The Journal Of Nutritional Biochemistry 

Resveratrol regulates muscle fiber type conversion via miR-22-3p and AMPK/SIRT1/PGC-1α pathway
Wanxue Wen, Xiaoling Chen, Zhiqing Huang, Daiwen Chen, Hong Chen, Yuheng Luo, Jun He, Ping Zheng, Jie Yu, Bing Yu

To appear in: The Journal of Nutritional Biochemistry

Please cite this article as: W. Wen, X. Chen, Z. Huang, et al., Resveratrol regulates muscle fiber type conversion via miR-22-3p and AMPK/SIRT1/PGC-1α pathway, The Journal of Nutritional Biochemistry(2019), https://doi.org/10.1016/j.jnutbio.2019.108297
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Resveratrol regulates muscle fiber type conversion via miR-22-3p and AMPK/SIRT1/PGC-1 pathway

Wanxue Wen 1,§, Xiaoling Chen 1,§, Zhiqing Huang 1,*, Daiwen Chen 1, Hong Chen 2,

Yuheng Luo 1, Jun He 1, Ping Zheng 1, Jie Yu 1, Bing Yu 1

1 Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, Sichuan 611130, P. R. China

2 College of Food Science, Sichuan Agricultural University, Yaan, Sichuan 625014, P.

R. China

 These authors contributed equally to this work.

Correspondence: Zhiqing Huang, Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu, Sichuan 611130, P. R. China; Tel/Fax: 86-28-86290976; E-mail: [email protected].

Abstract

This study investigated the effects of resveratrol and miR-22-3p on muscle fiber type conversion in mouse C2C12 myotubes. Here we showed that resveratrol significantly increased the protein level of slow myosin heavy chain (MyHC) and the activities of succinic dehydrogenase and malate dehydrogenase, as well as markedly decreased the protein level of fast MyHC and the activity of lactate dehydrogenase. Immunofluorescence staining showed that resveratrol remarkably upregulated the number of slow MyHC-positive myotubes and downregulated the number of fast MyHC-positive myotubes, suggesting that resveratrol promoted muscle fiber type conversion from fast-twitch to slow-twitch in C2C12 myotubes. We also showed that miR-22-3p had an opposite function on muscle fiber type conversion and resveratrol was able to repress the expression of miR-22-3p. Furthermore, AMP-activated protein kinase (AMPK) inhibitor Compound C and miR-22-3p mimics could attenuate and eliminate muscle fiber type conversion from fast-twitch to slow-twitch cause by resveratrol, respectively. Together, we provided the first evidence that resveratrol promotes muscle fiber type conversion from fast-twitch to slow-twitch via miR-22-3p and AMPK/SIRT1/PGC-1 pathway in C2C12 myotubes.
Keywords: Resveratrol; muscle fiber type conversion; C2C12 myotubes; miR-22-3p; AMPK/SIRT1/PGC-1 pathway

1. Introduction

Skeletal muscle is a multifunctional tissue contributed to various biological processes[1]. Skeletal muscle and nervous system jointly control body movement[2-4]. Skeletal muscle dysfunction is related with many diseases, such as muscle dystrophy, diabetes and other metabolic disorders[5-8]. It has been widely understood that skeletal muscle is composed of different types of muscle fibers. Based on its contractile property, skeletal muscle fiber types are divided into slow oxidative fiber [type I fiber expressing myosin heavy chain (MyHC) I] and fast glycolytic/oxidative fibers (type II fibers expressing MyHC IIa, MyHC IIx or MyHC IIb), and muscle fiber types can transform from type I fiber to type II fibers or vice versa. Type I fiber has higher oxidative ability and type II fibers have higher glycolysis, suggesting that skeletal muscle fiber conversion and metabolic change are important for body health[9-12].
Skeletal muscle development is regulated by nutrients, microRNAs and many other factors[1, 13-15]. Resveratrol, a natural polyphenolic compound, can directly affect skeletal muscle metabolism and development[16-18]. Resveratrol was reported to increase mitochondrial size, citrate synthase and muscle endurance ability in mice fed with high-fat diet[19]. Moreover, resveratrol induced more expression of the slow oxidative phenotype in mdx mice muscle [20]. Resveratrol supplementation promoted MyHC IIa mRNA level and inhibited MyHC IIb mRNA level in finishing pigs[21]. These results indicated that resveratrol regulated muscle fiber composition and affected muscle function. However, the role of resveratrol in muscle fiber transformation and its underlying mechanisms are still unclear.

