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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.The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6).

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

Inhibition of PARP-1 reduces subpopulations of apoptotic and necrotic cells induced by H2O2.C2C12 myoblasts were exposed to 0.8 mM H2O2 for 24h in the presence or absence of the PARP inhibitor PJ34 (10 μM). PJ34 reduced the oxidant-induced death of the myoblasts by decreasing the proportion of the necrotic, early and late apoptotic cell populations. Representative dot plots (A) and analysis (B) are shown. One-way ANOVA was used for determining significance between groups. * shows significant differences, p ≤ 0.05, in the cell response to H2O2 relative to controls, while # shows significant protective effect of PJ34, p≤ 0.05. Data representative of 3 different determinations conducted on different experimental days are shown. (Total cell number in each group was set to 100%).
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pone.0134227.g007: Inhibition of PARP-1 reduces subpopulations of apoptotic and necrotic cells induced by H2O2.C2C12 myoblasts were exposed to 0.8 mM H2O2 for 24h in the presence or absence of the PARP inhibitor PJ34 (10 μM). PJ34 reduced the oxidant-induced death of the myoblasts by decreasing the proportion of the necrotic, early and late apoptotic cell populations. Representative dot plots (A) and analysis (B) are shown. One-way ANOVA was used for determining significance between groups. * shows significant differences, p ≤ 0.05, in the cell response to H2O2 relative to controls, while # shows significant protective effect of PJ34, p≤ 0.05. Data representative of 3 different determinations conducted on different experimental days are shown. (Total cell number in each group was set to 100%).

Mentions: Our observation that myotubes are resistant to oxidant induced stress was further validated by flow cytometry, which showed that oxidative stress induces cell death of mixed type (necrotic and apoptotic), and that PARP inhibition with PJ34 reduces cell death primarily by decreasing the portion of necrotic and early apoptotic cell populations (Fig 7A and 7B). Taken together, these data indicate that myotubes are more resistant to oxidative stress than myoblasts, and that the oxidative stress-induced cell dysfunction/cell death in myoblasts involves a significant PARP-1dependent component.


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)

Inhibition of PARP-1 reduces subpopulations of apoptotic and necrotic cells induced by H2O2.C2C12 myoblasts were exposed to 0.8 mM H2O2 for 24h in the presence or absence of the PARP inhibitor PJ34 (10 μM). PJ34 reduced the oxidant-induced death of the myoblasts by decreasing the proportion of the necrotic, early and late apoptotic cell populations. Representative dot plots (A) and analysis (B) are shown. One-way ANOVA was used for determining significance between groups. * shows significant differences, p ≤ 0.05, in the cell response to H2O2 relative to controls, while # shows significant protective effect of PJ34, p≤ 0.05. Data representative of 3 different determinations conducted on different experimental days are shown. (Total cell number in each group was set to 100%).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134227.g007: Inhibition of PARP-1 reduces subpopulations of apoptotic and necrotic cells induced by H2O2.C2C12 myoblasts were exposed to 0.8 mM H2O2 for 24h in the presence or absence of the PARP inhibitor PJ34 (10 μM). PJ34 reduced the oxidant-induced death of the myoblasts by decreasing the proportion of the necrotic, early and late apoptotic cell populations. Representative dot plots (A) and analysis (B) are shown. One-way ANOVA was used for determining significance between groups. * shows significant differences, p ≤ 0.05, in the cell response to H2O2 relative to controls, while # shows significant protective effect of PJ34, p≤ 0.05. Data representative of 3 different determinations conducted on different experimental days are shown. (Total cell number in each group was set to 100%).
Mentions: Our observation that myotubes are resistant to oxidant induced stress was further validated by flow cytometry, which showed that oxidative stress induces cell death of mixed type (necrotic and apoptotic), and that PARP inhibition with PJ34 reduces cell death primarily by decreasing the portion of necrotic and early apoptotic cell populations (Fig 7A and 7B). Taken together, these data indicate that myotubes are more resistant to oxidative stress than myoblasts, and that the oxidative stress-induced cell dysfunction/cell death in myoblasts involves a significant PARP-1dependent component.

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.The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6).

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