Does protein kinase a-mediated phosphorylation of the cardiac ryanodine receptor play any role in adrenergic regulation of calcium handling in health and disease?

Cardiac myocyte ryanodine receptors (sarcoplasmic reticulum [SR] Ca release channel; cardiac ryanodine receptor [RyR2]) are localized in the junctional SR, in close proximity to L-type Ca channels (LTCCs) embedded in the membranes of the transverse (T)-tubules. This signaling microdomain has been termed the couplon,1 because it is here that excitation–contraction coupling takes place. As the heart fills with blood during diastole, RyR2 is stabilized in a closed state, allowing Ca uptake by the SR Ca ATPase to pump Ca from the cytoplasm into the SR, providing the primary source of Ca to activate the contractile apparatus during the next heart beat (systole). During systole, the cardiac action potential depolarizes the T-tubules and causes the opening of LTCCs. Ca influx through LTCCs elevates the [Ca] within the cytoplasmic space between the T-tubular and SR membrane, which promotes Ca binding to neighboring RyR2s, inducing some to open. Ca then moves out of the SR lumen into the subsarcolemmal, space and the additional elevation of [Ca] induces a regenerative opening of other RyRs in the couplon. The resulting localized Ca signal is called a Ca spark.2 The action potential synchronizes these local Ca release events throughout the cell (termed Ca-induced Ca release) to produce the global cardiac [Ca] transient.3 The stabilized closed state and Ca-dependent opening of RyR2 during diastole and systole are essential for normal cardiac diastolic and systolic function. Destabilizing the behavior of RyR2 would be expected to have a negative impact on the function of the heart. Approximately 10 years ago, the Marks laboratory4 reported that protein kinase (PK)A-mediated (hyper)phosphorylation of RyR2 at S2809 in the failing heart caused increased RyR Ca sensitivity and abnormal channel activity. Based on a series of provocative experiments,4–10 this group suggested that PKA-mediated RyR2-S2809 phosphorylation is involved in the normal increase in cardiac contractility produced by sympathetic stimulation (one example would be to increase cardiac contractility during exercise). They also proposed that in severe pathological stress, persistent adrenergic activity caused RyR2-S2809 hyperphosphorylation and destabilization of RyR2 function, resulting in diastolic leakage of Ca from the SR and a propensity for arrhythmias. The mechanism for these effects was shown to involve hyperphosphorylation of RyR2 at S2809, which leads to FK-binding protein (FKBP)12.6 (normally bound to RyR and proposed as a stabilizer of its function) dissociation from RyR, resulting in abnormal RyR activity.4 The RyR-S2809 hyperphosphorylation and loss of the putative FKBP12.6-stabilizing effect appeared to be critical to the development and progression of heart failure, because prevention of these processes significantly improved cardiac function after myocardial infarction9,10 and reduced stress-related arrhythmias.7,8 As would be expected, these reports sparked a host of studies to confirm the original observations and further define the role of RyR2 in heart failure and life threatening arrhythmias. Some of these studies have not been able to confirm critical features of the original RyR-S2809 hyperphosphorylation hypothesis.11–22 In this issue of Circulation Research, the Bers laboratory refutes an essential element of the PKA RyR-S2809 hyperphosphorylation hypothesis.23 Guo et al specifically explored the portion of the hypothesis that predicts that PKA hyperphosphorylation of RyR2 at S2809 causes FKBP12.6 to dissociate from RyR2 and thereby produce a leaky SR.23 In a superb series of experiments in cardiac myocytes, these authors show that FKBP12.6 (the putative RyR2 stabilizer) is found in much lower abundance than RyR. Therefore, FKBP12.6 was only bound to a small percentage ( 15%) of RyRs. Importantly, Guo et al23 convincingly demonstrate that FKBP12.6 does not dissociate from RyR2 with PKAmediated phosphorylation of RyR-S2809. They also show that FKBP12 is more abundant than FKBP12.6, is tightly bound to RyR2, does not dissociate with PKA phosphorylation of RyR2, and does not affect RyR2 function. To me, these are definitive experiments. The authors should be commended because they developed the novel, quantitative techniques that were needed to directly address critical controversies related to RyR. They used 2 or 3 independent techniques to assess FKBP12.6(and FKBP12)-RyR binding. All of their results support the conclusion that FKBP12.6 binding to RyR is not influenced by PKA phosphorylation of RyR-S2809. A particular strength of this new study is that the investigators developed techniques that allowed RyR binding and unbinding of FKBP12.6 (and FKBP12) to be directly visualized over time, in real cardiac myocytes, in the absence and presence of PKA activators. The quantitative approaches developed for these experiments were much more rigorous than have been used previously.4 To this reader, the authors The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Department of Physiology and Cardiovascular Research Center, School of Medicine, Temple University, Philadelphia, Pa. Correspondence to Steven R. Houser, PhD, School of Medicine, Temple University, 3500 N Broad St, Philadelphia, PA 19140. E-mail [email protected] (Circ Res. 2010;106:1672-1674.) © 2010 American Heart Association, Inc.