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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Heart Rhythm. Author manuscript; available in PMC Feb 1, 2012.
Published in final edited form as:
PMCID: PMC3020993
NIHMSID: NIHMS241597
CaMKII Regulation of the Cardiac Ryanodine Receptor and SR Calcium Release
Xander H.T. Wehrens
Address for correspondence: Xander H.T. Wehrens, MD, PhD, FHRS Dept. of Molecular Physiology and Biophysics Dept. of Medicine (in Cardiology) One Baylor Plaza, BCM 335 Houston, TX 77030 Tel.: 713-798-4261 ; wehrens/at/bcm.edu
Abstract
Spontaneous release of Ca2+ from the sarcoplasmic reticulum has emerged as a mechanism underlying triggered activity and cardiac arrhythmias. Recent studies suggest an important role for increased Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of ryanodine receptors (RyR2) in the induction of arrhythmias. This article briefly reviews the mechanisms underlying CaMKII regulation of RyR2 and discusses directions of current and future research.
Keywords: arrhythmias, calmodulin kinase II, heart failure, phosphorylation, ryanodine receptors
In recent years, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has emerged as an important regulator of excitation-contraction coupling, the cellular process by which an action potential triggers contraction of cardiac myocytes. It has become clear that CaMKII-mediated phosphorylation of Ca2+ cycling proteins plays an important role in intrinsic regulatory pathways in the heart, such as the force-frequency response1. Moreover, emerging data suggest that chronic activation of CaMKII in diseased hearts leads to abnormal intracellular Ca2+ handling, which could promote cardiac arrhythmias2, 3. This viewpoint article in the mini series on ‘CaMKII in the heart’ will focus on the effects of CaMKII on intracellular Ca2+ release channels known as ryanodine receptors type 2 (RyR2) in relationship to cardiac arrhythmia mechanisms.
Contraction of cardiac myocytes is preceded by the influx of Ca2+ into the cytosol via voltage-gated L-type Ca2+ channels, which triggers a larger release of Ca2+ from the sarcoplasmic reticulum (SR) through RyR24. The ensuing Ca2+ transient leads to myocyte contraction, which quickly ends when Ca2+ is removed from the cytoplasm due to SR Ca2+ reuptake through the SR Ca2+-ATPase (SERCA2a) and is extruded via the Na+/Ca2+-exchanger (NCX). In healthy hearts, RyR2 remain closed during diastole to facilitate SR refilling before the subsequent systolic Ca2+ release. In contrast, heart failure is associated with spontaneous openings of RyR2 during diastole, which can interfere with SR Ca2+ loading, decreased systolic Ca2+ release and cardiac contractility4. Moreover, it has been shown that diastolic Ca2+ releases from the SR can initiate arrhythmogenic afterdepolarizations, as will be discussed below.
The RyR2 channel comprises a macromolecular complex consisting of 4 RyR2 monomers each binding several accessory proteins (e.g., FKBP12.6, calmodulin, calsequestrin) that regulate channel activity5. Moreover, RyR2 open probability is also modulated by posttranslational modifications such as phosphorylation, nitrosylation, and oxidation. It has been demonstrated that phosphorylation of RyR2 increases the channel open probability, and thus SR Ca2+ release during both systole and diastole, although it remains somewhat controversial whether RyR2 phosphorylation alters SR Ca2+ release in a cellular environment1, 6. The first phosphorylation site identified on RyR2 was serine 2808 (S2808, or S2809 in some species), which is believed to be the primary target of protein kinase A6. Subsequently, it was shown that another serine (S2814) is the main substrate of CaMKII phosphorylation on RyR21. CaMKII phosphorylation of RyR2 increases the sensitivity to Ca2+-dependent activation and frequency of Ca2+ sparks7. Since CaMKII can decode the frequency of Ca2+ transients, it is believed that CaMKII phosphorylation of RyR2 plays a role in enhancing SR Ca2+ release in response to sudden increases in heart rate or adrenergic stress in normal hearts1, 8, 9. However, chronically increased CaMKII activity in diseased hearts can become maladaptive and lead to abnormal diastolic Ca2+ release from the SR, associated with triggered arrhythmias10.
