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. 2017 Dec:50:103-115.
doi: 10.1016/j.jnutbio.2017.08.008. Epub 2017 Aug 24.

Dietary resveratrol confers apoptotic resistance to oxidative stress in myoblasts

Satoshi Haramizu  1 , Shinichi Asano  1 , David C Butler  1 , David A Stanton  1 , Ameena Hajira  1 , Junaith S Mohamed  1 , Stephen E Alway  2
Affiliations

Dietary resveratrol confers apoptotic resistance to oxidative stress in myoblasts

Satoshi Haramizu et al. J Nutr Biochem. 2017 Dec.

Abstract

High levels of reactive oxygen species (ROS) contribute to muscle cell death in aging and disuse. We have previously found that resveratrol can reduce oxidative stress in response to aging and hindlimb unloading in rodents in vivo, but it was not known if resveratrol would protect muscle stem cells during repair or regeneration when oxidative stress is high. To test the protective role of resveratrol on muscle stem cells directly, we treated the C2C12 mouse myoblast cell line with moderate (100 μM) or very high (1 mM) levels of H2O2 in the presence or absence of resveratrol. The p21 promoter activity declined in myoblasts in response to high ROS, and this was accompanied a greater nuclear to cytoplasmic translocation of p21 in a dose-dependent matter in myoblasts as compared to myotubes. Apoptosis, as indicated by TdT-mediated dUTP nick-end labeling, was greater in C2C12 myoblasts as compared to myotubes (P<.05) after treatment with H2O2. Caspase-9, -8 and -3 activities were elevated significantly (P<.05) in myoblasts treated with H2O2. Myoblasts were more susceptible to ROS-induced oxidative stress than myotubes. We treated C2C12 myoblasts with 50 μM of resveratrol for periods up to 48 h to determine if myoblasts could be rescued from high-ROS-induced apoptosis by resveratrol. Resveratrol reduced the apoptotic index and significantly reduced the ROS-induced caspase-9, -8 and -3 activity in myoblasts. Furthermore, Bcl-2 and the Bax/Bcl-2 ratio were partially rescued in myoblasts by resveratrol treatment. Similarly, muscle stem cells isolated from mouse skeletal muscles showed reduced Sirt1 protein abundance with H2O2 treatment, but this could be reversed by resveratrol. Reduced apoptotic susceptibility in myoblasts as compared to myotubes to ROS is regulated, at least in part, by enhanced p21 promoter activity and nuclear p21 location in myotubes. Resveratrol confers further protection against ROS by improving Sirt1 levels and increasing antioxidant production, which reduces mitochondrial associated apoptotic signaling, and cell death in myoblasts.

Keywords: Apoptosis; Myoblasts; Myogenesis; Myotubes; Oxidative stress; Reactive oxygen species.

