Rationale:The cardiac sodium channel Na v 1.5 plays a key role in excitability and conduction. The 3 last residues of Na v 1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin-dystrophin complex. As dystrophin is absent at the intercalated discs, Na v 1.5 could potentially interact with other, yet unknown, proteins at this site.Objective: The aim of this study was to determine whether Na v 1.5 is part of distinct regulatory complexes at lateral membranes and intercalated discs. Methods and Results:Immunostaining experiments demonstrated that Na v 1.5 localizes at lateral membranes of cardiomyocytes with dystrophin and syntrophin. Optical measurements on isolated dystrophin-deficient mdx hearts revealed significantly reduced conduction velocity, accompanied by strong reduction of Na v 1.5 at lateral membranes of mdx cardiomyocytes. Pull-down experiments revealed that the MAGUK protein SAP97 also interacts with the SIV motif of Na v 1.5, an interaction specific for SAP97 as no pull-down could be detected with other cardiac MAGUK proteins (PSD95 or ZO-1). Furthermore, immunostainings showed that Na v 1.5 and SAP97 are both localized at intercalated discs. Silencing of SAP97 expression in HEK293 and rat cardiomyocytes resulted in reduced sodium current (I Na ) measured by patch-clamp. The I Na generated by Na v 1.5 channels lacking the SIV motif was also reduced. Finally, surface expression of Na v 1.5 was decreased in silenced cells, as well as in cells transfected with SIV-truncated channels. Conclusions:These data support a model with at least 2 coexisting pools of Na v 1.5 channels in cardiomyocytes: one targeted at lateral membranes by the syntrophin-dystrophin complex, and one at intercalated discs by SAP97. (Circ Res. 2011;108:294-304.) Key Words: sodium channel Ⅲ Na v 1.5 Ⅲ MAGUK proteins Ⅲ SAP97 Ⅲ dystrophin T he cardiac sodium channel Na v 1.5 initiates the cardiac action potential, thus playing a key role in cardiac excitability and impulse propagation. The physiological importance of this channel is illustrated by numerous cardiac pathologies caused by hundreds of mutations identified in SCN5A, the gene encoding Na v 1.5. 1 The Na v 1.5 channel is composed of one 220-kDa ␣-subunit that constitutes a functional channel, and 30-kDa ␤-subunits. In addition to these accessory ␤-subunits, several proteins have been shown to regulate and interact with Na v 1.5. 1,2 In most cases, the physiological relevance of these interactions is poorly understood, mainly because of a lack of appropriate animal models. Many of the interacting proteins bind to the C terminus of Na v 1.5, where several protein-protein interaction motifs are located. 1,2 We have shown that the ubiquitin-protein ligase Nedd4-2 binds the PY motif of Na v 1.5 and reduces the sodium current (I Na ) in HEK293 cells by promoting its internalization. 3 We have also demonstrated that Na v 1.5 associates with the dystrophin-syntrophin multiprotein complex (DMC) in cardiac cells. 4 In dystrophin-deficient mice (m...

Rationale: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. Objective: To test the hypothesis that cardiac myocyte–targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electric activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell–derived cardiac myocytes. Methods and Results: We first generated mice with cardiac myocyte–restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch clamping. The use of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiac myocytes. Optical cardiograms could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in induced pluripotent stem cell–derived cardiac myocytes. Conclusions: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof of concept for its use in optogenetic sensing of physiological and pathological excitation in mature and immature cardiac myocytes in vitro and in vivo.

Aims A mutation in the phospholamban (PLN) gene, leading to deletion of Arg14 (R14del), has been associated with malignant arrhythmias and ventricular dilation. Identifying pre-symptomatic carriers with vulnerable myocardium is crucial because arrhythmia can result in sudden cardiac death, especially in young adults with PLN-R14del mutation. This study aimed at assessing the efficiency and efficacy of in vivo genome editing, using CRISPR/Cas9 and a cardiotropic adeno-associated virus (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del. Methods and Results Humanized mice were generated expressing human wild-type (hPLN-WT) or mutant (hPLN-R14del) PLN in the heterozygous state, mimicking human carriers. Cardiac magnetic resonance imaging at 12 weeks of age showed bi-ventricular dilation and increased stroke volume in mutant vs. WT mice, with no deficit in ejection fraction or cardiac output. Challenge of ex vivo hearts with isoproterenol and rapid pacing unmasked higher propensity for sustained ventricular tachycardia (VT) in hPLN-R14del relative to hPLN-WT. Specifically, the VT threshold was significantly reduced (20.3±1.2 Hz in hPLN-R14del vs. 25.7±1.3 Hz in WT, p < 0.01) reflecting higher arrhythmia burden. To inactivate the R14del allele, mice were tail-vein-injected with AAV9.CRISPR/Cas9/gRNA or AAV9 empty capsid (controls). CRISPR-Cas9 efficiency was evaluated by droplet digital PCR and NGS-based amplicon sequencing. In vivo gene editing significantly reduced end diastolic and stroke volumes in hPLN-R14del CRISPR-treated mice compared to controls. Susceptibility to VT was also reduced, as the VT threshold was significantly increased relative to controls (30.9±2.3 Hz vs. 21.3±1.5 Hz; p < 0.01). Conclusions This study is the first to show that disruption of hPLN-R14del allele by AAV9- CRISPR/Cas9 improves cardiac function and reduces VT susceptibility in humanized PLN-R14del mice, offering preclinical evidence for translatable approaches to therapeutically suppress the arrhythmogenic phenotype in human patients with PLN-R14del disease. Translational Perspective The phospholamban R14del mutation causes dilated and arrhythmogenic cardiomyopathies, with increased risk of malignant ventricular arrhythmias in young adult carriers. With few available therapeutic options, heart transplantation is often the ultimate treatment. This study presents the first humanized mouse model of PLN-R14del disease, reveals the ability to detect abnormal cardiac function and increased arrhythmogenic vulnerability in pre-symptomatic hPLN-R14del mice, and demonstrates that allele-specific disruption of R14del using in vivo AAV9/CRISPR-Cas9 reverses the disease phenotype. This preclinical study offers promising translatable approaches to detect and therapeutically suppress the arrhythmogenic phenotype in patients with PLN-R14del disease and potentially other inherited cardiomyopathies.

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.