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.
Highlights
Duchenne muscular dystrophy (DMD) (OMIM 310200) is one of the most common inherited muscular diseases, affecting more than one in every 3500 live-born male children and characterized by progressive muscle wasting, succumbing at the second to fourth decades of life [1]
From the result, using an antibody against the C-terminal region, dystrophin Dp71 could be visualized in the MT fraction as double bands
We described here the identification of Dp71 subisoforms in MT fractions from human cancer cells
Summary
Duchenne muscular dystrophy (DMD) (OMIM 310200) is one of the most common inherited muscular diseases, affecting more than one in every 3500 live-born male children and characterized by progressive muscle wasting, succumbing at the second to fourth decades of life [1]. DMD is caused by dystrophin deficiency due to mutations in the DMD gene [2]. The gene spans more than 2.4 Mb on chromosome X and encodes a 14-kb transcript comprising 79 exons [3]. 7 alternative promoters/first exons that produce promoter- or tissue-specific dystrophin isoforms [3,4]. Another development-specific promoter/first exon was identified to produce a full-length transcript, Dystrophin protein (Dp) 412e, giving further complexity in transcript [5]. Dystrophin, Dp427m (muscle isoform), is localized beneath the skeletal muscle membrane and links the extracellular protein with intracellular actin by forming the dystrophin-dystroglycan complex [8,9]
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