Gene-corrected human iPSC-derived cardiomyocytes and skeletal muscles reveal partial dystrophin Dp427 preservation and cardiac Dp116 expression in Duchenne muscular dystrophy

Abstract

BackgroundDuchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disease caused by mutations in the DMD gene, leading to the absence or dysfunction of dystrophin. While cardiac and skeletal muscles are both affected, tissue-specific differences in disease manifestation and dystrophin regulation remain poorly understood. MethodsTo investigate these differences, we established a human induced pluripotent stem cell (hiPSC) model of DMD from peripheral blood mononuclear cells (PBMC) of a patient carrying a splice-site mutation in intron 68 (c.9975-1G>T). An isogenic control line was generated via CRISPR/Cas9 correction. Both repaired and DMD hiPSCs were differentiated into cardiomyocytes (hiPSC-CMs) and skeletal muscle cells (hiPSC-SMs). Transcript and protein analyses were performed, along with functional assessment using microelectrode array. ResultsTranscript analysis revealed an in-frame deletion of two amino acids (Tyr3325 and Arg3326) due to skipping of the first six nucleotides of exon 69. Despite this, near full-length Dp427 was detected by western blot, along with expression of Dp116 in hiPSC-CMs. Dystrophin levels were preserved in DMD hiPSC-CMs but markedly reduced in hiPSC-SMs, suggesting tissue-specific regulation. Functional analysis showed altered {beta}-adrenergic responsiveness in DMD hiPSC-CMs, with increased beating frequency and accelerated repolarization upon isoproterenol stimulation. ConclusionsOur study identifies a splice-site mutation that preserves high level of dystrophin expression in cardiac but reduced in skeletal muscle and reveals Dp116 expression in cardiomyocytes. These findings highlight the importance of tissue context in DMD and demonstrate the power of hiPSC-based systems for dissecting mutation-specific effects.