Arrhythmia Mechanism and Dynamics in a Humanized Mouse Model of Inherited Cardiomyopathy Caused by Phospholamban R14del Mutation.

BACKGROUND: Arginine (Arg) 14 deletion (R14del) in the calcium regulatory protein phospholamban (hPLNR14del) has been identified as a disease-causing mutation in patients with an inherited cardiomyopathy. Mechanisms underlying the early arrhythmogenic phenotype that predisposes carriers of this mutation to sudden death with no apparent structural remodeling remain unclear. METHODS: To address this, we performed high spatiotemporal resolution optical mapping of intact hearts from adult knock-in mice harboring the human PLNWT (wildtype [WT], n=12) or the heterozygous human PLNR14del mutation (R14del, n=12) before and after ex vivo challenge with isoproterenol and rapid pacing. RESULTS: Adverse electrophysiological remodeling was evident in the absence of significant structural or hemodynamic changes. R14del hearts exhibited increased arrhythmia susceptibility compared with wildtype. Underlying this susceptibility was preferential right ventricular action potential prolongation that was unresponsive to ß-adrenergic stimulation. A steep repolarization gradient at the left ventricular/right ventricular interface provided the substrate for interventricular activation delays and ultimately local conduction block during rapid pacing. This was followed by the initiation of macroreentrant circuits supporting the onset of ventricular tachycardia. Once sustained, these circuits evolved into high-frequency rotors, which in their majority were pinned to the right ventricle. These rotors exhibited unique spatiotemporal dynamics that promoted their increased stability in R14del compared with wildtype hearts. CONCLUSIONS: Our findings highlight the crucial role of primary electric remodeling caused by the hPLNR14del mutation. These inherently arrhythmogenic features form the substrate for adrenergic-mediated VT at early stages of PLNR14del induced cardiomyopathy.

Interplay between Triadin and Calsequestrin in the Pathogenesis of CPVT in the Mouse.

Recessive forms of catecholaminergic polymorphic ventricular tachycardia (CPVT) are induced by mutations in genes encoding triadin or calsequestrin, two proteins that belong to the Ca2+ release complex, responsible for intracellular Ca2+ release triggering cardiac contractions. To better understand the mechanisms of triadin-induced CPVT and to assay multiple therapeutic interventions, we used a triadin knockout mouse model presenting a CPVT-like phenotype associated with a decrease in calsequestrin protein level. We assessed different approaches to rescue protein expression and to correct intracellular Ca2+ release and cardiac function: pharmacological treatment with kifunensine or a viral gene transfer-based approach, using adeno-associated virus serotype 2/9 (AAV2/9) encoding the triadin or calsequestrin. We observed that the levels of triadin and calsequestrin are intimately linked, and that reduction of both proteins contributes to the CPVT phenotype. Different combinations of triadin and calsequestrin expression level were obtained using these therapeutic approaches. A full expression of each is not necessary to correct the phenotype; a fine-tuning of the relative re-expression of both triadin and calsequestrin is required to correct the CPVT phenotype and rescue the cardiac function. AAV-mediated gene delivery of calsequestrin or triadin and treatment with kifunensine are potential treatments for recessive forms of CPVT due to triadin mutations.

Intra-tracheal gene delivery of aerosolized SERCA2a to the lung suppresses ventricular arrhythmias in a model of pulmonary arterial hypertension.

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. RESULTS: Over 80% of MCT but none of the CTRL hearts were prone to sustained VT by rapid pacing (P < .01). Aerosolized gene delivery of AAV1.S2a to the lung suppressed the incidence of VT to <15% (P < .05). Investigation of the EP substrate revealed marked prolongation of AP duration (APD), increased APD heterogeneity, a reversal in the trans-epicardial APD gradient, and marked conduction slowing in untreated MCT compared to CTRL hearts. These myocardial EP changes coincided with major remodeling in the expression of K and Ca channel subunits, decreased expression of Cx43 and increased expression of pro-fibrotic and pro-hypertrophic markers. Intra-tracheal gene delivery of aerosolized AAV1 carrying S2a but not luciferase resulted in selective upregulation of the human isoform of SERCA2a in the lung but not the heart. This pulmonary intervention, in turn, ameliorated MCT-induced APD prolongation, reversed spatial APD heterogeneity, normalized myocardial conduction, and suppressed the incidence of pacing-induced VT. Comparison of the minimal conduction velocity (CV) generated at the fastest pacing rate before onset of VT or at the end of the protocol revealed significantly lower values in untreated compared to AAV1.S2a treated PAH and CTRL hearts. Reversal of EP remodeling by pulmonary AAV1.S2a gene delivery was accompanied by restored expression of key ion channel transcripts. Restored expression of Cx43 and collagen but not the pore-forming Na channel subunit Nav1.5 likely ameliorated VT by improving CV at rapid rates in PAH. CONCLUSION: Aerosolized AAV1.S2a gene delivery selectively to the lungs ameliorates myocardial EP remodeling and VT susceptibility at rapid heart rates. Our findings highlight for the first time the utility of a non-cardiac gene therapy approach for arrhythmia suppression.

