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

Oláh G, Szczesny B, Brunyánszki A, López-García IA, Gerö D, Radák Z, Szabo C - PLoS ONE (2015)

Bottom Line: We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Lack of the contact inhibitory effect on PARP-1 expression in myoblasts.(A) Western blot analysis of PARP-1 in myoblasts at day 1 and 2 after reaching 100% confluence. Actin was used as a loading control. PARP-1 densitometric analysis was normalized to actin (B) or PCNA (C); values obtained in Day 1 cells were set as 100%. The results show no significant difference in PARP-1 protein between cells kept for 1 or 2 days after reaching 100% confluence.
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pone.0134227.g003: Lack of the contact inhibitory effect on PARP-1 expression in myoblasts.(A) Western blot analysis of PARP-1 in myoblasts at day 1 and 2 after reaching 100% confluence. Actin was used as a loading control. PARP-1 densitometric analysis was normalized to actin (B) or PCNA (C); values obtained in Day 1 cells were set as 100%. The results show no significant difference in PARP-1 protein between cells kept for 1 or 2 days after reaching 100% confluence.

Mentions: In order to investigate whether PARP-1 downregulation is an effect of contact inhibition, confluent myoblast culture was maintained for an additional one or two days while the level of PARP-1 was monitored by Western blot. As shown in Fig 3, the level of PARP-1 was not changed under these conditions suggesting that reduction of the PARP-1 level is an effect of differentiation, not contact inhibition.


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

Oláh G, Szczesny B, Brunyánszki A, López-García IA, Gerö D, Radák Z, Szabo C - PLoS ONE (2015)

Lack of the contact inhibitory effect on PARP-1 expression in myoblasts.(A) Western blot analysis of PARP-1 in myoblasts at day 1 and 2 after reaching 100% confluence. Actin was used as a loading control. PARP-1 densitometric analysis was normalized to actin (B) or PCNA (C); values obtained in Day 1 cells were set as 100%. The results show no significant difference in PARP-1 protein between cells kept for 1 or 2 days after reaching 100% confluence.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4517814&req=5

pone.0134227.g003: Lack of the contact inhibitory effect on PARP-1 expression in myoblasts.(A) Western blot analysis of PARP-1 in myoblasts at day 1 and 2 after reaching 100% confluence. Actin was used as a loading control. PARP-1 densitometric analysis was normalized to actin (B) or PCNA (C); values obtained in Day 1 cells were set as 100%. The results show no significant difference in PARP-1 protein between cells kept for 1 or 2 days after reaching 100% confluence.
Mentions: In order to investigate whether PARP-1 downregulation is an effect of contact inhibition, confluent myoblast culture was maintained for an additional one or two days while the level of PARP-1 was monitored by Western blot. As shown in Fig 3, the level of PARP-1 was not changed under these conditions suggesting that reduction of the PARP-1 level is an effect of differentiation, not contact inhibition.

Bottom Line: We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus