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Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The musclespecific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies., Introduction Cardiac muscle mass adapts as a consequence of functional requirements. Increased workload, such as during exercise or chronic disease stresses causing pressure overload, induces cardiomyocyte hypertrophy, while hemodynamic unloading [...]

During the classic 'fight-or-flight' stress response, sympathetic nervous system activation leads to catechol-amine 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.sup.2+]). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-[S2808A.sup.+/+] mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte [Ca.sup.2+] transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-[S2808A.sup.+/+] mice. The blunted cardiac response to catecholamines in RyR2-[S2808A.sup.+/+] mice resulted in impaired exercise capacity. RyR2-[S2808A.sup.+/+] 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., Introduction During 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 [...]

Endoplasmic reticulum (ER) stress-induced apoptosis is involved in many diseases, but the mechanisms linking ER stress to apoptosis are incompletely understood. Based on roles for C/EPB homologous protein (CHOP) and ER calcium release in apoptosis, we hypothesized that apoptosis involves the activation of inositol 1,4,5-triphosphate (IP3) receptor (IP3R) via CHOP-induced ERO1-[alpha] (ER oxidase 1 [alpha]). In ER-stressed cells, ERO1-[alpha] is induced by CHOP, and small interfering RNA (siRNA) knockdown of ERO1-[alpha] suppresses apoptosis. IP3-induced calcium release (IICR)is increased during ER stress, and this response is blocked by siRNA-mediated silencing of EROI-[alpha] or IP3R1 and by loss-of-function mutations in Erola or Chop. Reconstitution of ER01-[alpha] in [Chop.sup.-/-] macrophages restores ER stress--induced IICR and apoptosis. In vivo, macrophages from wild-type mice but not [Chop.sup.-/-] mice have elevated IICR when the animals are challenged with the ER stressor tunicamycin. Macrophages from insulin-resistant ob/ob mice, another model of ER stress, also have elevated IICR. These data shed new light on how the CHOP pathway of apoptosis triggers calcium-dependent apoptosis through an ER01-[alpha]-IP3R pathway.

The [Ca.sup.2+] release channel ryanodine receptor 2 (RyR2) is required for excitation-contraction coupling in the heart and is also present in the brain. Mutations in RyR2 have been linked to exercise-induced sudden cardiac death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the channel. We found that mice heterozygous for the R2474S mutation in Ryr2 (Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and sudden cardiac death. Treatment with a novel RyR2-specific compound (S107) that enhances the binding of calstabin2 to the mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky Ryr2-R2474S channels in the brain can cause seizures in mice, independent of cardiac arrhythmias. Based on these data, we propose that CPVT is a combined neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy, and the same leaky channels in the heart cause exercise-induced sudden cardiac death., Introduction Pharmacological seizure models have implicated abnormalities in intracellular [Ca.sup.2+] cycling of inhibitory interneurons and/or astrocytes as a mechanism of seizure generation (1), (2), and the inositol 1,4,5-trisphosphate receptor (IP3R), [...]

Over the past century, understanding the mechanisms underlying muscle fatigue and weakness has been the focus of much investigation. However, the dominant theory in the field, that lactic acidosis causes muscle fatigue, is unlikely to tell the whole story. Recently, dysregulation of sarcoplasmic reticulum (SR) [Ca.sup.2+] release has been associated with impaired muscle function induced by a wide range of stressors, from dystrophy to heart failure to muscle fatigue. Here, we address current understandings of the altered regulation of SR [Ca.sup.2+] release during chronic stress, focusing on the role of the SR [Ca.sup.2+] releases channel known as the type 1 ryanodine receptor., Introduction Skeletal muscle contraction is initiated by depolarization of the surface membrane (the sarcolemma) of muscle cells. Depolarization of the sarcolemma leads to the formation of cross-bridges between myosin and [...]

Although the importance of mitochondria in pathophysiology has become increasingly evident, it remains unclear whether these organelles play a role in [Ca.sup.2+] handling by skeletal muscle. This undefined situation is mainly due to technical limitations in measuring [Ca.sup.2+] transients reliably during the contraction--relaxation cycle. Using two-photon microscopy and genetically expressed 'cameleon' [Ca.sup.2+] sensors, we developed a robust system that enables the measurement of both cytoplasmic and mitochondrial [Ca.sup.2+] transients in vivo. We show here for the first time that, in vivo and under highly physiological conditions, mitochondria in mammalian skeletal muscle take up [Ca.sup.2+] during contraction induced by motor nerve stimulation and rapidly release it during relaxation. The mitochondrial [Ca.sup.2+] increase is delayed by a few milliseconds compared with the cytosolic [Ca.sup.2+] rise and occurs both during a single twitch and upon tetanic contraction.