Cardiac Stim1 Silencing Impairs Adaptive Hypertrophy and Promotes Heart Failure Through Inactivation of mTORC2/Akt Signaling.

BACKGROUND: Stromal interaction molecule 1 (STIM1) is a dynamic calcium signal transducer implicated in hypertrophic growth of cardiomyocytes. 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 cardiomyocytes by using in vivo gene delivery of specific short hairpin RNAs. In 3 different models, we found that Stim1 silencing prevents the development of pressure overload-induced hypertrophy but also reverses preestablished 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 antihypertrophic and proapoptotic GSK-3ß molecule. Pharmacological inhibition of glycogen synthase kinase-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 Akt(S473), a hydrophobic motif of Akt that is directly phosphorylated by mTOR complex 2. We found that Stim1 silencing directly impaired mTOR complex 2 kinase activity, which was supported by a direct interaction between STIM1 and Rictor, a specific component of mTOR complex 2. CONCLUSIONS: These data support a model whereby STIM1 is critical to deactivate a key negative regulator of cardiac hypertrophy. In cardiomyocytes, STIM1 acts by tuning Akt kinase activity through activation of mTOR complex 2, which further results in repression of GSK-3ß activity.

Absence of triadin, a protein of the calcium release complex, is responsible for cardiac arrhythmia with sudden death in human.

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.

Quantitative Assessment of Mitochondrial Morphology and Electrophysiological Function in the Diabetic Heart.

Mitochondria within a cardiomyocyte form a highly dynamic network that undergoes fusion and fission events in response to acute and chronic stressors, such as hyperglycemia and diabetes mellitus. Changes in mitochondrial architecture and morphology not only reflect their capacity for oxidative phosphorylation and ATP synthesis but also impact their subcellular localization and interaction with other organelles. The role of these ultrastructural abnormalities in modulating electrophysiological properties and excitation-contraction coupling remains largely unknown and warrants direct investigation considering the growing appreciation of the functional and structural coupling between the mitochondrial network, the calcium cycling machinery, and sarcolemmal ion channels in the cardiac myocyte. In this Methods in Molecular Biology chapter, we provide a protocol that allows for a quantitative assessment of mitochondrial shape and morphology in control and diabetic hearts that had undergone detailed electrophysiological measurements using high resolution optical action potential (AP) mapping.

Role of AMPK in Atrial Metabolic Homeostasis and Substrate Preference.

Atrial fibrillation is the most common clinical arrhythmia and may be due in part to metabolic stress. Atrial specific deletion of the master metabolic sensor, AMP-activated protein kinase (AMPK), induces atrial remodeling culminating in atrial fibrillation in mice, implicating AMPK signaling in the maintenance of atrial electrical and structural homeostasis. However, atrial substrate preference for mitochondrial oxidation and the role of AMPK in regulating atrial metabolism are unknown. Here, using LC-MS/MS methodology combined with infusions of [ 13 C 6 ]glucose and [ 13 C 4 ]ß-hydroxybutyrate in conscious mice, we demonstrate that conditional deletion of atrial AMPK catalytic subunits shifts mitochondrial atrial metabolism away from fatty acid oxidation and towards pyruvate oxidation. LC-MS/MS-based quantification of acyl-CoAs demonstrated decreased atrial tissue content of long-chain fatty acyl-CoAs. Proteomic analysis revealed a broad downregulation of proteins responsible for fatty acid uptake (LPL, CD36, FABP3), acylation and oxidation. Atrial AMPK deletion reduced expression of atrial PGC1-α and downstream PGC1-α/PPARα/RXR regulated gene transcripts. In contrast, atrial [ 14 C]2-deoxyglucose uptake and GLUT1 expression increased with fasting in mice with AMPK deletion, while the expression of glycolytic enzymes exhibited heterogenous changes. Thus, these results highlight the crucial homeostatic role of AMPK in the atrium, with loss of atrial AMPK leading to downregulation of the PGC1-α/PPARα pathway and broad metabolic reprogramming with a loss of fatty acid oxidation, which may contribute to atrial remodeling and arrhythmia.

Right predominant electrical remodeling in a pure model of pulmonary hypertension promotes reentrant arrhythmias.

