Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease so far related to mutations in the cardiac ryanodine receptor (RYR2) or the cardiac calsequestrin (CASQ2) genes. Because mutations in RYR2 or in CASQ2 are not retrieved in all CPVT cases, we searched for mutations in the physiological protein partners of RyR2 and CSQ2 in a large cohort of CPVT patients with no detected mutation in these two genes. Based on a candidate gene approach, we focused our investigations on triadin and junctin, two proteins that link RyR2 and CSQ2. Mutations in the triadin (TRDN) and in the junctin (ASPH) genes were searched in a cohort of 97 CPVT patients. We identified three mutations in triadin which cosegregated with the disease on a recessive mode of transmission in two families, but no mutation was found in junctin. Two TRDN mutations, a 4 bp deletion and a nonsense mutation, resulted in premature stop codons; the third mutation, a p.T59R missense mutation, was further studied. Expression of the p.T59R mutant in COS-7 cells resulted in intracellular retention and degradation of the mutant protein. This was confirmed after in vivo expression of the mutant triadin in triadin knock-out mice by viral transduction. In this work, we identified TRDN as a new gene responsible for an autosomal recessive form of CPVT. The mutations identified in the two families lead to the absence of the protein, thereby demonstrating the importance of triadin for the normal function of the cardiac calcium release complex in humans.

Background STromal Interaction Molecule 1 (STIM1) is a dynamic calcium signal transducer implicated in hypertrophic growth of cardiac myocytes. STIM1 is thought to act as an initiator of cardiac hypertrophic response at the level of the sarcolemma but the pathways underpinning this effect have not been examined. Methods and Results To determine the mechanistic role of STIM1 in cardiac hypertrophy and during the transition to heart failure, we manipulated STIM1 expression in mice cardiac myocytes using in vivo gene delivery of specific short hairpin RNAs. In three different models, we found that Stim1 silencing prevents the development of pressure-overload induced hypertrophy but also reverses pre-established cardiac hypertrophy. Reduction in STIM1 expression promoted a rapid transition to heart failure. We further showed that Stim1 silencing resulted in enhanced activity of the anti-hypertrophic and pro-apoptotic GSK-3β molecule. Pharmacological inhibition of GSK-3 was sufficient to reverse the cardiac phenotype observed after Stim1 silencing. At the level of ventricular myocytes, Stim1 silencing or inhibition abrogated the capacity for phosphorylation of AktS473, a hydrophic motif of Akt that is directly phosphorylated by mTORC2. We found that Stim1 silencing directly impaired mTORC2 kinase activity, which was supported by a direct interaction between STIM1 and Rictor, a specific component of mTORC2 complex. Conclusions These data support a model whereby STIM1 is critical to deactivate a key negative regulator of cardiac hypertrophy. In cardiac myocytes, STIM1 acts by tuning Akt kinase activity through activation of mTOR complex 2 (mTORC2), which further results in repression of GSK-3β activity.

Background: Pulmonary arterial hypertension (PAH) results in right ventricular (RV) failure, electro-mechanical dysfunction and heightened risk of sudden cardiac death (SCD), although exact mechanisms and predisposing factors remain unclear. Because impaired chronotropic response to exercise is a strong predictor of early mortality in patients with PAH, we hypothesized that progressive elevation in heart rate can unmask ventricular tachyarrhythmias (VT) in a rodent model of monocrotaline (MCT)-induced PAH. We further hypothesized that intra-tracheal gene delivery of aerosolized AAV1.SERCA2a (AAV1.S2a), an approach which improves pulmonary vascular remodeling in PAH, can suppress VT in this model. Objective: To determine the efficacy of pulmonary AAV1.S2a in reversing electrophysiological (EP) remodeling and suppressing VT in PAH. Methods: Male rats received subcutaneous injection of MCT (60 mg/kg) leading to advanced PAH. Three weeks following MCT, rats underwent intra-tracheal delivery of aerosolized AAV1.S2a (MCT+S2a, N=8) or saline (MCT, N=9). Age-matched rats served as controls (CTRL, N=7). The EP substrate and risk of VT were determined using high-resolution optical action potential (AP) mapping ex vivo. The expression levels of key ion channel subunits, fibrosis markers and hypertrophy indices were measured by RT-PCR and histochemical analyses.

Background:Central Core Disease (CCD) is a congenital myopathy often resulting from a mutation in RYR1 gene. Mutations in RyR1 can increase or decrease channel activity, or induce a reduction in the amount of protein. The consequences of a single mutation are sometimes multiple and the analysis of the functional effects is complex.Objective:The consequences of the p.Y4864H mutation identified in a CCD patient have been studied regarding both RyR1 function and amount.Methods:The amount of RyR1 in human and mouse muscles was evaluated using qRT-PCR and quantitative Western blot, and calcium release was studied using calcium imaging on primary cultures. The results were compared between human and mouse.Results:The p.Y4864H mutation induced an alteration of calcium release, and in addition was associated to a reduction in the amount of RyR1 in the patient’s muscle. This suggests two possible pathophysiological mechanisms: the alteration of calcium release could result from a modification of the channel properties of RyR1 or from a RyR1 reduction. In order to discriminate between the two hypotheses, we used the heterozygous RyR1 knockout (RyR1+/–) mouse model showing a comparable RyR1 protein reduction. No reduction in calcium release was observed in primary muscle culture from these mice, and no muscle weakness was measured.Conclusions:Because the reduction in the amount of RyR1 protein has no functional consequences in the murine model, the muscle weakness observed in the patient is most likely the result of a modification of the calcium channel function of RyR1 due to the p.Y4864H mutation.