Over the past decade, extensive evidence has emerged linking diastolic RyR2 Ca2+ leakage to arrhythmogenesis in patients with atrial and ventricular arrhythmias11, 12. First, inherited mutations in RyR2 were identified in patients with a rare condition known as catecholaminergic polymorphic ventricular tachycardia (CPVT), providing direct evidence that defects in RyR2 can cause life-threatening arrhythmias and sudden cardiac death13. CPVT-linked mutations in RyR2 lead to an increased propensity toward spontaneous Ca2+ release from the SR, which under certain conditions (such as during exercise or beta-adrenergic stimulation) facilitates the development of delayed afterdepolarizations (DADs) and triggered arrhythmias14. Clinical observations suggest that CaMKII activation might increase the risk of arrhythmias in patients with CPVT, as arrhythmias typically only occur above specific threshold heart rates (and CaMKII activity is rate-dependent)15. However, it remains to be determined whether CaMKII plays a role in triggering ventricular arrhythmias in these patients, a hypothesis that could be studied in mouse models of CPVT16, 17. Drs. Priori and Napolitano will discuss these concepts in more detail in their future viewpoint article.
Some evidence suggests that increased CaMKII activity in heart failure could promote diastolic SR Ca2+ release and ventricular tachyarrhythmias. In an arrhythmogenic rabbit model of nonischemic heart failure, Ai et al.18 demonstrated that enhanced diastolic SR Ca2+ leak could be blocked by CaMKII inhibition. Because CaMKII phosphorylation of RyR2 was elevated in this model, it was proposed that CaMKII-dependent phosphorylation of RyR2 was involved in enhanced SR diastolic Ca leak and thus arrhythmogenesis in HF18. Recent studies provide further evidence for the involvement of CaMKII phosphorylation of RyR2 in arrhythmogenesis in heart failure, since pharmacological inhibition of CaMKII completely abolished spontaneous Ca2+ waves, which are known to activate inward NCX current and DADs19. Studies in CaMKII-δc transgenic mice also revealed that increased cytosolic CaMKII activation contributes to cardiac arrhythmogenesis20. Thus, it is likely that increased CaMKII phosphorylation of RyR2 contributes to arrhythmogenesis in failing hearts. However, it remains to be determined whether CaMKII phosphorylation of RyR2 by itself is sufficient and/or necessary to promote arrhythmias in the failing heart.
Defects in RyR2 regulation may also contribute to triggered activity and arrhythmogenesis in patients with atrial arrhythmias11. Several studies have provided evidence for defects in SR Ca2+ release in patients with chronic atrial fibrillation (AF)21, 22. Because the amplitude of the L-type Ca2+ channel is decreased and SR Ca2+ loading is typically unaltered, increased open probability of RyR2 emerged as the potential culprit for Ca2+-dependent arrhythmias in patients with AF21-23. Indeed, planar lipid bilayer studies provided direct evidence for enhanced RyR2 open probability in dogs with chronic AF11. Whether increased Ca2+ sensitivity of RyR2 leads to elevated cytosolic Ca2+ levels in atrial myocytes of patients with chronic AF is still controversial. Neef al al.22 recently reported that increased RyR2-dependent SR Ca2+ leak caused elevated cytosolic Ca2+ levels in myocytes from patients with chronic AF. On the other hand, Voigt et al.24 demonstrated unaltered cytosolic Ca2+ levels despite enhanced RyR2 Ca2+ leak in atrial myocytes isolated from patients with chronic AF.
Several alterations within the RyR2 macromolecular complex are thought to underlie the increased open probability of RyR2 in AF. Biochemical studies revealed decreased levels of FK506-binding protein 12.6 (FKBP12.6, or calstabin2), and increased phosphorylation levels of the PKA (S2808) and CaMKII (S2814) sites on RyR2 in dogs and patients with chronic AF3, 11. However, one potential problem with studies of human tissue samples obtained from patients undergoing coronary artery bypass or valve replacement surgery is the co-existent structural heart disease. It will be important but at the same time challenging to explore alternative means of obtaining human atrial tissue from patients with paroxysmal or chronic AF in the absence of ischemic or structural heart disease.
