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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.However, the deleterious effects of H2O2 (1000 µM) on cell migration were alleviated after preconditioning with 10 µM-RS.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 did not improve myoblast metabolic state and further enhanced the inhibitory effect of H2O2.(a) Effects of 24 and 48 h of resveratrol treatment on C2C12 myoblast energy metabolism. Cells were cultured 24 h and 48 h in the absence (CT) or presence of RS (10, 20, 40 and 60 µM), or DM vehicle. (ai) Phase contrast images showing a representative cellular formazan staining in CT and cell treated with 60 µM RS for 24 h. (b) In the experiment of preconditioning, 24 h treatment with scalar amount of resveratrol (day1) was followed by 24 h treatment with 100, 500 or 1000 µM H2O2 (day2). Treatment with H2O2 alone was also performed at day2. (b) Data from H2O2 alone and RS preconditioning were compared to CT at day2. (a,b) Irrespective of the period of incubation, 10 and 20 µM RS did not affect myoblast energy metabolism, while it was significantly reduced by 60 µM RS. Only 1000 and 500 µM H2O2 caused a reduction in energy metabolism, which was further hampered in a dose-dependent manner by RS-preconditioning. (b) Data are expressed as mean ± s.e.m. of biological triplicate. P-values calculated using a two-tailed Student's t-test. *: P<0.01 vs CT; #: P<0.001 vs H2O2 1000 µM; ‡: P<0.01 vs H2O2 500 µM; ¤: P<0.01 vs H2O2 100 µM. Original magnification, x 50. Bars 20 µm.
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f2: Resveratrol did not improve myoblast metabolic state and further enhanced the inhibitory effect of H2O2.(a) Effects of 24 and 48 h of resveratrol treatment on C2C12 myoblast energy metabolism. Cells were cultured 24 h and 48 h in the absence (CT) or presence of RS (10, 20, 40 and 60 µM), or DM vehicle. (ai) Phase contrast images showing a representative cellular formazan staining in CT and cell treated with 60 µM RS for 24 h. (b) In the experiment of preconditioning, 24 h treatment with scalar amount of resveratrol (day1) was followed by 24 h treatment with 100, 500 or 1000 µM H2O2 (day2). Treatment with H2O2 alone was also performed at day2. (b) Data from H2O2 alone and RS preconditioning were compared to CT at day2. (a,b) Irrespective of the period of incubation, 10 and 20 µM RS did not affect myoblast energy metabolism, while it was significantly reduced by 60 µM RS. Only 1000 and 500 µM H2O2 caused a reduction in energy metabolism, which was further hampered in a dose-dependent manner by RS-preconditioning. (b) Data are expressed as mean ± s.e.m. of biological triplicate. P-values calculated using a two-tailed Student's t-test. *: P<0.01 vs CT; #: P<0.001 vs H2O2 1000 µM; ‡: P<0.01 vs H2O2 500 µM; ¤: P<0.01 vs H2O2 100 µM. Original magnification, x 50. Bars 20 µm.

Mentions: Mitochondrial depolarization may lead to a reduction in the number of mitochondria. Succinate dehydrogenase (SDH) activity has a fundamental function in oxidative energy metabolism35 and may be indicative of mitochondrial content and cell viability36. We examined the effect of RS on the active metabolic state in myoblasts (Fig. 2) and oxidative capacity in myocytes (SDH activity; Fig. 3). The active metabolic state was examined in cell myoblasts and not in myotubes, since the method required the accurate seeding of equal cell density for each condition, which is not possible for fused myocytes after 8 days of differentiation. C2C12 myoblasts (for active metabolic state; Fig. 2a) and myotubes (for SDH activity; Fig. 3a) were cultured 24 h and 48 h in the absence or presence of RS scalar amount. Figure 2a, shows that, irrespective of the period of incubation, 10 and 20 µM RS did not significantly affect energy metabolism, but higher doses (40 or 60 µM) caused a reduction in metabolic state in myoblasts (60 µM; Fig. 2a) and oxidative capacity in myotubes (40 or 60 µM; Fig. 3a).