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Figures

Figure 1
Figure 1. Myotubes are more resistant to oxidative stress than myotubes
A. The percent of total protein loss is shown relative to control cells (0 μM) for each H2O2 treatment. C2C12 myoblasts (Mb) or myotubes (Mt) were incubated for 6, 12, or 24 h in 100 μM or 1.0 mM of H2O2. B. Examples of myoblasts and myotubes that were incubated with 0 μM or 1.0 mM of H2O2 for 24 hours. TUNEL positive nuclei (green) are noted especially in myoblasts after treatment with H2O2. Examples of TUNEL positive cells are shown by the white arrows. Myoblasts and myotubes were incubated in antibodies to desmin and myosin heavy chain respectively, followed by incubation and staining with a secondary Alexa 546 conjugated anti-mouse IgG (red). C. The apoptotic index is expressed as the number of positive TUNEL to the number of total cells. *, different from the corresponding treatment conditions (P< 0.05). †, difference from the control cells (P < 0.05).
Figure 2
Figure 2. Altered caspase activities of myoblasts and myotubes following indicated H2O2 treatment
Caspase activity is shown for caspase-9 (A,B), caspase-8 (C,D) and caspase-3 (E,F) after 6 h (A,C,E) and 24 h (D,E,F) of incubation in 0 μM, 0.1 mM or 1.0 mM H2O2. H2O2-induced caspase activity is presented as a fold change relative to the corresponding control values. Note the different axes scales for 6 and 24 h *, myoblasts are different from myotubes (P<0.05). †, different from control cells (P < 0.05).
Figure 3
Figure 3. The effect of H2O2 treatment on p21 promoter activity and protein abundance
A. Luciferase activity is shown as relative luciferase light units (firefly/Renilla) and represents means ± SEM for four samples 24 h after incubation in 0 μM, 100 μM or 1.0 mM of H2O2. *, myoblasts are different from myotubes in the same group (P<0.05). †, different from control cells (P < 0.05). B. Quantification of protein abundance by western blots for p21 nuclear content after 24 hours of incubation after 0 μM, 100 μM or 1.0 mM of H2O2. Results are presented relative to the corresponding controls. *, myoblasts are different from myotubes in the same group (P<0.05). †, different from control cells (P < 0.05). C. Representative immunoblots of p21 content in nuclear protein fractions performed after 6 hours of H2O2 treatment.
Figure 4
Figure 4. Localization of p21 protein in the myonuclei
Immunohistochemical staining showing localization of p21 (red) in nuclei and cytoplasm of myoblasts (MB) and myotubes (MT) after treatment with H2O2 for 24 hours. Nuclei are counterstained with DAPI (blue). The data show a loss of p21 (red staining) over the nuclei after treatment and several nuclei with no p21 after treatment with H2O2. Less p21 staining is associated with the nucleus (DAPI) after 24 hours of treatment.
Figure 5
Figure 5. Effects of resveratrol on protein loss and apoptotic signaling in response to oxidative stress
A. Total protein loss in myoblasts (expressed relative to control untreated cells) after 24 h or 48 h of incubation in 0 or 0.1 mM (white bars) and 0 or 1.0 mM (black bars) of H2O2 and 0–50 μM of resveratrol. *P<0.05 significantly different from non-treatment control myoblasts (0 mM resveratrol and 0 mM and H2O2) at 24 h or 48 h of incubation, respectively. †, P<0.05, Treatment group is significantly different from 0.1 mM or 1 mM H2O2 + 0 mM resveratrol at 24 or 48 h of incubation. B. Apoptotic index (expressed as a percent of total nuclei) myoblasts after 24 h of incubation in 0 or 1 mM (white bars) or 1.0 mM (black bars) of H2O2 and 0–50 μM of resveratrol. *P<0.05 significantly different from non-treatment control myoblasts (0 mM resveratrol and 0 mM and H2O2) at 6 or 12 h of incubation, respectively. †, P<0.05, Treatment group is significantly different from 0.1 mM or 1 mM H2O2 + 0 mM resveratrol at 6 or 12 h of incubation. Bax (C), Bcl-2 (D) and AIF (F) protein levels expressed relative to untreated control cells incubated in 0 or 1 mM of H2O2 and 0–50 μM of resveratrol for 24 h. E. The Bax/Bcl-2 ratio. P<0.05 significantly different from non-treatment control myoblasts (0 mM resveratrol and 0 mM and H2O2) at 6 or 12 h of incubation, respectively. †, P<0.05, Treatment group is significantly different from 1 mM H2O2 + 0 mM resveratrol at 6 or 12 h of incubation.
Figure 6
Figure 6. Resveratrol reduces caspase activity in myoblasts after 6 or 12 hours of H2O2-induced oxidative stress
The activity of caspase-9 (A,B), caspase-8 (C,D) and caspase-3 (E,F), was determined after 6 (A,C,E) or 12 (B,D,F) hours of incubation in 0–1 mM of H2O2 and 0–50 μM of resveratrol. *P<0.05 significantly different from non-treatment control myoblasts (0 mM resveratrol and 0 mM and H2O2) at 6 or 12 h of incubation, respectively. †, P<0.05, Treatment group is significantly different from 1 mM H2O2 + 0 mM resveratrol at 6 or 12 h of incubation.
Figure 7
Figure 7. Resveratrol suppresses caspase activity in myoblasts after 24 or 48 hours of oxidative stress induced by H2O2
The activity of caspase-9 (A,B), caspase-8 (C,D) and caspase-3 (E,F), was determined after 24 (A,C,E) or 48 (B,D,F) hours of incubation in 0–1 mM of H2O2 and 0–50 μM of resveratrol.*P<0.05 significantly different from non-treatment control myoblasts (0 mM resveratrol and 0 mM and H2O2) at 6 or 12 h of incubation, respectively. †, P<0.05, Treatment group is significantly different from 1 mM H2O2 + 0 mM resveratrol at 24 or 48 h of incubation.
Figure 8
Figure 8. Regulation of Sirt1 protein by oxidative stress
The figure shows representative blots of Sirt1 protein abundance after 24 h incubation in 0 mM or 1 mM of H2O2 and 0–50 μM of resveratrol. The protein abundance (expressed relative to untreated control cells) of Sirt1 was determined by western blots. The myoblasts were incubated in 0 or 1 mM of H2O2 and 0–50 μM of resveratrol. The protein bands was normalized to GAPDH/*P<0.05 significantly different from non-treatment control myoblasts (0 mM resveratrol and 0 mM and H2O2) at 6 or 12 h of incubation, respectively. †, P<0.05, Treatment group is significantly different from 0.1 mM or 1 mM H2O2 + 0 mM resveratrol at 24 or 48 hours of incubation.
Figure 9
Figure 9. Fluorescence Activated Cell Sorting (FACS) separation of the Syn4 population of MSCs from mouse muscle
A. The pre-sort FACS data show that 20.2% of the gated population of MSC cells isolated from mouse gastrocnemius muscles expressed syndecan-4 (Syn4). The post-sort data show that the population was 97% pure. These represent a relevant muscle stem cell (MSC) population. Post-sort verification showed that 98.2% of the cells were negative for Syn4 in population sorted to be syndecan-4 negative (data not shown). Post-sort analysis of sorted cells which were identified from the 20.2% population of pre-sorted cells) as Syn4 positive, were 97.0% positive for Syn4. These data show that we have established the methods to isolate and evaluate Syn4 MSC cells from mouse skeletal muscles. B. Sirt1 activity in Syn4 myotubes. Syn4 cells were examined in response to H2O2 (1 mM), and/or FK866 (50nM), and/or resveratrol for 48 hours. * P<0.05, significantly different from control untreated conditions. **, NMN treatment was greater than all other conditions. †, P<0.05, significantly different from FK866 treated cells.
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References