Whereas it is likely that aforementioned modifications of RyR2 indeed contribute to an increased open probability and atrial arrhythmogenesis in patients, studies in genetically-modified mice provided additional insights into potential causal relationships between individual molecular changes associated with AF and an increased arrhythmia risk. Knock-in mice with a gain-of-function mutation in RyR2 (R176Q) were found to be more susceptible to atrial arrhythmias following rapid atrial pacing3. On the other hand, genetic or pharmacological inhibition of CaMKII prevented inducibility of AF in R176Q/+ mice. The specific contribution of CaMKII phosphorylation of RyR2 was studied in RyR2-S2814A knock-in mice, in which the CaMKII site S2814 was genetically inactivated due to alanine substitution. We demonstrated that inhibition of S2814 phosphorylation was sufficient to prevent the induction of pacing-induced AF following carbachol injection in S2814A mice3. Taken together, our studies revealed that increased CaMKII activity is likely to be a pro-arrhythmogenic factor in AF, and that CaMKII phosphorylation of RyR2 may be an important but probably not exclusive downstream target of CaMKII in AF.
Some have argued that increased RyR2 open probability could not be pro-arrhythmogenic because enhanced diastolic SR Ca2+ release would deplete SR Ca2+ content and prevent subsequent spontaneous SR Ca2+ releases25. This is probably true if RyR2 ‘leak’ occurs as an isolated event. However, AF is characterized by extensive remodeling of various ion channels and Ca2+ handling proteins26. For example, activated CaMKII also increases the level of PLN phosphorylation at Thr17, which promotes SR Ca2+ refilling and could support chronic diastolic SR Ca2+ leak, which may cause afterdepolarizations and triggered activity27.
Finally, it has been shown that diastolic SR Ca2+ release may activate a transient inward current (ITI) that can trigger delayed afterdepolarizations (DADs) in mouse ventricular myocytes14. Pharmacological inhibition of RyR2 with JTV519 inhibited ITI and DADs in this particular study, suggesting that excessive Ca2+ release via RyR2 can initiate arrhythmogenic DADs. In AF, increased activity of the NCX could further increase the likelihood that spontaneous SR Ca2+ release events initiate pro-arrhythmic events28. Thus, several modifications of Ca2+ handling proteins might synergistically promote the induction of AF, including increased CaMKII phosphorylation of RyR2, increased SERCA2a activity due to increased PLN phosphorylation by CaMKII, and enhanced NCX activity. It remains to be determined if CaMKII plays a role in stimulating NCX in patients with AF.
In conclusion, there is emerging evidence that increased CaMKII activity and in particular CaMKII phosphorylation of RyR2 may promote triggered activity and arrhythmia induction in patients with heart failure and atrial fibrillation. Genetically modified mice have emerged as important models to probe the specific contributions of particular CaMKII isoforms or CaMKII phosphorylation sites on downstream targets, including the RyR2 Ca2+ release channel. Although increased CaMKII phosphorylation of RyR2 increases diastolic SR Ca2+ leak, it is likely that alterations in other Ca2+ handling proteins in conjunction with RyR2 modifications are necessary to enable the development of delayed afterdepolarizations that could trigger arrhythmias. These observations suggest that pharmacological targeting of RyR2 and/or CaMKII signaling in the heart may constitute a promising avenue for the treatment of cardiac arrhythmias.
Acknowledgments
Funding
X.H.T.W. is a W.M. Keck Foundation Distinguished Young Scholar in Medical Research, and is supported by NIH/NHLBI grants R01-HL089598 and R01-HL091947. The lab is also supported by a Fondation Leducq Award to the Alliance for Calmodulin Kinase Signaling in Heart Disease (08CVD01).
Footnotes
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