Investigation of genotype-phenotype correlation in 2D cell models of Duchenne muscular dystrophy patients shows mutation-dependent defects in mitochondrial respiration and delay in myogenic program

Duchenne Muscular Dystrophy is a genetic X-linked neuromuscular disorder with no cure. It is due to heterogeneous mutations in dystrophin gene, that commonly lead to the absence of the protein and to a complex cascade of events. Whether the heterogeneity of the mutations could contribute to different clinical phenotype is still an open point. Phenotype-genotype correlations have never been investigated in patient-derived muscle precursors, that represent a simplified platform to investigate personalized therapies. We used two dystrophic immortalised satellite cell lines (Myoline), HDMD1 with a stop codon mutation at exon 59 and HDMD2 with deletion of 48-52 exon. We also used a third immortalised cell line from a healthy control (Hwt). Our results showed a remarkable difference in the Oxygen Consumption Rate (OCR) between HDMD2 and HDMD1 cells, assessed via mitostress assays. We observed a significant reduction of all OCR parameters (basal respiration, ATP turnover/oligomycin-sensitive respiration, maximal respiration) in HDMD1 during differentiation at 48, 96 hours and 17 days, compared to HDMD2 and HWT, suggesting that the respiratory defect is exacerbated by the differentiation process in a mutation-related manner. An electrophysiological investigation by means of automated patch clamp (Patchliner-Nanion) showed a marked and general reduction of membrane capacitance in mutant myocytes until 6 days of differentiation and a slight delay in maturation of inward and outward currents. Molecular biology experiments confirmed mutation-specific delay in myogenic cascade, and in key players of mitochondrial function and of ion channels, with HDMD1 showing the less differentiated phenotype. This characterization shed light into genotype-phenotype correlation at cellular level, helping to identify molecular mechanisms underlying patients-specific mutations and new druggable targets.