MicroRNAs are a conserved class of noncoding RNAs involved in a variety of biological processes, such as cell proliferation, differentiation and apoptosis[22-24]. In skeletal muscle development, miR-22-3p overexpression promoted myoblast differentiation and repressed myoblast proliferation[25, 26]. However, the effect of miR-22-3p on muscle fiber type conversion is still unknown. A recent study indicated that a total of 26 miRNAs expression were increased in resveratrol-supplemented group, while a total of 20 miRNAs expression were decreased[27]. Moreover, resveratrol down-regulated miR-133 level, contributed to myoblast differentiation by targeted the transcript encoding the serum-controlled factor[28]. Nevertheless, whether miR-22-3p attributes to the effect of resveratrol on skeletal muscle fiber type conversion is unclear.
In this study, we investigated the effect and potential mechanism of resveratrol on muscle fiber type transformation in C2C12 myotubes. To the best of our knowledge, this study revealed for the first time the mechanism of resveratrol promoting the conversion of muscle fiber type from fast-twitch to slow-twitch and illustrated the relationship between resveratrol and miR-22-3p.
2. Materials and methods

2.1 Ethics statement

The experimental procedures were approved by the Animal Care Advisory Committee of Sichuan Agricultural University.
2.2 Tissue samples collection

Eight-week-old male C57/BL6J mice were purchased from Dashuo Experimental

Animal Co. Ltd (Chengdu, China). The heart, liver, spleen, lung, kidney, skeletal longissimus dorsi muscle, tibialis anterior (TA) muscle, extensor digitorum longus (EDL) muscle and soleus (SOL) muscle were harvested for miR-22-3p expression analysis.
2.3 Cell culture and treatment

The mouse C2C12 myoblast cell line was maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Paisley, Scotland, UK) at 37 ℃ and 5% CO2. Cells were induced to differentiate in differentiation medium containing 2% horse serum (HS) (Gibco) after the density reached about 80% fusion. C2C12 myotubes were treated with different concentrations of resveratrol (0 µM, 12.5 µM, 25 µM, 50 µM) (purity≥99%, Sigma, St. Louis, MO, USA) for 3 days. The differentiation medium and resveratrol were strictly exchanged every day.
To illuminate the mechanism of resveratrol on skeletal muscle transformation, C2C12 myotubes were treated with AMP-activated protein kinase (AMPK) inhibitor compound C (50 µM, Calbiochem, Germany) prior to adding resveratrol as described above.
2.4 Cell transfection

C2C12 cells were induced to differentiate when the cells reached about 80% confluence. After three days of differentiation, C2C12 cells were transfected using Lipofectamine 3000 (Invitrogen, USA) according to the standard protocol. To examine the effect of miR-22-3p on skeletal muscle fiber type conversion, C2C12

myotubes were transfected with 200 nM miR-22-3p mimics or 400 nM miR-22-3p inhibitor (GenePharma, Shanghai, China). Twenty-four hours after transfection, the medium was renewed to fresh differentiation medium. Myoblasts were continued to differentiate for 3 days and then harvested for further analysis.
2.5 Western blotting

Radio Immunoprecipitation Assay lysis buffer (Beyotime, China) was used to extracted protein samples from C2C12 myotubes according to the manufacturer’s instructions. Firstly, C2C12 myotubes were lysed in lysis buffer for 30 min and the liquid supernatant was collected by centrifugation at 14,000 g for 20 min at 4 ℃. Cell protein concentrations were determined by BCA protein assay kit (Pierce, USA) and Nano-Drop ND 2000c Spectrophotometer (Thermo Scientific, USA). Lysates (30 µg) were separated by 8% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore Eschborn, Germany) using wet Trans-Blot system (Bio-Rad, Hercules, CA, USA). After blocking with 5% bovine serum albumin for 3 hours, membranes were incubated with the following primary antibodies in a shaker at 4 ℃ overnight: slow MyHC (Sigma, Cat. No. M8421), fast MyHC (Sigma, Cat. No. M4276), MyHC IIa (DSHB, Cat. No. SC-71), MyHC IIb (DSHB, Cat. No. 10F5),
p-AMPK (Cell Signaling, Cat. No. 2535), AMPK (Cell Signaling, Cat. No. 5831), SIRT1 (Cell Signaling, Cat. No. 8469), PGC-1 (Cell Signaling, Cat. No. 2178), β-actin (Cell Signaling, Cat. No. 4967) and β-tubulin (Cell Signaling, Cat. No. 2128). Membranes were then washed and incubated with second antibodies for one hour and a half. The signals were visualized with BeyoECL Plus (Beyotime, China). Protein

expressions were calculated by Gel-Pro Analyzer and were normalized to β-actin or β-tubulin.
2.6 Immunofluorescence