BACKGROUND: Electrophysiological (EP) properties have been studied mainly in the monocrotaline model of pulmonary arterial hypertension (PAH). Findings are confounded by major extrapulmonary toxicities, which preclude the ability to draw definitive conclusions regarding the role of PAH per se in EP remodeling. OBJECTIVE: The purpose of this study was to investigate the EP substrate and arrhythmic vulnerability of a new model of PAH that avoids extracardiopulmonary toxicities. METHODS: Sprague-Dawley rats underwent left pneumonectomy (Pn) followed by injection of the vascular endothelial growth factor inhibitor Sugen-5416 (Su/Pn). Five weeks later, cardiac magnetic resonance imaging was performed in vivo, optical action potential (AP) mapping ex vivo, and molecular analyses in vitro. RESULTS: Su/Pn rats exhibited right ventricular (RV) hypertrophy and were highly prone to pacing-induced ventricular tachycardia/fibrillation (VT/VF). Underlying this susceptibility was disproportionate RV-sided prolongation of AP duration, which promoted formation of right-sided AP alternans at physiological rates. While propagation was impaired at all rates in Su/Pn rats, the extent of conduction slowing was most severe immediately before the emergence of interventricular lines of block and onset of VT/VF. Measurement of the cardiac wavelength revealed a decrease in Su/Pn relative to control. Nav1.5 and total connexin 43 expression was not altered, while connexin 43 phosphorylation was decreased in PAH. Col1a1 and Col3a1 transcripts were upregulated coinciding with myocardial fibrosis. Once generated, VT/VF was sustained by multiple reentrant circuits with a lower frequency of RV activation due to wavebreak formation. CONCLUSION: In this pure model of PAH, we document RV-predominant remodeling that promotes multiwavelet reentry underlying VT. The Su/Pn model represents a severe form of PAH that allows the study of EP properties without the confounding influence of extrapulmonary toxicity.

Atrial AMP-activated protein kinase is critical for prevention of dysregulation of electrical excitability and atrial fibrillation.

Metabolic stress is an important cause of pathological atrial remodeling and atrial fibrillation. AMPK is a ubiquitous master metabolic regulator, yet its biological function in the atria is poorly understood in both health and disease. We investigated the impact of atrium-selective cardiac AMPK deletion on electrophysiological and structural remodeling in mice. Loss of atrial AMPK expression caused atrial changes in electrophysiological properties and atrial ectopic activity prior to the onset of spontaneous atrial fibrillation. Concomitant transcriptional downregulation of connexins and atrial ion channel subunits manifested with delayed left atrial activation and repolarization. The early molecular and electrophysiological abnormalities preceded left atrial structural remodeling and interstitial fibrosis. AMPK inactivation induced downregulation of transcription factors (Mef2c and Pitx2c) linked to connexin and ion channel transcriptional reprogramming. Thus, AMPK plays an essential homeostatic role in atria, protecting against adverse remodeling potentially by regulating key transcription factors that control the expression of atrial ion channels and gap junction proteins.

Caveolin 3 is associated with the calcium release complex and is modified via in vivo triadin modification.

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.

Cardiomyocyte-Specific STIM1 (Stromal Interaction Molecule 1) Depletion in the Adult Heart Promotes the Development of Arrhythmogenic Discordant Alternans.

BACKGROUND: STIM1 (stromal interaction molecule 1) is a calcium (Ca2+) sensor that regulates cardiac hypertrophy by triggering store-operated Ca2+ entry. Because STIM1 binding to phospholamban increases sarcoplasmic reticulum Ca2+ load independent of store-operated Ca2+ 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 STIM1flox/flox-Cretg/- (STIM1-KD) and littermate controls for STIM1flox/flox (referred to as STIM1-Ctl) and for Cretg/- 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.

Primary Effect of SERCA 2a Gene Transfer on Conduction Reserve in Chronic Myocardial Infarction.

Background SERCA 2a gene transfer ( GT ) improves mechano-electrical function in animal models of nonischemic heart failure Whether SERCA 2a 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. SERCA 2a GT following myocardial infarction ( MI ). Methods and Results Pigs developed mechanical dysfunction 1 month after anterior MI , at which point they received intracoronary adeno-associated virus serotype 1. SERCA 2a ( MI + SERCA 2a) 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 + SERCA 2a 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 + SERCA 2a pigs relative to controls. SERCA 2a GT reduced in in vivo arrhythmias in response to dobutamine. Ex vivo preparations from MI but not MI + SERCA 2a or control pigs were prone to pacing-induced ventricular tachycardia and fibrillation. Underlying these arrhythmias was pronounced conduction velocity slowing in MI versus MI + SERCA 2a at elevated rates leading to ventricular tachycardia and fibrillation. Reduced susceptibility to ventricular tachycardia and fibrillation in MI + SERCA 2a pigs was not related to hemodynamic function, contractile reserve, fibrosis, or the expression of Cx43 and Nav1.5. Rather, SERCA 2a GT decreased phosphoactive CAMKII -delta levels by >50%, leading to improved excitability at fast rates. Conclusions SERCA 2a GT increases conduction velocity reserve, likely by preventing CAMKII overactivation. Our findings suggest a primary effect of SERCA 2a GT on myocardial excitability, independent of altered mechanical function.

Functional Characterization of a Central Core Disease RyR1 Mutation (p.Y4864H) Associated with Quantitative Defect in RyR1 Protein.

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.