Dystrophin Dp71 Subisoforms Localize to the Mitochondria of Human Cells

Abstract

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by deficiency in dystrophin, a protein product encoded by the DMD gene. Mitochondrial dysfunction is now attracting much attention as a central player in DMD pathology. However, dystrophin has never been explored in human mitochondria. Here, we analyzed dystrophin in cDNAs and mitochondrial fractions of human cells. Mitochondrial fraction was obtained using a magnetic-associated cell sorting (MACS) technology. Dystrophin was analyzed by reverse transcription (RT)-PCR and western blotting using an antibody against the dystrophin C-terminal. In isolated mitochondrial fraction from HEK293 cells, dystrophin was revealed as a band corresponding to Dp71b and Dp71ab subisoforms. Additionally, in mitochondria from HeLa, SH-SY5Y, CCL-136 and HepG2 cells, signals for Dp71b and Dp71ab were revealed as well. Concomitantly, dystrophin mRNAs encoding Dp71b and Dp71ab were disclosed by RT-PCR in these cells. Primary cultured myocytes from three dystrophinopathy patients showed various levels of mitochondrial Dp71 expression. Coherently, levels of mRNA were different in all cells reflecting the protein content, which indicated predominant accumulation of Dp71. Dystrophin was demonstrated to be localized to human mitochondrial fraction, specifically as Dp71 subisoforms. Myocytes derived from dystrophinopathy patients manifested different levels of mitochondrial Dp71, with higher expression revealed in myocytes from Becker muscular dystrophy (BMD) patient-derived myocytes.