C2C12 myotubes were washed three times with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (Beyotime, China) for 15 min. After the following washes, C2C12 myotubes were permeabilized with 0.5% Triton X-100 for 20 min and then blocked with blocking buffer (Beyotime, China) for 3 hours at 37 ℃. Cells were incubated with the primary antibodies including slow MyHC (1:50, Sigma, Cat. No. M8421), fast MyHC (1:50, Sigma, Cat. No. M4276) for 16 hours at 4℃. After washing three times with PBS, cells were incubated with the fluorescent secondary antibody (1:1000, Cell Signaling, USA) for 2 hours at 37℃, followed by 4,6-diamidino-2-phenylindole (DAPI) (Beyotime, China) for 1 hour at room temperature. Images were captured using a fluorescence microscope (version 6.0.0.260; Media Cybernetics, Inc.).
2.7 Metabolic enzyme assay

Briefly, C2C12 myotubes were taken using scraper, lysed in PBS, centrifuged at 2000 rpm for 20 min and collected the liquid supernatant for further analysis. Cell protein concentrations were determined by BCA protein assay kit (Pierce, USA) and Nano-Drop ND 2000c Spectrophotometer (Thermo Scientific, USA). Succinic dehydrogenase (SDH) activity, malate dehydrogenase (MDH) activity and lactate dehydrogenase (LDH) activity were measured using commercial kits according to the manufacturers’ instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing,

China). AMPK ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd, Shanghai) was used to test phosphorylated AMPK (p-AMPK) concentration in mouse C2C12 myoblasts. The activities of specific enzyme were defined as U/mg of protein units.
2.8 RNA isolation and real-time quantitative PCR

Total RNA was fully collected from mouse C2C12 myotubes using RNAiso Plus reagent (TaKaRa, China). Nano-Drop ND 2000c Spectrophotometer (Thermo Scientific, USA) was also used to measure the concentrations of RNA. Reverse transcription of mRNA and miRNA was done with a PrimeScript ® RT reagent Kit with gDNA Eraser (TaKaRa, China). Relative quantification of mRNA and miRNA was performed on a 7900HT real-time PCR system (384-cell standard block) (Applied Biosystem, Foster, CA, USA) with SYBR select Master Mix (Applied Biosystem). The primer sequences used are listed in Table 1. Relative gene expression was assessed by 2-ΔΔCt method and normalized to GAPDH or U6 snRNA.
2.9 Statistical analysis

All data are expressed as means and standard errors. SPSS 21.0 software (Chicago, IL, USA) was used for statistical analysis. One-way analysis of variance and Tukey’s
test were used to analyze the significance of results. P＜.05 was defined as a
significant difference.

3. Results

3.1 Resveratrol regulates muscle fiber type conversion in C2C12 myotubes

To investigate whether resveratrol regulates muscle fiber type conversion, we

firstly used mouse C2C12 myotubes to verify its effects. As shown in Fig. 1A, resveratrol significantly increased the slow MyHC protein level and markedly decreased the fast MyHC protein level. Immunofluorescence staining showed that resveratrol remarkably upregulated the number of slow MyHC-positive myotubes and downregulated the number of fast MyHC-positive myotubes (Fig. 1B). We also got similar results by real-time quantitative PCR (Fig. 1C). Collectively, these findings suggested that resveratrol regulated muscle fiber type conversion from fast-twitch to slow-twitch in C2C12 myotubes.
3.2 Effect of resveratrol on metabolic enzyme activities

Moreover, we examined the oxidative metabolic enzyme activities of SDH and MDH and the glycolytic metabolic enzyme activity of LDH in C2C12 myotubes. As shown in Fig. 2, resveratrol increased SDH and MDH activities and decreased LDH activity in a dose-dependent manner, further demonstrating that resveratrol promoted muscle fiber type conversion from fast-twitch to slow-twitch in C2C12 myotubes.
3.3 Resveratrol regulates muscle fiber type conversion through AMPK/SIRT1/PGC-1 pathway in C2C12 myotubes
To further confirm the effects of resveratrol on AMPK/SIRT1/PGC-1 pathway,