    1. Alamdari N, Aversa Z, Castillero E, Gurav A, Petkova V, Tizio S, et al. Resveratrol prevents dexamethasone-induced expression of the muscle atrophy-related ubiquitin ligases atrogin-1 and MuRF1 in cultured myotubes through a SIRT1-dependent mechanism. Biochem Biophys Res Commun. 2012;417:528–533. - PMC - PubMed
    1. Allen RG, Tresini M. Oxidative stress and gene regulation. Free Radic Biol Med. 2000;28:463–499. - PubMed
    1. Alway SE, Bennett BT, Wilson JC, Sperringer J, Mohamed JS, Edens NK, et al. Green tea extract attenuates muscle loss and improves muscle function during disuse, but fails to improve muscle recovery following unloading in aged rats. J App Physiol. 2015;118:319–330. - PMC - PubMed
    1. Alway SE, Degens H, Krishnamurthy G, Smith CA. Potential role for Id myogenic repressors in apoptosis and attenuation of hypertrophy in muscles of aged rats. Am J Physiol Cell Physiol. 2002;283:C66–C76. - PubMed
    1. Alway SE, Degens H, Lowe DA, Krishnamurthy G. Increased myogenic repressor Id mRNA and protein levels in hindlimb muscles of aged rats. Am J Physiol Regul Integr Comp Physiol. 2002;282:R411–R422. - PMC - PubMed

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