Purpose: The technologydriven,rapid lifestyle changes over the past century exceed the evolutionary and biological adaptability of the human heart, which renders it susceptible to degenerative diseases. Loss of ventricular cardiomyocytes (CMCs) is inadequately compensated by electromechanically incompetent fibroblasts. This maladaptive and proarrhythmic cellular response raised our interest in creating a new, cardiomyocytelike cell type by forced fusion between fibroblasts and surrounding fibroblasts. We hypothesized such forced heterocellular fusion (FHF) transfers desirable electromechanical properties to fibroblasts and thereby ameliorates fibrosisassociated proarrhythmic effects on cardiac tissue. Methods: Human ventricular scar cells (hVSCs) were isolated from myocardial scars of heart failure patients and cocultured (1:4) with neonatal rat CMCs (nrCMCs) into confluent monolayers. Prior to coculture, hVSCs were transduced with lentiviral vectors encoding the enhanced green fluorescent protein (eGFP, control cultures) or eGFP and the fusogenic vesicular stomatitis virus G protein (fused cultures). The structural and functional effects of FHF were investigated by (humanspecific) immunocytological staining, patchclamp and optical mapping. Results: hVSCnrCMC heterokaryons were only observed in fused (VSVG expressing) cultures. Such heterokaryons contained 6±3 nuclei (46±18% of human origin). These new, excitable and contractile cells expressed αactinin and Cx43. Nuclear expression of NKX2.5 was absent in control hVSCs, while hVSC nuclei in heterokaryons stained positive. Expression levels of Cav1.2 and Cx43 did not relate to the percentage of human nuclei (R2=0.05), suggesting phenotypical dominance of CMCs. Additionally, FHF strongly reduced action potential duration (APD80, 313.1±6.3ms vs. 510.7±11.8ms, p<0.05 and dispersion of repolarization (75.1±4.3ms vs. 125.9±9.4ms, p<0.05). Importantly, early afterdepolarizations (EADs) rarely occurred in fused cultures (4.6% [n=65] vs. 43.4% [n=60], p<0.0001). Mechanistically, this enhanced repolarization force was due to an increased outward Kv current, as partial inhibition by tetraethylammonium chloride (TEA) reverted the antiarrhythmic effects of fusion towards control values (7.9% to 34.2% EADs (p<0.001).Conclusions: FHF between nrCMCs and hVSCs represents a novel approach to counteract proarrhythmogeneity of hVSCs by forcing a CMClike phenotype that increases repolarization reserve. These results provide proofofconcept for a previously unexplored therapeutic potential of heterocellular fusion.

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