Human iPSC-Based Model Reveals NOX4 as Therapeutic Target in Duchenne Cardiomyopathy

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder, caused by mutations in the Dystrophin gene. Cardiomyopathy is one of the major causes of early death. In this study, we used DMD patient-specific induced pluripotent stem cells (iPSCs) to model cardiomyopathic features in DMD and unravel novel pathological mechanistic insights. Cardiomyocytes (CMs) differentiated from DMD iPSCs showed enhanced premature cell death, due to significantly elevated intracellular reactive oxygen species (ROS) concentrations, as a result of depolarized mitochondria and high NADPH oxidase 4 (NOX4) protein levels. Genetic correction of Dystrophin through CRISPR/Cas9 editing restored normal ROS levels. Application of ROS reduction by N-acetyl-L-cysteine (NAC), partial Dystrophin re-expression by ataluren (PTC124) and enhancing mitochondrial electron transport chain function by idebenone improved cell survival of DMD iPSC-CMs. We show applications that could counteract the detrimental oxidative stress environment in DMD iPSC-CMs by stimulating adenosine triphosphate (ATP) production. ATP could bind to the ATP-binding domain in the NOX4 enzyme, and we demonstrate that ATP resulted in partial inhibition of the NADPH-dependent ROS production of NOX4. Considering the complexity and the early cellular stress responses in DMD cardiomyopathy, we propose to target ROS production and prevent the detrimental effects of NOX4 on DMD CMs as a promising therapeutic strategy.

Long-Term Culture of Patient-Derived Cardiac Organoids Recapitulated Duchenne Muscular Dystrophy Cardiomyopathy and Disease Progression

“…Duchenne Muscular Dystrophy iPSC (DMD-hiPSC) was obtained from DMD patient’s fibroblasts carrying a point mutation in exon 35 (c.4 996C>T; p.Arg1,666X) of the Dystrophin gene that leads to a premature stop codon (Duelen et al, 2021). Human DMD isogenic control (DMD-Iso iPSC) was generated through CRISPR/Cas9 gene editing from the S. pyogenes system (5’-NGG PAM) as previously described (Ran et al, 2013; Duelen et al, 2021).…”

“…Duchenne Muscular Dystrophy iPSC (DMD-hiPSC) was obtained from DMD patient’s fibroblasts carrying a point mutation in exon 35 (c.4 996C>T; p.Arg1,666X) of the Dystrophin gene that leads to a premature stop codon (Duelen et al, 2021). Human DMD isogenic control (DMD-Iso iPSC) was generated through CRISPR/Cas9 gene editing from the S. pyogenes system (5’-NGG PAM) as previously described (Ran et al, 2013; Duelen et al, 2021). Human iPSC lines were cultured feeder-free on Geltrex LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix and maintained in Essential 8 Flex Basal Medium (Thermo Fisher Scientific) supplemented with Essential 8 Flex Supplement (50x, Thermo Fisher Scientific) and penicillin–streptomycin (0.1%, Thermo Fisher Scientific), at 37 °C under normoxic conditions (21% O 2 and 5% CO 2 ).…”