Abstract
Reactive oxygen species (ROS), generated as a by-product of mitochondrial oxidative phosphorylation, are particularly damaging to the genome of skeletal muscle because of their high oxygen consumption. Proliferating myoblasts play a key role during muscle regeneration by undergoing myogenic differentiation to fuse and restore damaged muscle. This process is severely impaired during aging and in muscular dystrophies. In this study, we investigated the role of oxidatively damaged DNA and its repair in the mitochondrial genome of proliferating skeletal muscle progenitor myoblasts cells and their terminally differentiated product, myotubes. Using the C2C12 cell line as a well-established model for skeletal muscle differentiation, we show that myoblasts are highly sensitive to ROS-mediated DNA damage, particularly in the mitochondrial genome, due to deficiency in 5’ end processing at the DNA strand breaks. Ectopic expression of the mitochondrial-specific 5’ exonuclease, EXOG, a key DNA base excision/single strand break repair (BER/SSBR) enzyme, in myoblasts but not in myotubes, improves the cell’s resistance to oxidative challenge. We linked loss of myoblast viability by activation of apoptosis with deficiency in the repair of the mitochondrial genome. Moreover, the process of myoblast differentiation increases mitochondrial biogenesis and the level of total glutathione. We speculate that our data may provide a mechanistic explanation for depletion of proliferating muscle precursor cells during the development of sarcopenia, and skeletal muscle dystrophies.
Highlights
The mitochondria generate the majority of the cellular energy via oxidative phosphorylation (OXPHOS) but are involved in other important cellular functions including cell death signaling [1,2,3], calcium signaling [4,5] and various biosynthetic pathways [6,7,8]
The DNA base excision repair (BER) is the primary DNA repair pathway that maintains integrity of the nuclear and mitochondrial genomes by repairing damage arising from oxidation, alkylation, deamination, depurination/depirimidination and the repair of abasic (AP) sites and single-strand breaks (SSBs) resulting from spontaneous hydrolysis and oxidation, respectively [18,19,20,21]
We monitored the expression of the transcription factor paired-box 7 (Pax7) and proliferating cell nuclear antigen (PCNA), known to be inhibited during myoblast differentiation or by repression of cellular proliferation, respectively [65,66], as well as expression of myogenin, known to be positively associated with differentiation [67]; these were accomplished by Western analysis of myoblasts at days 0-4 after induction of differentiation
Summary
The mitochondria generate the majority of the cellular energy via oxidative phosphorylation (OXPHOS) but are involved in other important cellular functions including cell death signaling [1,2,3], calcium signaling [4,5] and various biosynthetic pathways [6,7,8]. Repair of a base lesion is initiated with its excision by a DNA glycosylase to generate an AP site which is cleaved by AP-endonuclease 1 (APE1) in mammalian cells, leaving the 3’ OH group and a nonligatable 5’ deoxyribose phosphate (dRP) residue. This 5’ blocking group is removed by DNA polymerase β (Pol β) or Polγ via their intrinsic dRP lyase activity in the nucleus and mitochondria, respectively [22]. We hypothesized that proliferating myoblasts are highly sensitive to oxidative damage due to a deficiency in the repair of the mitochondrial genome, which, in turn causes a loss of their viability by activation of the apoptotic pathway
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