Abstract-Cardiac] i transients Ⅲ ryanodine receptor Ⅲ excitation-contraction coupling Ⅲ CPVT C atecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by stress-induced, adrenergically mediated bidirectional or polymorphic ventricular tachycardia occurring in structurally normal hearts. 1 During exercise or acute emotions, CPVT patients develop life-threatening ventricular arrhythmias, leading to syncope or sudden death. The first cardiac ryanodine receptor (RyR2) mutation identified in a CPVT family was R4497C. 2 Today, more than 70 RyR2 mutations have been reported (http://www.fsm.it/cardmoc), and they comprise the most common genetic subtype of CPVT, 3-7 although mutations in the calsequestrin gene can also cause CPVT. 8,9 Diverging results and conclusions have been generated from expression studies of RyR2 R4496C in heterologous systems. Jiang et al showed that RyR2 R4496C (the mouse equivalent of the human RyR2 R4497C mutation), when expressed in human embryonic kidney (HEK) cells, exhibits increased basal activity and increased sensitivity to luminal Ca 2ϩ . 10 However, other authors found no difference in the basal activity of RyR2 R4497C but, instead, showed increased activity and gating frequency after protein kinase A phosphorylation 11 or sarcoplasmic reticulum (SR) Ca 2ϩ overload. 12 The expression studies were carried out in a variety of models, which may explain the inhomogeneous findings. Furthermore, heterologous systems lack cardiac intracellular environment with all the RyR2 accessory proteins 13 and most Ca 2ϩ -handling proteins, so analysis in native cardiac myocytes is now critical to elucidate the mechanisms by which the mutation leads to cardiac arrhythmia.Recently, a knock-in mouse model carrier of the RyR2 R4496C mutation was developed. 14 Their phenotype presents extraordinary similarity with the clinical manifestations of patients carrying the RyR2 R4497C mutation, including the development of bidirectional ventricular tachycardia. When exposed to adrenaline and caffeine, the RyR2 R4496C cardiomyocytes develop delayed afterdepolarizations (DADs), 15 suggesting that triggered arrhythmias are elicited by adrenergic activation. 16 Here we demonstrate that untreated RyR2 R4496C myocytes have increased spontaneous Ca 2ϩ release in diastole during electric pacing, because of the enhanced Ca 2ϩ sensitivity of mutant RyR2; this abnormality is further augmented by exposure to isoproterenol and increasing pacing rates. Materials and Methods Ventricular cardiomyocytes from male and female RyR2R4496Cϩ/Ϫ mice (RyR2 R4496C ) and their wild-type (WT) RyR2 R4496CϪ/Ϫ littermates were isolated using a standard enzymatic digestion. 17 [Ca 2ϩ ] i transients and Ca 2ϩ sparks were viewed in isolated myocytes by confocal microscopy and analyzed using homemade routines. All experiments were carried out according to the ethical principles laid down by the French (Ministry of Agriculture) and European Union

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Epac is a guanine nucleotide exchange protein that is directly activated by cAMP, but whose cardiac cellular functions remain unclear. It is important to understand cardiac Epac signaling, because it is activated in parallel to classical cAMP-dependent signaling via protein kinase A. In addition to activating contraction, Ca2+ is a key cardiac transcription regulator (excitation-transcription coupling). It is unknown how myocyte Ca2+ signals are decoded in cardiac myocytes to control nuclear transcription. We examine Epac actions on cytosolic ([Ca2+]i) and intranuclear ([Ca2+]n) Ca2+ homeostasis, focusing on whether Epac alters [Ca2+]n and activates a prohypertrophic program in cardiomyocytes. Adult rat cardiomyocytes, loaded with fluo-3 were viewed by confocal microscopy during electrical field stimulation at 1 Hz. Acute Epac activation by 8-pCPT increased Ca2+ sparks and diastolic [Ca2+]i, but decreased systolic [Ca2+]i. The effects on diastolic [Ca2+]i and Ca2+ spark frequency were dependent on phospholipase C (PLC), inositol 1,3,5 triphosphate receptor (IP3R) and CaMKII activation. Interestingly, Epac preferentially increased [Ca2+]n during both diastole and systole, correlating with the perinuclear expression pattern of Epac. Moreover, Epac activation induced histone deacetylase 5 (HDAC5) nuclear export, with consequent activation of the prohypertrophic transcription factor MEF2. These data provide the first evidence that the cAMP-binding protein Epac modulates cardiac nuclear Ca2+ signaling by increasing [Ca2+]n through PLC, IP3R and CaMKII activation, and initiates a prohypertrophic program via HDAC5 nuclear export and subsequent activation of the transcription factor MEF2.

