Background-TGF-β 1 (transforming growth factor-β 1 ) importantly contributes to cardiac fibrosis by controlling differentiation, migration, and collagen secretion of cardiac myofibroblasts. It is still elusive, however, to which extent TGF-β 1 alters the electrophysiological phenotype of myofibroblasts and cardiomyocytes and whether it affects proarrhythmic myofibroblast-cardiomyocyte crosstalk observed in vitro. Methods and Results-Patch-clamp recordings of cultured neonatal rat ventricular myofibroblasts revealed that TGF-β 1 , applied for 24 to 48 hours at clinically relevant concentrations (≤2.5 ng/mL), causes substantial membrane depolarization concomitant with a several-fold increase of transmembrane currents. Transcriptome analysis revealed TGF-β 1 -dependent changes in 29 of 63 ion channel/pump/connexin transcripts, indicating a pleiotropic effect on the electrical phenotype of myofibroblasts. Whereas not affecting cardiomyocyte membrane potentials and cardiomyocyte-cardiomyocyte gap junctional coupling, TGF-β 1 depolarized cardiomyocytes coupled to myofibroblasts by ≈20 mV and increased gap junctional coupling between myofibroblasts and cardiomyocytes >5-fold as reflected by elevated connexin 43 and consortin transcripts. TGF-β 1 -dependent cardiomyocyte depolarization resulted from electrotonic crosstalk with myofibroblasts as demonstrated by immediate normalization of cardiomyocyte electrophysiology after targeted disruption of coupled myofibroblasts and by cessation of ectopic activity of cardiomyocytes coupled to myofibroblasts during pharmacological gap junctional uncoupling. In cardiac fibrosis models exhibiting slow conduction and ectopic activity, block of TGF-β 1 signaling completely abolished both arrhythmogenic conditions. Conclusions-TGF-β 1 profoundly alters the electrophysiological phenotype of cardiac myofibroblasts. Apart from possibly contributing to the control of cell function in general, the changes proved to be pivotal for proarrhythmic myofibroblastcardiomyocyte crosstalk in vitro, which suggests that TGF-β 1 may play a potentially important role in arrhythmogenesis of the fibrotic heart. (Circ Arrhythm Electrophysiol. 2017;10:e004567.

Rationale: Catecholaminergic polymorphic ventricular tachycardia is an inherited disease that predisposes to cardiac arrest and sudden death. The disease is associated with mutations in the genes encoding for the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2). CASQ2 mutations lead to a major loss of CASQ2 monomers, possibly because of enhanced degradation of the mutant protein. The decrease of CASQ2 is associated with a reduction in the levels of Triadin (TrD) and Junctin (JnC), two proteins that form, with CASQ2 and RyR2, a macromolecular complex devoted to control of calcium release from the sarcoplasmic reticulum.Objective: We intended to evaluate whether viral gene transfer of wild-type CASQ2 may rescue the broad spectrum of abnormalities caused by mutant CASQ2. Methods and Results:We used an adeno-associated serotype 9 viral vector to express a green fluorescent protein-tagged CASQ2 construct. Twenty weeks after intraperitoneal injection of the vector in neonate CASQ2 KO mice, we observed normalization of the levels of calsequestrin, triadin, and junctin, rescue of electrophysiological and ultrastructural abnormalities caused by CASQ2 ablation, and lack of life-threatening arrhythmias. Conclusions:

Non-excitable cells (NECs) such as cardiac myofibroblasts that are electrotonically coupled to cardiomyocytes affect conduction velocity (θ) by representing a capacitive load (CL: increased membrane to be charged) and a resistive load (RL: partial depolarization of coupled cardiomyocytes). In this study, we untangled the relative contributions of both loading modalities to NEC-dependent arrhythmogenic conduction slowing. Discrimination between CL and RL was achieved by reversibly removing the RL component by light activation of the halorhodopsin-based hyperpolarizing membrane voltage actuator eNpHR3.0-eYFP (enhanced yellow fluorescent protein) expressed in communication-competent fibroblast-like NIH3T3 cells (3T3 HR cells) that served as a model of coupled NECs. Experiments were conducted with strands of neonatal rat ventricular cardiomyocytes coated at increasing densities with 3T3 HR cells. Impulse conduction along preparations stimulated at 2.5 Hz was assessed with multielectrode arrays. The relative density of 3T3 HR cells was determined by dividing the area showing eYFP fluorescence by the area covered with cardiomyocytes [coverage factor (CF)]. Compared to cardiomyocytes, 3T3 HR cells exhibited a depolarized membrane potential (−34 mV) that was shifted to −104 mV during activation of halorhodopsin. Without illumination, 3T3 HR cells slowed θ along the preparations from ∼330 mm/s (control cardiomyocyte strands) to ∼100 mm/s (CF = ∼0.6). Illumination of the preparation increased the electrogram amplitudes and induced partial recovery of θ at CF > 0.3. Computer simulations demonstrated that the θ deficit observed during illumination was attributable in full to the CL represented by coupled 3T3 HR cells with θ showing a power-law relationship to capacitance with an exponent of −0.78 (simulations) and −0.99 (experiments). The relative contribution of CL and RL to conduction slowing changed as a function of CF with CL dominating at CF ≤ ∼0.3, both mechanisms being equally important at CF = ∼0.5, and RL dominating over CL at CF > 0.5.