Abstract 213: Dystrophic iPSC-derived Cardiomyocytes Have Mislocalization of eNOS and Increased Susceptibility to Cell Death which is Reversed by the Nitrate-like Properties of Nicorandil.

Abstract

Background: Cardiomyopathy is a leading cause of death in Duchenne and Becker muscular dystrophy (D/BMD) patients and specific therapies are lacking. Dystrophin deficiency is associated with mislocalization of nitric oxide synthase (NOS) whose dysregulation contributes to the pathogenesis of dystrophic cardiomyopathy. We have modeled dystrophic cardiomyopathy using patient-specific induced pluripotent stem cells (iPSCs) and show that DMD- and BMD- iPSC derived cardiomyocytes have mislocalization of endothelial (eNOS) but not neuronal NOS (nNOS). In this study, we determined whether nicorandil, a drug with nitrate-like properties, protects against stress-induced dystrophic cardiomyocyte injury. Methods/Results: DMD-iPSC (exon 3-6 deletion), BMD-iPSC (exon 45-53 deletion) and non-dystrophic (N)-iPSC lines were differentiated into cardiomyocytes (iCMs) and matured for 35-38 days. Immunofluorescence revealed eNOS co-localized with dystrophin along the sarcolemma in N-iCMs but not in DMD- or BMD-iCMs. eNOS also co-localized with caveolin-1 in all groups. nNOS did not co-localize with dystrophin but with the ryanodine receptor 2 in all groups. Both DMD- and BMD-iCMs have increased cell death as determined by TUNEL + staining when subject to 2 hours of metabolic stress and 4 hours of recovery (DMD: 66±3%, BMD: 29±4% vs. 1.5±0.04%; n=3-6). This was associated with dissipation of the mitochondrial membrane potential as monitored by laser-scanning confocal microscopy (DMD: complete loss, BMD: 441±168 vs. N: 891±183 AU; n=3-5). Nicorandil pretreatment was able to restore mitochondrial membrane potential and decrease cell death. The protective effects of nicorandil were abolished by ODQ, a selective inhibitor of NO-sensitive guanylyl cyclase. Conclusion: Nicorandil protects against stress-induced cell death and preserves mitochondrial function in dystrophic cardiomyocytes through its nitrite-like properties. This suggests a potential therapeutic role of nicorandil in the treatment of dystrophic cardiomyopathy.