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

Bosutti A, Degens H - Sci Rep (2015)

Resveratrol did not improve myoblast metabolic state and further enhanced the inhibitory effect of H2O2.(a) Effects of 24 and 48 h of resveratrol treatment on C2C12 myoblast energy metabolism. Cells were cultured 24 h and 48 h in the absence (CT) or presence of RS (10, 20, 40 and 60 µM), or DM vehicle. (ai) Phase contrast images showing a representative cellular formazan staining in CT and cell treated with 60 µM RS for 24 h. (b) In the experiment of preconditioning, 24 h treatment with scalar amount of resveratrol (day1) was followed by 24 h treatment with 100, 500 or 1000 µM H2O2 (day2). Treatment with H2O2 alone was also performed at day2. (b) Data from H2O2 alone and RS preconditioning were compared to CT at day2. (a,b) Irrespective of the period of incubation, 10 and 20 µM RS did not affect myoblast energy metabolism, while it was significantly reduced by 60 µM RS. Only 1000 and 500 µM H2O2 caused a reduction in energy metabolism, which was further hampered in a dose-dependent manner by RS-preconditioning. (b) Data are expressed as mean ± s.e.m. of biological triplicate. P-values calculated using a two-tailed Student's t-test. *: P<0.01 vs CT; #: P<0.001 vs H2O2 1000 µM; ‡: P<0.01 vs H2O2 500 µM; ¤: P<0.01 vs H2O2 100 µM. Original magnification, x 50. Bars 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4308712&req=5

f2: Resveratrol did not improve myoblast metabolic state and further enhanced the inhibitory effect of H2O2.(a) Effects of 24 and 48 h of resveratrol treatment on C2C12 myoblast energy metabolism. Cells were cultured 24 h and 48 h in the absence (CT) or presence of RS (10, 20, 40 and 60 µM), or DM vehicle. (ai) Phase contrast images showing a representative cellular formazan staining in CT and cell treated with 60 µM RS for 24 h. (b) In the experiment of preconditioning, 24 h treatment with scalar amount of resveratrol (day1) was followed by 24 h treatment with 100, 500 or 1000 µM H2O2 (day2). Treatment with H2O2 alone was also performed at day2. (b) Data from H2O2 alone and RS preconditioning were compared to CT at day2. (a,b) Irrespective of the period of incubation, 10 and 20 µM RS did not affect myoblast energy metabolism, while it was significantly reduced by 60 µM RS. Only 1000 and 500 µM H2O2 caused a reduction in energy metabolism, which was further hampered in a dose-dependent manner by RS-preconditioning. (b) Data are expressed as mean ± s.e.m. of biological triplicate. P-values calculated using a two-tailed Student's t-test. *: P<0.01 vs CT; #: P<0.001 vs H2O2 1000 µM; ‡: P<0.01 vs H2O2 500 µM; ¤: P<0.01 vs H2O2 100 µM. Original magnification, x 50. Bars 20 µm.
Mentions: Mitochondrial depolarization may lead to a reduction in the number of mitochondria. Succinate dehydrogenase (SDH) activity has a fundamental function in oxidative energy metabolism35 and may be indicative of mitochondrial content and cell viability36. We examined the effect of RS on the active metabolic state in myoblasts (Fig. 2) and oxidative capacity in myocytes (SDH activity; Fig. 3). The active metabolic state was examined in cell myoblasts and not in myotubes, since the method required the accurate seeding of equal cell density for each condition, which is not possible for fused myocytes after 8 days of differentiation. C2C12 myoblasts (for active metabolic state; Fig. 2a) and myotubes (for SDH activity; Fig. 3a) were cultured 24 h and 48 h in the absence or presence of RS scalar amount. Figure 2a, shows that, irrespective of the period of incubation, 10 and 20 µM RS did not significantly affect energy metabolism, but higher doses (40 or 60 µM) caused a reduction in metabolic state in myoblasts (60 µM; Fig. 2a) and oxidative capacity in myotubes (40 or 60 µM; Fig. 3a).

Bottom Line: RS did not increase oxidative capacity.However, the deleterious effects of H2O2 (1000 µM) on cell migration were alleviated after preconditioning with 10 µM-RS.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