During exercise, defects in calcium (Ca 2؉ ) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca 2؉ release channel required for excitationcontraction coupling (ECC) in skeletal muscle, the ryanodine receptor (RyR1), is progressively PKA-hyperphosphorylated, S-nitrosylated, and depleted of the phosphodiesterase PDE4D3 and the RyR1 stabilizing subunit calstabin1 (FKBP12), resulting in ''leaky'' channels that cause decreased exercise tolerance in mice. Mice with skeletal musclespecific calstabin1 deletion or PDE4D deficiency exhibited significantly impaired exercise capacity. A small molecule (S107) that prevents depletion of calstabin1 from the RyR1 complex improved force generation and exercise capacity, reduced Ca 2؉ -dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca 2؉ leak via calstabin1-depleted RyR1 channels leads to defective Ca 2؉ signaling, muscle damage, and impaired exercise capacity. muscle fatigue ͉ calcium channel ͉ calstabin ͉ exitation-contraction coupling ͉ rycals

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.

Macrophages in advanced atherosclerotic lesions accumulate large amounts of unesterified, or "free," cholesterol (FC). FC accumulation induces macrophage apoptosis, which likely contributes to plaque destabilization. Apoptosis is triggered by the enrichment of the endoplasmic reticulum (ER) with FC, resulting in depletion of ER calcium stores, and induction of the unfolded protein response. To explain the mechanism of ER calcium depletion, we hypothesized that FC enrichment of the normally cholesterol-poor ER membrane inhibits the macrophage ER calcium pump, sarcoplasmic-endoplasmic reticulum calcium ATPase-2b (SERCA2b). FC enrichment of ER membranes to a level similar to that occuring in vivo inhibited both the ATPase activity and calcium sequestration function of SERCA2b. Enrichment of ER with ent-cholesterol or 14:0 -18:0 phosphatidylcholine, which possess the membrane-ordering properties of cholesterol, also inhibited SERCA2b. Moreover, at various levels of FC enrichment of ER membranes, there was a very close correlation between increasing membrane lipid order, as monitored by 16-doxyl-phosphatidycholine electron spin resonance, and SERCA2b inhibition. In view of these data, we speculate that SERCA2b, a conformationally active protein with 11 membrane-spanning regions, loses function due to decreased conformational freedom in FC-ordered membranes. This biophysical model may underlie the critical connection between excess cholesterol, unfolded protein response induction, macrophage death, and plaque destabilization in advanced atherosclerosis.

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BACKGROUND-Sudden infant death syndrome (SIDS) is the leading cause of postneonatal mortality in the United States. Mutations in the RyR2-encoded cardiac ryanodine receptor cause the highly lethal catecholaminergic polymorphic ventricular tachycardia (CPVT1) in the young.

Rationale Mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT-associated RyR2 mutations cause fatal ventricular arrhythmias in young individuals during beta-adrenergic stimulation. Objective This study sought to determine the effects of a novel identified RyR2-G230C mutation and whether this mutation and RyR2-P2328S alter the sensitivity of the channel to luminal calcium (Ca2+). Methods Functional characterizations of recombinant human RyR2-G230C channels were performed under conditions mimicking stress. Human RyR2 mutant channels were generated by site-directed mutagenesis and heterologously expressed in HEK293 cells together with calstabin2 (FKBP12.6). RyR2 channels were measured in order to examine the regulation of the channels by cytosolic vs. luminal SR Ca2+. Results A 50 year-old Caucasian male with repeated syncopal episodes after exercise had a cardiac arrest and harbored the mutation RyR2-G230C. PKA-phosphorylated RyR2-G230C channels exhibited a significantly higher open probability (Po) at diastolic Ca2+ concentrations, associated with a depletion of calstabin2. The luminal Ca2+ sensitivities of RyR2-G230C and RyR2-P2328S channels were WT-like. Conclusions The RyR2-G230C mutant exhibits similar biophysical defects compared to previously characterized CPVT mutations: decreased binding of the stabilizing subunit calstabin2 and a leftward shift in the Ca2+-dependence for activation under conditions that simulate exercise, consistent with a “leaky” channel. Both RyR2-G230C and RyR2-P2328S channels exhibit normal luminal Ca2+ activation. Thus, diastolic SR Ca2+ leak caused by reduced calstabin2 binding and a leftward shift in the Ca2+-dependence for activation by diastolic levels of cytosolic Ca2+ is a common mechanism underlying CPVT.

Zahradníková , A., M. Dura, I. Gyö rke, A. L. Escobar, I. Zahradník, and S. Gyö rke. Regulation of dynamic behavior of cardiac ryanodine receptor by Mg 2ϩ under simulated physiological conditions.

The most common histological subtypes of cutaneous melanoma include superficial spreading and nodular melanoma. However, the spectrum of somatic mutations developed in those lesions and all potential druggable targets have not yet been fully elucidated. We present the results of a sequence capture NGS analysis of 114 primary nodular and superficial spreading melanomas identifying driver mutations using biostatistical, immunohistochemical and/or functional approach. The spectrum and frequency of pathogenic or likely pathogenic variants were identified across 54 evaluated genes, including 59 novel mutations, and the newly identified TP53 loss-of-function mutations p.(L194P) and p.(R280K). Frequently mutated genes most commonly affected the MAPK pathway, followed by chromatin remodeling, and cell cycle regulation. Frequent aberrations were also detected in the genes coding for proteins involved in DNA repair and the regulation and modification of cellular tight junctions. Furthermore, relatively frequent mutations were described in KDR and MET, which represent potential clinically important targets. Those results suggest that with the development of new therapeutic possibilities, not only BRAF testing, but complex molecular testing of cutaneous melanoma may become an integral part of the decision process concerning the treatment of patients with melanoma.