Differentiation-Associated Downregulation of Poly(ADP-Ribose) Polymerase-1 Expression in Myoblasts Serves to Increase Their Resistance to Oxidative Stress.

Gábor Oláh,Bartosz Szczesny,Csaba Szabo,Zsolt Radák,Attila Brunyánszki,Domokos Gerö,Isabel A. López-García,Shaida A. Andrabi

doi:10.1371/journal.pone.0134227

Abstract

Poly(ADP-ribose) polymerase 1 (PARP-1), the major isoform of the poly (ADP-ribose) polymerase family, is a constitutive nuclear and mitochondrial protein with well-recognized roles in various essential cellular functions such as DNA repair, signal transduction, apoptosis, as well as in a variety of pathophysiological conditions including sepsis, diabetes and cancer. Activation of PARP-1 in response to oxidative stress catalyzes the covalent attachment of the poly (ADP-ribose) (PAR) groups on itself and other acceptor proteins, utilizing NAD+ as a substrate. Overactivation of PARP-1 depletes intracellular NAD+ influencing mitochondrial electron transport, cellular ATP generation and, if persistent, can result in necrotic cell death. Due to their high metabolic activity, skeletal muscle cells are particularly exposed to constant oxidative stress insults. In this study, we investigated the role of PARP-1 in a well-defined model of murine skeletal muscle differentiation (C2C12) and compare the responses to oxidative stress of undifferentiated myoblasts and differentiated myotubes. We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes. This alteration correlated with an increased resistance to oxidative stress of the myotubes, as measured by MTT and LDH assays. Mitochondrial function, assessed by measuring mitochondrial membrane potential, was preserved under oxidative stress in myotubes compared to myoblasts. Moreover, basal respiration, ATP synthesis, and the maximal respiratory capacity of mitochondria were higher in myotubes than in myoblasts. Inhibition of the catalytic activity of PARP-1 by PJ34 (a phenanthridinone PARP inhibitor) exerted greater protective effects in undifferentiated myoblasts than in differentiated myotubes. The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6). Forced overexpression of PARP1 in C2C12 myotubes sensitized the cells to oxidant-induced injury. Taken together, our data indicate that the reduction of PARP-1 expression during the process of the skeletal muscle differentiation serves as a protective mechanism to maintain the cellular functions of skeletal muscle during oxidative stress.

Highlights

Poly(ADP-ribose) polymerase 1 (PARP-1), the major member of the PARP family, is a constitutive nuclear and mitochondrial enzyme that plays important roles in DNA repair, gene transcription, and chromatin remodeling [1,2,3,4]
We monitored the expression of transcription factor paired-box 7 (Pax7), proliferating cell nuclear antigen (PCNA), which is known to be inhibited during myoblast differentiation or repression of cellular proliferation, [34,35] and myogenin, which is known to be expressed in differentiated myotubes [23]
To confirm our observation that skeletal muscle cell differentiation is accompanied by reduction in PARP-1 expression, we performed similar Western blot analyses using another well-defined model of skeletal muscle differentiation, namely, rat-derived L6 cells [16]

Summary

Introduction

Poly(ADP-ribose) polymerase 1 (PARP-1), the major member of the PARP family, is a constitutive nuclear and mitochondrial enzyme that plays important roles in DNA repair, gene transcription, and chromatin remodeling [1,2,3,4]. Adult muscle is a relatively stable metabolic tissue under resting conditions, but its oxygen consumption markedly increases during exercise Under these conditions (as well as in various pathophysiological conditions and during physiological aging), intracellular production of oxidative free radicals is enhanced, mainly due to the leakage of superoxide from the mitochondrial electron transport chain [5,6,7,8]. Myogenic differentiation is a highly orchestrated sequence of events that produces mature skeletal muscle Very often this process is induced by muscle injury (e.g. caused by extensive exercise), or by other pathophysiological conditions that leads to muscle loss, e.g. in patients with muscular dystrophy, advanced cancer, AIDS or burn [17,18,19,20]. Satellite cells can re-enter the cell cycle and, after proliferation, irreversibly withdraw from the cell cycle, differentiate, and with existing myofibrils to form muscle fiber [21,22,23]

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Conclusion