Background Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is characterized by stress-triggered syncope and sudden death. CPVT patients manifest sino-atrial node (SAN) dysfunction, the mechanisms of which remain unexplored. Methods and Results We investigated SAN [Ca2+]i handling in mice carrying the CPVT-linked mutation of ryanodine receptor (RyR2R4496C) and on their wild-type (WT) littermates. In vivo telemetric recordings showed impaired SAN automaticity in RyR2R4496C mice following Isoproterenol (ISO) injection, analogous to what was observed in CPVT patients after exercise. Pacemaker activity was explored by measuring spontaneous [Ca2+]i transients in SAN cells within the intact SAN by confocal microscopy. RyR2R4496C SAN presented significantly slower pacemaker activity and impaired chronotropic response under β-adrenergic stimulation, accompanied by the appearance of pauses (in spontaneous [Ca2+]i transients and action potentials) in 75% of the cases. Ca2+ spark frequency was increased by 2-fold in RyR2R4496C SAN. Whole-cell patch-clamp experiments performed on isolated RyR2R4496C SAN cells showed that L-type Ca2+ current (ICa,L) density was reduced by ~50%, an effect blunted with internal Ca2+ buffering. ISO dramatically increased the frequency of Ca2+ sparks and waves by ~5 and ~10-fold, respectively. Interestingly, the sarcoplasmic reticulum (SR) Ca2+ content was significantly reduced in RyR2R4496C SAN cells in the presence of ISO, which may contribute to stopping the “Ca2+-clock” rhythm generation, originating SAN pauses. Conclusions The increased activity of RyR2R4496C in SAN leads to an unanticipated decrease on SAN automaticity by Ca2+-dependent decrease of ICa,L and SR Ca2+ depletion during diastole, identifying subcellular pathophysiologic alterations contributing to the SAN dysfunction in CPVT patients.

Background-Cardiac hypertrophy underlies arrhythmias and sudden death, for which mineralocorticoid receptor (MR) activity has recently been implicated. We sought to establish the sequence of ionic events that link the initiating insult and MR to hypertrophy development. Methods and Results-Using whole-cell, patch-clamp and quantitative reverse transcription-polymerase chain reaction techniques on right ventricular myocytes of a myocardial infarction (MI) rat model, we examined the cellular response over time. One week after MI, no sign of cellular hypertrophy was found, but action potential duration (APD) was lengthened. Both an increase in Ca 2ϩ current (I Ca ) and a decrease in K ϩ transient outward current (I to ) underlay this effect. Consistently, the relative expression of mRNA coding for the Ca 2ϩ channel ␣1C subunit (Ca v 1.2) increased, and that of the K ϩ channel K v 4.2 subunit decreased. Three weeks after MI, AP prolongation endured, whereas cellular hypertrophy developed. I Ca density, Ca v 1.2, and K v 4.2 mRNA levels regained control levels, but I to density remained reduced. Long-term treatment with RU28318, an MR antagonist, prevented this electrical remodeling. In a different etiologic model of abdominal aortic constriction, we confirmed that APD prolongation and modifications of ionic currents precede cellular hypertrophy. Conclusions-Electrical remodeling, which is triggered at least in part by MR activation, is an initial, early cellular response to hypertrophic insults.

Background-The mineralocorticoid pathway is involved in cardiac arrhythmias associated with heart failure through mechanisms that are incompletely understood. Defective regulation of the cardiac ryanodine receptor (RyR) is an important cause of the initiation of arrhythmias. Here, we examined whether the aldosterone pathway might modulate RyR function. Methods and Results-Using the whole-cell patch clamp method, we observed an increase in the occurrence of delayed afterdepolarizations during action potential recordings in isolated adult rat ventricular myocytes exposed for 48 hours to aldosterone 100 nmol/L, in freshly isolated myocytes from transgenic mice with human mineralocorticoid receptor expression in the heart, and in wild-type littermates treated with aldosterone. Sarcoplasmic reticulum Ca 2ϩ load and RyR expression were not altered; however, RyR activity, visualized in situ by confocal microscopy, was increased in all cells, as evidenced by an increased occurrence and redistribution to long-lasting and broader populations of spontaneous Ca

The pores of ion channel proteins are often modeled as static structures. In this view, selectivity reflects rigidly constrained backbone orientations. Such a picture is at variance with the generalization that biological proteins are flexible, capable of major internal motions on biologically relevant time scales. We tested for motions in the sodium channel pore by systematically introducing pairs of cysteine residues throughout the pore-lining segments. Two distinct pairs of residues spontaneously formed disulfide bonds bridging domains I and II. Nine other permutations, involving all four domains, were capable of disulfide bonding in the presence of a redox catalyst. The results are inconsistent with a single fixed backbone structure for the pore; instead, the segments that line the permeation pathway appear capable of sizable motions.

Store-operated Ca2؉ entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca 2؉ influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum-and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca 2؉ ] i , which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum-and glucocorticoid-regulated kinase 1 pathway.The last discovered Ca 2ϩ channel family, transient receptor potential canonical (TRPC) 2 and Orai1 channels, is widely expressed, but the role of these channels and the modulation of their expression are not completely understood. Notably, the dysregulation of these important mediators of Ca 2ϩ -dependent signal transduction has been involved in a broad range of human diseases such as adenocarcinomas, type 2 diabetes and diabetic nephropathy, human proteinuric kidney disease focal and segmental glomerulosclerosis, severe immunodeficiency, congenital myopathy, chronic pulmonary disease, and ectodermal dysplasia (1, 2). Although predominantly thought of in the context of non-excitable cells, store-operated Ca 2ϩ entry (SOCE) by store-operated channels (SOCs) has recently emerged as a potential mechanism to alter Ca 2ϩ in the diseased cardiomyocyte. Although still under debate, the prime candidate proteins for SOCs encompass the TRPC channel(s) as non-selective cation channels as well as Orai1, the Ca 2ϩ selective pore-forming unit of the Ca 2ϩ release-activated Ca 2ϩ channel (3). Stromal interaction molecule 1 (STIM1) serves as a Ca 2ϩ sensor in the endoplasmic reticulum/sarcoplasmic reticulum (SR), clustering proximal to the plasma membrane to activate Orai1 (4) and TRPC channel(s) (5) when Ca 2ϩ stores are depleted. The seven isoforms of the T...