DNA damage response by single-strand breaks in terminally differentiated muscle cells and the control of muscle integrity

Abstract

DNA single-strand breaks (SSB) formation coordinates the myogenic program, and defects in SSB repair in post-mitotic cells have been associated with human diseases. However, the DNA damage response by SSB in terminally differentiated cells has not been explored yet. Here we show that mouse post-mitotic muscle cells accumulate SSB after alkylation damage, but they are extraordinarily resistant to the killing effects of a variety of SSB-inducers. We demonstrate that, upon SSB induction, phosphorylation of H2AX occurs in myotubes and is largely ataxia telangiectasia mutated (ATM)-dependent. However, the DNA damage signaling cascade downstream of ATM is defective as shown by lack of p53 increase and phosphorylation at serine 18 (human serine 15). The stabilization of p53 by nutlin-3 was ineffective in activating the cell death pathway, indicating that the resistance to SSB inducers is due to defective p53 downstream signaling. The induction of specific types of damage is required to activate the cell death program in myotubes. Besides the topoisomerase inhibitor doxorubicin known for its cardiotoxicity, we show that the mitochondria-specific inhibitor menadione is able to activate p53 and to kill effectively myotubes. Cell killing is p53-dependent as demonstrated by full protection of myotubes lacking p53, but there is a restriction of p53-activated genes. This new information may have important therapeutic implications in the prevention of muscle cell toxicity. Cells respond to genotoxic stress by activating a signaling cascade known as the DNA damage response (DDR). The DDR is a complex interlaced network comprised of DNA damage repair factors and cell cycle regulators. 1 Our knowledge of the mechanisms of DDR mainly relies on studies conducted in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signaling. Much less is known in post-mitotic cells that undergo irreversible cell cycle withdrawal. DNA repair is strongly affected by the exit from the cell cycle as revealed by downregulation of the major DNA repair pathways. 2 This occurs during differentiation-associated gene reprogramming at transcriptional level as in the case of genes coding for proteins shared by DNA repair and replication (e.g., replicative DNA polymerases, Flap structure-specific endonuclease 1, proliferating cell nuclear antigen and DNA ligase 1) 3 or repair proteins that are cell-cycle related (e.g., XRCC1 (X-ray repair complementing defective repair in Chinese hamster cells 1), uracil-DNA glycosylase). 4,5 Alternatively, post-translational modifications may modify the efficiency of specific DNA repair components as in the case of transcription factor II H that, because of reduced ubiquitination, may lead to decreased global genomic nucleotide excision repair typical of differentiated cells. 6 Exposure of single-stranded (ss) DNA and/or the generation of double-strand breaks (DSB) are powerful activators of DDR by recruiting and activating two protein kinases, ataxia telangiectasia and Rad3-related (ATR)...

Age-related change in γH2AX of Drosophila muscle: its significance as a marker for muscle damage and longevity

“…Moreover, aged Drosophila ISCs show increased proliferative activity despite increased cH2AX formation (Park et al 2012;Na et al 2013). In differentiated muscle cells, however, factors involved in DNA damage repair are downregulated compared to their counterparts in proliferative cells (Fortini and Dogliotti 2010;Szczesny et al 2010) and muscle cells have slow kinetics of cH2AX formation after DNA damage (Fortini et al 2012). Our finding that irradiation-induced cH2AX remained present for 168 h in Drosophila muscle (Fig.…”

DNA Damage Response and DNA Repair in Skeletal Myocytes From a Mouse Model of Spinal Muscular Atrophy

“…Differentiated myocytes can efficiently activate p53 after DNA damage but these cells possess a downstream blockade of p53-mediated apoptosis and thus evade death (12). Although seminal studies are emerging on DDR and DNA repair mechanisms in differentiated skeletal muscle cells (10)(11)(12)(13), there is limited knowledge on whether myopathy-causing gene mutations can alter DDR and DNA repair mechanisms in skeletal muscle.…”

“…Much less is known about the intrinsic properties of DDR and DNA repair in postmitotic, terminally differentiated cells such as neurons and multinucleated myofibers in skeletal muscle (6)(7)(8)(9)(10). Terminally differentiated skeletal muscle cells have prominent down-regulation of DNA repair mechanisms and a low capacity for DNA base excision repair (11), although these cells are resistant to the cell killing effects of DNA single-strand break (SSB) inducers (10). Differentiated myocytes can efficiently activate p53 after DNA damage but these cells possess a downstream blockade of p53-mediated apoptosis and thus evade death (12).…”

Molecular dynamics of the full-length p53 monomer

“…Still now, after so many years and publications on the p53-DNA damage connection, new pathways are emerging. Just as an example, p53, with different degrees depending on its isoforms or its polymorphism at codon 72, 53 plays a crucial role in single-strand breaks during muscle function, 54 interacts with PRAP1, 55 as well as with DNA damage response. 56,57 The regulation of cell death by the p53 protein is quite complex, acting both at the level of autophagy, 58 lysosomes, 59 or at the core machinery of programmed cell death.…”