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

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.

Figure 1. The effects of Resveratrol on C2C12 myoblast remodelling.

Phase contrast images showing the impact of different concentrations of resveratrol (RS) on (a) C2C12 myoblast migration at t = 0 (basal) and at t = 24 h, (b) cell fusion to pluri-nucleated cells (myotubes) and (c and ci) sprout formation on spheroids. Figure ai, bi, cii and ciii show the number of migrated cells, pluri-nucleated cells, sprouts and cumulative sprout length, respectively. The effect of RS was dose-dependent. (a, ai) RS (10 µM) enhanced cell motility, while it was increasingly inhibited by increasing doses. A low dose of RS (10 µM) also enhanced sprout formation (cii, ciii), while high doses of RS (20–60 µM) markedly impaired the regenerative capacity as reflected by a lower number of pluri-nucleated cells (b and bi) and cell sprouts (cii) and sprout length (cii). Data are expressed as mean ± s.e.m. *: P<0.01 vs. CT; ‡: P<0.001 vs. RS 10 µM. DM vs. CT: in none of the cases significant. P-value calculated using a two-tailed Student's t-test. Bars 20 µm. Original magnification in ci, x 200. Each experiment was performed in triplicate.

Figure 2. 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.

Figure 3. Resveratrol did not improve myotube oxidative capacity.

(a) Effects of 24 and 48 h of resveratrol treatment on C2C12 myotube succinate dehydrogenase (SDH) activity. Myotubes at the 8^th day of differentiation were cultured 24 or 48 h in the absence or presence of RS (10, 20, 40, 60 µM) or vehicle (DM). (b) For the experiment of RS preconditioning, 24 h of treatment with scalar amount of resveratrol (day1) was followed by 24 h of treatment with 100, 500 or 1000 µM H₂O₂ (day2). Treatment with H₂O₂ alone was also performed at day2. In the graphs: day1 and day2 corresponding to the 9^th and the 10^th day of differentiation. Data from H₂O₂ alone and RS preconditioning experiment were compared to controls (CT) analysed at day2. (c) Representative phase contrast images showing SDH staining in CT and RS 40–60 µM or 1000, 500 and 100 µM H₂O₂-treated cells (24 h). (a) Ten and 20 µM RS alone did not significantly affect myotube SDH activity, but at higher doses (40 or 60 µM) it caused a reduced SDH activity. (b) 1000 µM H₂O₂ did reduce SDH activity, which was further aggravated by pre-treatment with increasing doses of RS. Data are mean ± s.e.m. of biological triplicate. P-values calculated using a two-tailed Student's t-test. *: P<0.01 vs CT;.‡: P<0.01 vs H₂O₂ 500 µM; ¤: P<0.01 vs H₂O₂ 100 µM. DM vs. CT in none of the cases significant. Original magnification x50. Bars 20 µm.

Figure 4. Resveratrol modulates myosin type 1 and total myosin ATPase activity.

Representative phase contrast images showing the effect of 24 h (a) and 48 h (b) of 10, 20, 40 and 60 µM resveratrol (RS) on intracellular myosin type 1 and total myosin (type 1 plus type 2) ATPase activity in C2C12 myotubes after 8 days in differentiation media (2% FBS). RS 10–20 µM did not significantly affect myosin ATPase activity either after 24 h (ai;aii) or 48 h (bi;bii). Forty and 60 µM RS caused a transient decrease in both type 1 (60 µM) and total (40 and 60 µM) myosin ATPase after 24 h incubation (ai;aii) and an increase in total myosin (bii), but not type 1 myosin (above normal levels) after 48 h incubation. P-values calculated using a two-tailed Student's t-test.*: P<0.01 vs CT; ¤: P<0.05 vs CT. DM vs. CT: in none of the cases significant. Bars 20 µm. Original magnification 100×. Data are expressed as mean ± s.e.m. of biological triplicates.

Figure 5. Resveratrol (10 µM) prevented the deleterious effect of H₂O₂ on cell migration but not cell fusion.

(a) Phase contrast images showing the effect of RS alone (10 and 20 µM) on cell migration (24 h). (b, c) Phase contrast images showing the effects of different pre-conditioning resveratrol (RS) concentrations and H₂O₂ on cell migration (b) and fusion (c). The number of migrated cells and myotubes in the different conditions are summarized in (d) and (e), respectively. (b and graph in d) show that the effects of 24 h H₂O₂ on cell migration were dose-dependent. High doses of H₂O₂ (500–1000 µM) blocked cell motility, while it was increased with 100 µM H₂O₂. (b and graph in d) shows that RS pre-conditioning (10 µM) abolished the deleterious effects of 500 µM, attenuated that of 1000 µM H₂O₂ and further enhanced cell migration induced by H₂O₂ 100 µM. (c,e) 500 and 1000 µM H₂O₂ did block cell fusion, which was not rescued with RS preconditioning. Data are expressed as mean ± s.e.m of biological quadruplicate. *: P<0.01 vs. CT; ‡: P<0.01 vs. RS 10 µM; ¤: P<0.01 vs. RS 20 µM. P-value calculated using a two-tailed Student's t-test. Bars 20 µm. Original magnification, x 50.

Figure 6. Resveratrol attenuated the impact of 1000 µM H₂O₂ on type 1 and total myosin ATPase activities in C2C12 myotubes.

(a) Representative phase contrast images showing the effect of different concentrations of resveratrol (RS) preconditioning and 1000 µM H₂O₂ on myosin type 1 and total myosin ATPase activity in C2C12 myotubes that have been 8 days in differentiation media (2% FBS). H₂O₂ (1000 µM) did reduce both myosin type 1 (b) and total (c) myosin ATPase activities. Ten µM RS pre-conditioning further reduced the myosin type 1 activity (b), but abolished the impact of H₂O₂ on total ATPase activity (c). Higher RS concentrations did attenuate the impact of H₂O₂ on both myosin type 1 and total ATPase activity (b,c). DM+1000 µM H₂O₂ vs. CT+1000 µM H₂O₂ in none of the cases significant. Data are expressed as mean ± s.e.m. of biological quadruplicate. P-values calculated using two-tailed Student's t-tests. *: P<0.01 vs. NT (untreated); ‡: P<0.05 vs. 1000 µM H₂O₂. Bars 10 µm.

Figure 7. 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.

Figure 8. Resveratrol induced p66Shc-Ser36 phosphorylation and up-regulation of Heat Shock Protein (Hsp)-70.

(a) Western blot analysis showing the effect of resveratrol (RS; 10, 20, 40 and 60 µM), H₂O₂ (100 and 1000 µM) and RS pre-conditioning (10 and 20 µM) on p66Shc-Ser36 phosphorylation and Hsp-70 protein levels. Dose dependent increases in Ser36-p66 phosphorylation and in Hsp-70 protein contents were observed in resveratrol-treated cells. 1000 µM, but not 100 µM H₂O₂ induced p66Shc(A)-Ser36 phosphorylation (ai); both concentrations of H₂O₂ elevated Hsp-70 protein levels, but more so in 1000 than 100 µM H₂O₂ (aii). RS (10 and 20 µM) pre-conditioning attenuated the effects of 1000 µM H₂O₂ on Hsp-70 (aii), but not on p66Shc-Ser36 phosphorylation (ai). DM vs. CT: in none of the cases was significant. 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 100 µM. Data are mean ± s.e.m.