Phosphorylation of the ryanodine receptor mediates the cardiac fight or flight response in mice

Abstract

During the classic "fight-or-flight" stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium (Ca 2+ ). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-S2808A +/+ mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte Ca 2+ transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-S2808A +/+ mice. The blunted cardiac response to catecholamines in RyR2-S2808A +/+ mice resulted in impaired exercise capacity. RyR2-S2808A +/+ mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation. IntroductionDuring exercise, heart rate (chronotropy) and cardiac contractility (inotropy) increase to meet the metabolic demands of the organs. Stress-induced activation of the sympathetic nervous system (SNS) results in catecholamine release, stimulation of β-adrenergic receptors (β-ARs), generation of cAMP, and activation of cAMP-dependent protein kinase (PKA) in cardiac myocytes. Catecholaminergic stimulation of the heart increases both heart rate and contractility (1). The essential role of β-ARs in the stress-induced enhancement of cardiac function has been demonstrated using β 1 -AR knockout mice that are unable to develop normal responses to stress (2). However, the complexity of the β-AR signaling cascade has made it difficult to elucidate specific contributions of downstream targets to the physiologic responses to stress.β-AR stimulation and downstream activation of PKA enhances calcium (Ca 2+ ) signaling in myocytes (3). During excitation-contraction (EC) coupling in the heart, depolarization of the sarcolemmal membrane activates the voltage-gated calcium channel (Ca V 1.2), causing a small Ca 2+ influx into the cell. This in turn triggers the opening of ryanodine receptor/calcium release channel (RyR2) and the release of Ca 2+ from the sarcoplasmic reticu-

Is ryanodine receptor phosphorylation key to the fight or flight response and heart failure?

“…Conversely, S2808D mice would be expected to exhibit spontaneous FKBP12.6 depletion from the RyR2 complex, initial hypercontractility (because of facilitated Ca 2+ release from the SR), spontaneous development of a cardiomyopathic phenotype, and exaggerated pathology under chronic stress. The results obtained by Marks and colleagues almost exactly matched these expectations and go beyond (25,26). S2808A mice also exhibited blunted chronotropic responses (i.e., blunted acceleration of the heart rate) to isoprenaline and reduced exercise capacity, suggesting that RyR2 phosphorylation at S2808 regulates not only contractile force but heart rate too and that it is a critical determinant of physical fitness.…”

“…Yet, this is only partially the case, as shown by two studies from the Marks laboratory published in this issue of the JCI (25,26). The two papers report on two new mouse models: one in which the S2808 site was mutated to a non-phosphorylatable alanine (S2808A; ref.…”

“…The two papers report on two new mouse models: one in which the S2808 site was mutated to a non-phosphorylatable alanine (S2808A; ref. 25), and another in which S2808 was replaced by aspartic acid (S2808D), a "phosphomimetic" amino acid (26). The expectation was that S2808A mice would be less sensitive to the forceincreasing acute effects of isoprenaline (blunted fight or flight response) but also protected from adverse long-term consequences of b-adrenergic stimulation, such as cardiac dilatation and dysfunction (as seen in mice lacking phosphatase inhibitor-1; ref.…”

Mitochondrial calcium overload is a key determinant in heart failure

“…Mice harboring the IP3R2 flox/flox allele were bred with MHC-Cre transgenic mice to obtain a cardiac ventricularspecific ablation of IP3R2. A detailed description of materials and methods for in vivo experiments (31)(32)(33)(34)(35), isolation of adult cardiomyocytes (34,36), isolation of mitochondria (37), assessment of mitochondrial dynamics, Ca 2+ content, and membrane potential (34,37), real-time RT-qPCR (38,39), immunoprecipitation/immunoblot, and electron microscopy (40) can be found in SI Materials and Methods.…”

PDE4B mediates local feedback regulation of β1-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes

“…2+ levels and myocyte contraction rate after bAR stimulation It is well established that stimulation of b 1 AR signaling increases heart rate (Devic et al, 2001;Xiang et al, 2005) and that this effect is due in part to increased PKA-mediated phosphorylation and activation of Ca V 1.2 and RyR2 (Bers, 2008;Shan et al, 2010). The fact that PDE4B controls the PKA-mediated phosphorylation of LTCC and RyR2 prompted us to compare the intracellular Ca 2+ levels and the contraction rate of wild-type and PDE4BKO NCMs.…”