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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

PARylation induced by oxidative stress is reduced in myotubes.(A) Representative Western blot shows a maximal amount of PAR signal at 30 minutes after H2O2 treatment. (B) Comparison of PAR formation of myoblasts and myotubes in response to exposure to 0.4 mM H2O2 in the presence or absence of PJ34 at 30 min post-treatment. (C) Densitometric analysis of PAR level in myoblasts and myotubes. One-way ANOVA was used for statistical analysis and determination of significance. * indicates p<0.05 relative to untreated myoblasts.
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pone.0134227.g005: PARylation induced by oxidative stress is reduced in myotubes.(A) Representative Western blot shows a maximal amount of PAR signal at 30 minutes after H2O2 treatment. (B) Comparison of PAR formation of myoblasts and myotubes in response to exposure to 0.4 mM H2O2 in the presence or absence of PJ34 at 30 min post-treatment. (C) Densitometric analysis of PAR level in myoblasts and myotubes. One-way ANOVA was used for statistical analysis and determination of significance. * indicates p<0.05 relative to untreated myoblasts.

Mentions: In order to study the effect of PARP-1 inhibition, we first determined the maximum non-toxic concentration of well-known PARP inhibitor, PJ34 [36]. For our subsequent studies, we selected 10μM as the highest, non-toxic concentration of PJ34 based on preliminary studies with MTT conversion and LDH release cell-viability assays (Fig 4). Consistent with our observations that PARP-1 expression is downregulated in myotubes as compared to myoblasts, H2O2 insult induced a lesser degree of PARP-1 activation in myotubes than in myoblasts, as determined by Western blot analysis of PAR adducts in whole-cell extracts of each cell type (Fig 5). Next, we compared the changes in the viability of myoblasts and myotubes exposed to various concentrations of H2O2 by monitoring the LDH release into the culture medium, measuring the capacity of the cells to convert MTT to formazan, and quantifying cellular NAD+ levels. As expected, increasing concentration of H2O2 caused an increase in LDH release (Fig 6A). 200 μM H2O2 resulted in a ~5-fold increase in LDH release by myoblasts but not myotubes (Fig 6A). As expected, pre-treatment with the PARP inhibitor, PJ34, significantly reduced H2O2-induced LDH release in myoblasts (Fig 6A). Similarly, we observed significant reduction of both MTT conversion capability and NAD+ levels in myoblasts exposed to increasing concentrations of H2O2, but not in myotubes (Fig 6B and 6C). Similarly, PJ34 pre-treatment attenuated the deleterious effect of H2O2 in myoblasts, with only relatively minor effects in myotubes (Fig 6B and 6C). The cytotoxic effects of glucose oxidase (GOx), an alternative oxidative stressor that generates constant, low levels of H2O2 in culture media, were also attenuated in PJ34-treated myoblasts in a concentration-dependent manner (assessed by measurement of MTT reduction, Fig 6D). Myotubes were affected only by the highest concentration of GOx and experienced no beneficial effect from PJ34 pre-treatment (Fig 6D).


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)

PARylation induced by oxidative stress is reduced in myotubes.(A) Representative Western blot shows a maximal amount of PAR signal at 30 minutes after H2O2 treatment. (B) Comparison of PAR formation of myoblasts and myotubes in response to exposure to 0.4 mM H2O2 in the presence or absence of PJ34 at 30 min post-treatment. (C) Densitometric analysis of PAR level in myoblasts and myotubes. One-way ANOVA was used for statistical analysis and determination of significance. * indicates p<0.05 relative to untreated myoblasts.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134227.g005: PARylation induced by oxidative stress is reduced in myotubes.(A) Representative Western blot shows a maximal amount of PAR signal at 30 minutes after H2O2 treatment. (B) Comparison of PAR formation of myoblasts and myotubes in response to exposure to 0.4 mM H2O2 in the presence or absence of PJ34 at 30 min post-treatment. (C) Densitometric analysis of PAR level in myoblasts and myotubes. One-way ANOVA was used for statistical analysis and determination of significance. * indicates p<0.05 relative to untreated myoblasts.
Mentions: In order to study the effect of PARP-1 inhibition, we first determined the maximum non-toxic concentration of well-known PARP inhibitor, PJ34 [36]. For our subsequent studies, we selected 10μM as the highest, non-toxic concentration of PJ34 based on preliminary studies with MTT conversion and LDH release cell-viability assays (Fig 4). Consistent with our observations that PARP-1 expression is downregulated in myotubes as compared to myoblasts, H2O2 insult induced a lesser degree of PARP-1 activation in myotubes than in myoblasts, as determined by Western blot analysis of PAR adducts in whole-cell extracts of each cell type (Fig 5). Next, we compared the changes in the viability of myoblasts and myotubes exposed to various concentrations of H2O2 by monitoring the LDH release into the culture medium, measuring the capacity of the cells to convert MTT to formazan, and quantifying cellular NAD+ levels. As expected, increasing concentration of H2O2 caused an increase in LDH release (Fig 6A). 200 μM H2O2 resulted in a ~5-fold increase in LDH release by myoblasts but not myotubes (Fig 6A). As expected, pre-treatment with the PARP inhibitor, PJ34, significantly reduced H2O2-induced LDH release in myoblasts (Fig 6A). Similarly, we observed significant reduction of both MTT conversion capability and NAD+ levels in myoblasts exposed to increasing concentrations of H2O2, but not in myotubes (Fig 6B and 6C). Similarly, PJ34 pre-treatment attenuated the deleterious effect of H2O2 in myoblasts, with only relatively minor effects in myotubes (Fig 6B and 6C). The cytotoxic effects of glucose oxidase (GOx), an alternative oxidative stressor that generates constant, low levels of H2O2 in culture media, were also attenuated in PJ34-treated myoblasts in a concentration-dependent manner (assessed by measurement of MTT reduction, Fig 6D). Myotubes were affected only by the highest concentration of GOx and experienced no beneficial effect from PJ34 pre-treatment (Fig 6D).

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