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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

PARP1 level is reduced in myotubes.(A) Representative phase-contrast microscopy images of myoblasts and myotubes showing typical morphological differences. (B) Immunocytochemistry of the differentiation marker, myogenin, in myoblasts and myotubes. DAPI was used for nuclear counterstaining. Increased myogenin signal was detectable in the fully differentiated myotubes. (C) The effect of differentiation on protein expression of PARP-1 and the differentiation markers, PCNA, Pax7, and myogenin were monitored on Days 0–7 using whole cell extracts. Actin was used as a loading control. The relative quantity of proteins was calculated by densitometry and normalized to actin based on the analysis of three independent Western blots. * indicates p<0.05 relative to myoblasts at Day 0 (100%). (D) PARP-1 distribution in undifferentiated myoblasts and differentiated myotubes. DAPI was used for nuclear counterstaining. Decreased PARP-1 signal was detectable in the fully differentiated myotubes.
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pone.0134227.g001: PARP1 level is reduced in myotubes.(A) Representative phase-contrast microscopy images of myoblasts and myotubes showing typical morphological differences. (B) Immunocytochemistry of the differentiation marker, myogenin, in myoblasts and myotubes. DAPI was used for nuclear counterstaining. Increased myogenin signal was detectable in the fully differentiated myotubes. (C) The effect of differentiation on protein expression of PARP-1 and the differentiation markers, PCNA, Pax7, and myogenin were monitored on Days 0–7 using whole cell extracts. Actin was used as a loading control. The relative quantity of proteins was calculated by densitometry and normalized to actin based on the analysis of three independent Western blots. * indicates p<0.05 relative to myoblasts at Day 0 (100%). (D) PARP-1 distribution in undifferentiated myoblasts and differentiated myotubes. DAPI was used for nuclear counterstaining. Decreased PARP-1 signal was detectable in the fully differentiated myotubes.

Mentions: The C2C12 cell line is a well-defined model for skeletal muscle differentiation that recapitulates the in vivo process through irreversible withdrawal from the cell cycle, repression of proliferation-associated genes, and expression of terminally differentiated muscle-specific genes [24–26]. Proliferating myoblasts differ from terminally differentiated, non-proliferating myotubes in morphology and protein expression profiles (Fig 1A and 1B). To confirm proper differentiation, 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]. Time-course Western blot analysis of myoblast differentiation, from day 0 through 7, is shown in Fig 1C. These results confirm that the process of myoblast differentiation is accurately recapitulated, as shown by decreased expression of Pax7 and PCNA, and increased expression of myogenin. Moreover, we observed a marked decrease in PARP-1 expression in myotubes (Fig 1C). PARP-1 expression was ten-fold greater in myoblasts than in myotubes (Fig 1C). Immunocytochemical analysis showed that in myoblasts, PARP-1 is localized mostly in the nucleus with little cytoplasmic staining, whereas terminally differentiated myotubes showed a global reduction of signal intensity (Fig 1D). 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]. The obtained data clearly indicate that myotubes of L6 cells have reduced expression of PARP-1 (Fig 2A). Moreover, differentiation of U937 cells, induced by PMA, also showed a reduction in PARP1 expression (Fig 2B).


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)

PARP1 level is reduced in myotubes.(A) Representative phase-contrast microscopy images of myoblasts and myotubes showing typical morphological differences. (B) Immunocytochemistry of the differentiation marker, myogenin, in myoblasts and myotubes. DAPI was used for nuclear counterstaining. Increased myogenin signal was detectable in the fully differentiated myotubes. (C) The effect of differentiation on protein expression of PARP-1 and the differentiation markers, PCNA, Pax7, and myogenin were monitored on Days 0–7 using whole cell extracts. Actin was used as a loading control. The relative quantity of proteins was calculated by densitometry and normalized to actin based on the analysis of three independent Western blots. * indicates p<0.05 relative to myoblasts at Day 0 (100%). (D) PARP-1 distribution in undifferentiated myoblasts and differentiated myotubes. DAPI was used for nuclear counterstaining. Decreased PARP-1 signal was detectable in the fully differentiated myotubes.
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pone.0134227.g001: PARP1 level is reduced in myotubes.(A) Representative phase-contrast microscopy images of myoblasts and myotubes showing typical morphological differences. (B) Immunocytochemistry of the differentiation marker, myogenin, in myoblasts and myotubes. DAPI was used for nuclear counterstaining. Increased myogenin signal was detectable in the fully differentiated myotubes. (C) The effect of differentiation on protein expression of PARP-1 and the differentiation markers, PCNA, Pax7, and myogenin were monitored on Days 0–7 using whole cell extracts. Actin was used as a loading control. The relative quantity of proteins was calculated by densitometry and normalized to actin based on the analysis of three independent Western blots. * indicates p<0.05 relative to myoblasts at Day 0 (100%). (D) PARP-1 distribution in undifferentiated myoblasts and differentiated myotubes. DAPI was used for nuclear counterstaining. Decreased PARP-1 signal was detectable in the fully differentiated myotubes.
Mentions: The C2C12 cell line is a well-defined model for skeletal muscle differentiation that recapitulates the in vivo process through irreversible withdrawal from the cell cycle, repression of proliferation-associated genes, and expression of terminally differentiated muscle-specific genes [24–26]. Proliferating myoblasts differ from terminally differentiated, non-proliferating myotubes in morphology and protein expression profiles (Fig 1A and 1B). To confirm proper differentiation, 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]. Time-course Western blot analysis of myoblast differentiation, from day 0 through 7, is shown in Fig 1C. These results confirm that the process of myoblast differentiation is accurately recapitulated, as shown by decreased expression of Pax7 and PCNA, and increased expression of myogenin. Moreover, we observed a marked decrease in PARP-1 expression in myotubes (Fig 1C). PARP-1 expression was ten-fold greater in myoblasts than in myotubes (Fig 1C). Immunocytochemical analysis showed that in myoblasts, PARP-1 is localized mostly in the nucleus with little cytoplasmic staining, whereas terminally differentiated myotubes showed a global reduction of signal intensity (Fig 1D). 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]. The obtained data clearly indicate that myotubes of L6 cells have reduced expression of PARP-1 (Fig 2A). Moreover, differentiation of U937 cells, induced by PMA, also showed a reduction in PARP1 expression (Fig 2B).

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