Feeds:
Posts
Comments

Posts Tagged ‘CaMKII’


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
fibrillation. Heart Rhythm. 2009;6:935-940.
5. Neuman RB, Bloom HL, Shukrullah I, Darrow LA, Kleinbaum D, Jones DP, Dudley SC. Oxidative stress markers are associated with persistent atrial fibrillation. Clin Chem.
2007;53:1652-1657.
 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).

 

CIRCULATIONAHA_oxCaMKII_AF_AR_Image_520 CIRCULATIONAHA_oxCaMKII_AF_AR_Image_523
CIRCULATIONAHA_oxCaMKII_AF_AR_Image_539    CIRCULATIONAHA_oxCaMKII_AF_AR_Image_538
CIRCULATIONAHA_oxCaMKII_AF_AR_Image_535                  CIRCULATIONAHA_oxCaMKII_AF_AR_Image_534
CIRCULATIONAHA_oxCaMKII_AF_AR_Image_522       CIRCULATIONAHA_oxCaMKII_AF_AR_Image_496

Read Full Post »