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The impact of resveratrol and hydrogen peroxide on muscle cell plasticity shows a dose-dependent interaction.

Bosutti A, Degens H - Sci Rep (2015)

Bottom Line: RS did not increase oxidative capacity.In conclusion, low resveratrol doses promoted in vitro muscle regeneration and attenuated the impact of ROS, while high doses augmented the reduced plasticity and metabolism induced by oxidative stress.Thus, the effects of resveratrol depend on its dose and degree of oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: School of Healthcare Science, Manchester Metropolitan University, Manchester, United Kingdom.

ABSTRACT
While reactive oxygen species (ROS) play a role in muscle repair, excessive amounts of ROS for extended periods may lead to oxidative stress. Antioxidants, as resveratrol (RS), may reduce oxidative stress, restore mitochondrial function and promote myogenesis and hypertrophy. However, RS dose-effectiveness for muscle plasticity is unclear. Therefore, we investigated RS dose-response on C2C12 myoblast and myotube plasticity 1. in the presence and 2. absence of different degrees of oxidative stress. Low RS concentration (10 μM) stimulated myoblast cell cycle arrest, migration and sprouting, which were inhibited by higher doses (40-60 μM). RS did not increase oxidative capacity. In contrast, RS induced mitochondria loss, reduced cell viability and ROS production, and activated stress response pathways [Hsp70 and pSer36-p66(ShcA) proteins]. However, the deleterious effects of H2O2 (1000 µM) on cell migration were alleviated after preconditioning with 10 µM-RS. This dose also enhanced cell motility mediated by 100 µM-H2O2, while higher RS-doses augmented the H2O2-induced impaired myoblast regeneration and mitochondrial dehydrogenase activity. In conclusion, low resveratrol doses promoted in vitro muscle regeneration and attenuated the impact of ROS, while high doses augmented the reduced plasticity and metabolism induced by oxidative stress. Thus, the effects of resveratrol depend on its dose and degree of oxidative stress.

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Resveratrol enhanced mitochondrial membrane depolarisation induced by H2O2.(ai) C2C12 myoblasts were exposed to 1000 µM H2O2 with or without RS preconditioning for 24 h as described in the text. Appropriate amounts of DMSO (DM pre-conditioning), followed by incubation with 1000 µM H2O2 (24 h), were used to ensure that the effects were resveratrol-specific. A positive control of mitochondrial depolarisation was obtained by two hours treatment with 5 µM of the apoptotic inducer staurosporine. The total % JC-1 green fluorescence cell population, including shift in depolarisation (gate UR+LR) was calculated. (a) representative images showing each condition and cell percentages in each gate from independent experiments. (aii) Percentage of depolarised cells (gate UR+LR) under different conditions. H2O2 with/without RS pre-conditioning induced mitochondrial depolarisation. Notably, with respect to H2O2 alone, RS pre-conditioning enhanced the effect of H2O2 toward the depolarised state (ai, LR panels), in a dose-dependent manner. DM+1000 µM H2O2 vs. CT+1000 µM H2O2 in none of the cases significant. Data are expressed as mean ± s.e.m. P-values calculated using a two-tailed Student's t-test. *: P = 0.01 vs NT (untreated); ¤: P<0.05 vs H2O2 1000 µM. Each experiment was performed in quadruplicate.
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f7: Resveratrol enhanced mitochondrial membrane depolarisation induced by H2O2.(ai) C2C12 myoblasts were exposed to 1000 µM H2O2 with or without RS preconditioning for 24 h as described in the text. Appropriate amounts of DMSO (DM pre-conditioning), followed by incubation with 1000 µM H2O2 (24 h), were used to ensure that the effects were resveratrol-specific. A positive control of mitochondrial depolarisation was obtained by two hours treatment with 5 µM of the apoptotic inducer staurosporine. The total % JC-1 green fluorescence cell population, including shift in depolarisation (gate UR+LR) was calculated. (a) representative images showing each condition and cell percentages in each gate from independent experiments. (aii) Percentage of depolarised cells (gate UR+LR) under different conditions. H2O2 with/without RS pre-conditioning induced mitochondrial depolarisation. Notably, with respect to H2O2 alone, RS pre-conditioning enhanced the effect of H2O2 toward the depolarised state (ai, LR panels), in a dose-dependent manner. DM+1000 µM H2O2 vs. CT+1000 µM H2O2 in none of the cases significant. Data are expressed as mean ± s.e.m. P-values calculated using a two-tailed Student's t-test. *: P = 0.01 vs NT (untreated); ¤: P<0.05 vs H2O2 1000 µM. Each experiment was performed in quadruplicate.

Mentions: The effects of H2O2 on mitochondrial membrane potential (ΔΨm) and its implication in the activation of the mitochondrial apoptosis cascade are well recognized37. Resveratrol has been suggested to counteract apoptosis induced by H2O2. Therefore, we analysed whether RS was able to counteract H2O2-induced mitochondria depolarisation. RS pre-conditioning did not prevent, but even enhanced the depolarization induced by 24 h exposure to 1000 µM H2O2 (Fig. 7). Notably, this was associated with increased phosphorylation of p66Shc(A)-Ser36 (Fig. 8ai), and elevated Hsp-70 protein levels (Fig. 8aii), two key players in the mitochondrial and cellular stress response3738. In particular, 1000 µM, but not 100 µM-H2O2 induced p66Shc(A)-Ser36 phosphorylation (Fig. 8ai) and both elevated Hsp-70 protein levels (Fig. 8aii). Although pre-incubation with RS reduced the 1000 µM H2O2-induced elevation in Hsp70, it did not alleviate the increased p66Shc(A) phosphorylation.


