Summary Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24 months) rodents were oxidized, cysteine-nitrosylated, and depleted of the channel stabilizing subunit calstabin1, compared to RyR1 from younger (3–6 months) adults. This RyR1 channel complex remodeling resulted in “leaky” channels with increased open probability leading to intracellular calcium leak in skeletal muscle. Similarly, six-month old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month old wild type mice. Treating aged mice with S107, stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca2+ release, muscle specific force and exercise capacity. Taken together these data indicate that leaky RyR1 contribute to age-related loss of muscle function.

Increased sarcoplasmic reticulum (SR) Ca 2+ leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-S2808D +/+ mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-S2808D +/+ mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR Ca 2+ store content, and increased diastolic SR Ca 2+ leak. After myocardial infarction, RyR2-S2808D +/+ mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR Ca 2+ leak and reduced HF progression in WT and RyR2-S2808D +/+ mice. In contrast, β-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-S2808D +/+ mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of β-blocker therapy in HF.

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-

Objectives The purpose of this study was to test the hypothesis that SCN10A variants contribute to the development of Brugada syndrome (BrS). Background BrS is an inherited sudden cardiac death syndrome. Fewer than 35% of BrS probands have genetically identified pathogenic variants. Recent evidence has implicated SCN10A, a neuronal sodium channel gene encoding Nav1.8 in the electrical function of the heart. Methods Clinical analysis and direct sequencing of BrS-susceptibility genes were performed on 150 probands, family members and >200 healthy controls. Expression and co-immunoprecipitation studies were performed to functionally characterize the putative pathogenic mutations. Results We identified 17 SCN10A mutations in 25 probands (20 M/5 F); 23 of the 25 (92.0%) displayed overlapping phenotypes. SCN10A mutations were found in 16.7% of BrS probands, approaching our yield for SCN5A mutations (20.1%). BrS patients with SCN10A mutations were more symptomatic and displayed significantly longer PR and QRS intervals than SCN10A negative BrS probands. The majority of mutations localized to the transmembrane-spanning regions. Heterologous co-expression of wild-type (WT) SCN10A with WT-SCN5A in HEK cells caused a near doubling of sodium channel current (INa) compared with WT-SCN5A alone. In contrast, co-expression of SCN10A mutants (R14L and R1268Q) with WT-SCN5A caused a 79.4% and 84.4% reduction in INa, respectively. Co-immunoprecipitation studies performed provide evidence for co-association of Nav1.8 and Nav1.5 in the plasma membrane. Conclusions Our study identifies SCN10A as a major susceptibility gene for BrS, thus greatly enhancing our ability to genotype and risk stratify probands and family members.

Rationale Atrial fibrillation (AF) is the most common cardiac arrhythmia, however the mechanism(s) causing AF remain poorly understood and therapy is suboptimal. The ryanodine receptor (RyR2) is the major calcium (Ca2+) release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction coupling in cardiac muscle. Objective In the present study we sought to determine whether intracellular diastolic SR Ca2+ leak via RyR2 plays a role in triggering AF and whether inhibiting this leak can prevent AF. Methods and Results We generated three knock-in mice with mutations introduced into RyR2 that result in leaky channels and cause exercise induced polymorphic ventricular tachycardia in humans [catecholaminergic polymorphic ventricular tachycardia (CPVT)]. We examined AF susceptibility in these three CPVT mouse models harboring RyR2 mutations to explore the role of diastolic SR Ca2+ leak in AF. AF was stimulated with an intra-esophageal burst pacing protocol in the three CPVT mouse models (RyR2-R2474S+/−, 70%; RyR2-N2386I+/−, 60%; RyR2-L433P+/−, 35.71%), but not in wild type (WT) mice (P<0.05). Consistent with these in vivo results, there was a significant diastolic SR Ca2+ leak in atrial myocytes isolated from the CPVT mouse models. Calstabin2 (FKBP12.6) is an RyR2 subunit that stabilizes the closed state of RyR2 and prevents a Ca2+ leak through the channel. Atrial RyR2 from RyR2-R2474S+/− mice were oxidized and the RyR2 macromolecular complex was depleted of calstabin2. The Rycal drug S107 stabilizes the closed state of RyR2 by inhibiting the oxidation/phosphorylation induced dissociation of calstabin2 from the channel. S107 reduced the diastolic SR Ca2+ leak in atrial myocytes and decreased burst pacing-induced AF in vivo. S107 did not reduce the increased prevalence of burst pacing-induced AF in calstabin2-deficient mice, confirming that calstabin2 is required for the mechanism of action of the drug. Conclusions The present study demonstrates that RyR2-mediated diastolic SR Ca2+ leak in atrial myocytes is associated with AF in CPVT mice. Moreover, the Rycal S107 inhibited diastolic SR Ca2+ leak through RyR2 and pacing-induced AF associated with CPVT mutations.