Background SERCA2a gene transfer (GT) improves mechano‐electrical function in animal models of nonischemic heart failure Whether SERCA2a GT reverses pre‐established remodeling at an advanced stage of ischemic heart failure is unclear. We sought to uncover the electrophysiological effects of adeno‐associated virus serotype 1.SERCA2a GT following myocardial infarction (MI).Methods and ResultsPigs developed mechanical dysfunction 1 month after anterior MI, at which point they received intracoronary adeno‐associated virus serotype 1.SERCA2a (MI+SERCA2a) or saline (MI) and were maintained for 2 months. Age‐matched naive pigs served as controls (Control). In vivo ECG‐and‐hemodynamic properties were assessed before and after dobutamine stress. The electrophysiological substrate was measured using optical action potential (AP) mapping in controls, MI, and MI+SERCA2a preparations. In vivo ECG measurements revealed comparable QT durations between groups. In contrast, prolonged QRS duration and increased frequency of R′ waves were present in MI but not MI+SERCA2a pigs relative to controls. SERCA2a GT reduced in in vivo arrhythmias in response to dobutamine. Ex vivo preparations from MI but not MI+SERCA2a or control pigs were prone to pacing‐induced ventricular tachycardia and fibrillation. Underlying these arrhythmias was pronounced conduction velocity slowing in MI versus MI+SERCA2a at elevated rates leading to ventricular tachycardia and fibrillation. Reduced susceptibility to ventricular tachycardia and fibrillation in MI+SERCA2a pigs was not related to hemodynamic function, contractile reserve, fibrosis, or the expression of Cx43 and Nav1.5. Rather, SERCA2a GT decreased phosphoactive CAMKII‐delta levels by >50%, leading to improved excitability at fast rates.Conclusions SERCA2a GT increases conduction velocity reserve, likely by preventing CAMKII overactivation. Our findings suggest a primary effect of SERCA2a GT on myocardial excitability, independent of altered mechanical function.

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Background: STIM1 (stromal interaction molecule 1) is a calcium (Ca 2+ ) sensor that regulates cardiac hypertrophy by triggering store-operated Ca 2+ entry. Because STIM1 binding to phospholamban increases sarcoplasmic reticulum Ca 2+ load independent of store-operated Ca 2+ entry, we hypothesized that it controls electrophysiological function and arrhythmias in the adult heart. Methods: Inducible myocyte-restricted STIM1-KD (STIM1 knockdown) was achieved in adult mice using an αMHC (α-myosin heavy chain)-MerCreMer system. Mechanical and electrophysiological properties were examined using echocardiography in vivo and optical action potential (AP) mapping ex vivo in tamoxifen-induced STIM1 flox/flox -Cre tg /− (STIM1-KD) and littermate controls for STIM1 flox/flox (referred to as STIM1-Ctl) and for Cre tg/− without STIM deletion (referred to as Cre-Ctl). Results: STIM1-KD mice (N=23) exhibited poor survival compared with STIM1-Ctl (N=22) and Cre-Ctl (N=11) with >50% mortality after only 8-days of cardiomyocyte-restricted STIM1-KD. STIM1-KD but not STIM1-Ctl or Cre-Ctl hearts exhibited a proclivity for arrhythmic behavior, ranging from frequent ectopy to pacing-induced ventricular tachycardia/ventricular fibrillation (VT/VF). Examination of the electrophysiological substrate revealed decreased conduction velocity and increased AP duration (APD) heterogeneity in STIM1-KD. These features, however, were comparable in VT/VF(+) and VT/VF(−) hearts. We also uncovered a marked increase in the magnitude of APD alternans during rapid pacing, and the emergence of a spatially discordant alternans profile in STIM1-KD hearts. Unlike conduction velocity slowing and APD heterogeneity, the magnitude of APD alternans was greater (by 80%, P <0.05) in VT/VF(+) versus VT/VF(−) STIM1-KD hearts. Detailed phase mapping during the initial beats of VT/VF identified one or more rotors that were localized along the nodal line separating out-of-phase alternans regions. Conclusions: In an adult murine model with inducible and myocyte-specific STIM1 depletion, we demonstrate for the first time the regulation of spatially discordant alternans by STIM1. Early mortality in STIM1-KD mice is likely related to enhanced susceptibility to VT/VF secondary to discordant APD alternans.

The triadin isoforms Trisk 95 and Trisk 51 are both components of the skeletal muscle calcium release complex. To investigate the specific role of Trisk 95 and Trisk 51 isoforms in muscle physiology, we overexpressed Trisk 95 or Trisk 51 using adenovirus-mediated gene transfer in skeletal muscle of newborn mice. Overexpression of either Trisk 95 or Trisk 51 alters the muscle fiber morphology, while leaving unchanged the expression of the ryanodine receptor, the dihydropyridine receptor, and calsequestrin. We also observe an aberrant expression of caveolin 3 in both Trisk 95- and Trisk 51-overexpressing skeletal muscles. Using a biochemical approach, we demonstrate that caveolin 3 is associated with the calcium release complex in skeletal muscle. Taking advantage of muscle and non-muscle cell culture models and triadin null mouse skeletal muscle, we further dissect the molecular organization of the caveolin 3-containing calcium release complex. Our data demonstrate that the association of caveolin 3 with the calcium release complex occurs via a direct interaction with the transmembrane domain of the ryanodine receptor. Taken together, these data suggest that caveolin 3-containing membrane domains and the calcium release complex are functionally linked and that Trisk 95 and Trisk 51 are instrumental to the regulation of this interaction, the integrity of which may be crucial for muscle physiology.