 2814 on RyR2 is associated with an increased susceptibility to acquired arrhythmias, including AF
- Proarrhythmic actions of ox-CaMKII require access to RyR2 serine 2814.
- Mutant S2814A knock-in mice (lacking serine 2814) were highly resistant to Ang II mediated AF
- AC3-I mice with transgenic myocardial expression of a CaMKII inhibitory peptide were also resistant to the proarrhythmic effects of Ang II infusion on pacing-induced AF
- S2814A, AC3-I and WT mice, all developed similar BP increases and cardiac hypertrophy in response to Ang II, indicating that these mice were not resistant to the hemodynamic effects of Ang II, but were nevertheless protected from AF.
- selectively targeted antioxidant therapies could be effective in preventing or reducing AF
- half of patients enrolled in the Mode Selection Trial (MOST) with sinus node dysfunction had a history of AF
- Ang II and diabetes-induced CaMKII oxidation caused sinus node dysfunction by increased pacemaker cell death and fibrosis
- ox-CaMKII increases susceptibility for AF via increased diastolic sarcoplasmic reticulum Ca2+ release
- clinical association between sinus node dysfunction and AF might have a mechanistic basis because sinus node dysfunction and AF are downstream consequences of elevated ox-CaMKII.
We refer to the following related articles published in pharmaceutical Intelligence:
Contributions to cardiomyocyte interactions and signaling
Author and Curator: Larry H Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN
http://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
http://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
http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/
Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/
Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/08/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
http://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
http://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
http://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
http://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
http://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
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
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).
Mouse Models and Experimental Methods
Statistics
Results
Oxidized CaMKII is increased in AF
Ang II treatment enhances AF susceptibility
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
- 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),
- 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
- 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
- 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).
- 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
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.
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.
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
http://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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