Upregulation of utrophin improves the phenotype of Duchenne muscular dystrophy hiPSC-derived CMs

Abstract

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease. Although it leads to muscle weakness, affected individuals predominantly die from cardiomyopathy, which remains uncurable. Accumulating evidence suggests that an overexpression of utrophin may counteract some of the pathophysiological outcomes of DMD. The aim of this study was to investigate the role of utrophin in dystrophin-deficient human cardiomyocytes and to test whether an overexpression of utrophin, implemented via the CRISPR/deadCas9-VP64 system, can improve their phenotype. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) lacking either dystrophin (DMD) or both dystrophin and utrophin (DMD KO/UTRN(+/-)). We carried out proteome analysis, which revealed considerable differences in the proteins related to muscle contraction, cell–cell adhesion and extracellular matrix organisation. Furthermore, we evaluated the role of utrophin in maintaining the physiological properties of DMD hiPSC-CM using atomic force microscopy, patch-clamp and Ca2+ oscillation analysis. Our results showed higher values of afterhyperpolarisation and altered patterns of cytosolic Ca2+ oscillations in DMD; the latter was further disturbed in DMD KO/UTRN(+/-) hiPSC-CM. Utrophin upregulation improved both parameters. Our findings demonstrate for the first time that utrophin maintains the physiological functions of DMD hiPSC-CM, and that its upregulation can compensate for the loss of dystrophin.