The impact of resveratrol and hydrogen peroxide on muscle cell plasticity shows a dose-dependent interaction.

Bosutti A, Degens H - Sci Rep (2015)

Resveratrol enhanced mitochondrial membrane depolarisation induced by H2O2.(ai) C2C12 myoblasts were exposed to 1000 µM H2O2 with or without RS preconditioning for 24 h as described in the text. Appropriate amounts of DMSO (DM pre-conditioning), followed by incubation with 1000 µM H2O2 (24 h), were used to ensure that the effects were resveratrol-specific. A positive control of mitochondrial depolarisation was obtained by two hours treatment with 5 µM of the apoptotic inducer staurosporine. The total % JC-1 green fluorescence cell population, including shift in depolarisation (gate UR+LR) was calculated. (a) representative images showing each condition and cell percentages in each gate from independent experiments. (aii) Percentage of depolarised cells (gate UR+LR) under different conditions. H2O2 with/without RS pre-conditioning induced mitochondrial depolarisation. Notably, with respect to H2O2 alone, RS pre-conditioning enhanced the effect of H2O2 toward the depolarised state (ai, LR panels), in a dose-dependent manner. DM+1000 µM H2O2 vs. CT+1000 µM H2O2 in none of the cases significant. Data are expressed as mean ± s.e.m. P-values calculated using a two-tailed Student's t-test. *: P = 0.01 vs NT (untreated); ¤: P<0.05 vs H2O2 1000 µM. Each experiment was performed in quadruplicate.
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Related In: Results  -  Collection

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Show All Figures
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f7: Resveratrol enhanced mitochondrial membrane depolarisation induced by H2O2.(ai) C2C12 myoblasts were exposed to 1000 µM H2O2 with or without RS preconditioning for 24 h as described in the text. Appropriate amounts of DMSO (DM pre-conditioning), followed by incubation with 1000 µM H2O2 (24 h), were used to ensure that the effects were resveratrol-specific. A positive control of mitochondrial depolarisation was obtained by two hours treatment with 5 µM of the apoptotic inducer staurosporine. The total % JC-1 green fluorescence cell population, including shift in depolarisation (gate UR+LR) was calculated. (a) representative images showing each condition and cell percentages in each gate from independent experiments. (aii) Percentage of depolarised cells (gate UR+LR) under different conditions. H2O2 with/without RS pre-conditioning induced mitochondrial depolarisation. Notably, with respect to H2O2 alone, RS pre-conditioning enhanced the effect of H2O2 toward the depolarised state (ai, LR panels), in a dose-dependent manner. DM+1000 µM H2O2 vs. CT+1000 µM H2O2 in none of the cases significant. Data are expressed as mean ± s.e.m. P-values calculated using a two-tailed Student's t-test. *: P = 0.01 vs NT (untreated); ¤: P<0.05 vs H2O2 1000 µM. Each experiment was performed in quadruplicate.
Mentions: The effects of H2O2 on mitochondrial membrane potential (ΔΨm) and its implication in the activation of the mitochondrial apoptosis cascade are well recognized37. Resveratrol has been suggested to counteract apoptosis induced by H2O2. Therefore, we analysed whether RS was able to counteract H2O2-induced mitochondria depolarisation. RS pre-conditioning did not prevent, but even enhanced the depolarization induced by 24 h exposure to 1000 µM H2O2 (Fig. 7). Notably, this was associated with increased phosphorylation of p66Shc(A)-Ser36 (Fig. 8ai), and elevated Hsp-70 protein levels (Fig. 8aii), two key players in the mitochondrial and cellular stress response3738. In particular, 1000 µM, but not 100 µM-H2O2 induced p66Shc(A)-Ser36 phosphorylation (Fig. 8ai) and both elevated Hsp-70 protein levels (Fig. 8aii). Although pre-incubation with RS reduced the 1000 µM H2O2-induced elevation in Hsp70, it did not alleviate the increased p66Shc(A) phosphorylation.

Bottom Line: RS did not increase oxidative capacity.In conclusion, low resveratrol doses promoted in vitro muscle regeneration and attenuated the impact of ROS, while high doses augmented the reduced plasticity and metabolism induced by oxidative stress.Thus, the effects of resveratrol depend on its dose and degree of oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: School of Healthcare Science, Manchester Metropolitan University, Manchester, United Kingdom.

ABSTRACT
While reactive oxygen species (ROS) play a role in muscle repair, excessive amounts of ROS for extended periods may lead to oxidative stress. Antioxidants, as resveratrol (RS), may reduce oxidative stress, restore mitochondrial function and promote myogenesis and hypertrophy. However, RS dose-effectiveness for muscle plasticity is unclear. Therefore, we investigated RS dose-response on C2C12 myoblast and myotube plasticity 1. in the presence and 2. absence of different degrees of oxidative stress. Low RS concentration (10 μM) stimulated myoblast cell cycle arrest, migration and sprouting, which were inhibited by higher doses (40-60 μM). RS did not increase oxidative capacity. In contrast, RS induced mitochondria loss, reduced cell viability and ROS production, and activated stress response pathways [Hsp70 and pSer36-p66(ShcA) proteins]. However, the deleterious effects of H2O2 (1000 µM) on cell migration were alleviated after preconditioning with 10 µM-RS. This dose also enhanced cell motility mediated by 100 µM-H2O2, while higher RS-doses augmented the H2O2-induced impaired myoblast regeneration and mitochondrial dehydrogenase activity. In conclusion, low resveratrol doses promoted in vitro muscle regeneration and attenuated the impact of ROS, while high doses augmented the reduced plasticity and metabolism induced by oxidative stress. Thus, the effects of resveratrol depend on its dose and degree of oxidative stress.

Show MeSH
Related in: MedlinePlus