Western blotting and real-time quantitative PCR were used for detection. As shown in Fig. 3A-C, resveratrol treatment observably enhanced p-AMPK, SIRT1 and PGC-1 protein and mRNA levels at a final concentration of 50µM and concurrently increased AMPK activity.
Previous findings illustrated that AMPK may be a biological target of resveratrol[29,

30]. To further assure the role of AMPK in the regulation of muscle fiber type transformation by resveratrol, mouse C2C12 myotubes were treated with AMPK inhibitor Compound C and resveratrol. We found that Compound C efficiently attenuated the effect of resveratrol on promoting slow MyHC protein level and inhibiting fast MyHC protein level (Fig. 3D). Moreover, Compound C attenuated resveratrol-induced increase in the number of slow MyHC-positive myotubes (Fig. 3E). Collectively, these results suggested that resveratrol regulated muscle fiber type conversion through AMPK/SIRT1/PGC-1 pathway in C2C12 myotubes.
3.4 miR-22-3p regulates muscle fiber type conversion in C2C12 myotubes

First, we analyzed the miR-22-3p level in various tissues as well as fast-twitch and slow-twitch muscles of mice. We found that miR-22-3p expression was most abundant in the skeletal muscle and the expression level of miR-22-3p in mouse fast-twitch TA and EDL muscles was significantly higher than that in slow-twitch SOL muscle (Fig. 4), indicating that miR-22-3p might be associated with skeletal muscle fiber type conversion. To investigate the effects of miR-22-3p on muscle fiber type transformation, we performed overexpression experiments and knockdown experiments in C2C12 myotubes. Real-time quantitative PCR assay showed that miR-22-3p mimics increased while miR-22-3p inhibitor decreased the level of miR-22-3p (Fig. 5A). Western blotting assay showed that overexpression of miR-22-3p markedly decreased MyHC I (also known as slow MyHC) and MyHC IIa protein levels and increased MyHC IIb protein level, while inhibition of miR-22-3p led to the opposite results (Fig. 5B). Immunofluorescence staining showed that

overexpression of miR-22-3p reduced the number of slow MyHC-positive myotubes, whereas inhibition of miR-22-3p got the opposite result (Fig. 5C).
3.5 Effect of resveratrol on miR-22-3p expression

The miR-22-3p level in resveratrol-treated C2C12 myotubes was detected by real-time quantitative PCR. As shown in Fig. 6, resveratrol markedly decreased miR-22-3p level.
3.6 miR-22-3p contributes to resveratrol-induced muscle fiber type conversion in C2C12 myotubes
Next, we investigated whether miR-22-3p contributes to resveratrol-induced muscle fiber type conversion in C2C12 myotubes. To accomplish this aim, C2C12 myotubes were transfected with miR-22-3p mimics followed by resveratrol treatment. As shown in Fig. 7, resveratrol increased MyHC I and MyHC IIa protein levels as well as decreased MyHC IIb protein level, whereas miR-22-3p mimics attenuated the effect of resveratrol on MyHC I, MyHC IIa and MyHC IIb expressions.
4. Discussion

It has been reported that resveratrol regulates energy metabolism and skeletal muscle function[31-33]. Change of skeletal muscle fiber types contributed to muscle metabolism and function[34, 35]. Extensive research papers to date highlight the truth that resveratrol is one of the major factors influencing skeletal muscle phenotype[36, 37]. In this study, we found that resveratrol increased the number of slow MyHC-positive myotubes and decreased the number of fast MyHC-positive myotubes, accompanied by increased slow MyHC protein level and decreased fast MyHC protein level.

Additionally, the remodeling of diverse muscle fiber types is essential for muscle energy metabolism. Slow-twitch oxidative muscle fibers contain higher oxidative enzymes (SDH, MDH) activities, while fast-twitch glycolytic muscle fibers have higher glycolytic enzyme (LDH) activity. Alteration in muscle fiber component is indirectly subject to the alteration by enzyme activities in energy metabolism[20, 38, 39]. Our data in vitro experiment showed that resveratrol increased SDH and MDH activities, but decreased LDH activity, confirming that resveratrol altered muscle fiber type in C2C12 myotubes.
It is well established that both AMPK and SIRT1 play a vital role in muscle fiber types conversion and muscle energy metabolism[40-42]. Resveratrol could activate AMPK and SIRT1. Knockdown of AMPK neutralized the effect of resveratrol on triggering SIRT1 activity[30, 43]. Furthermore, SIRT1 regulated cellular energy metabolism based on NAD+ availability. NAD+, a rate-limiting factor, was initiated by AMPK activation[44]. The above results illustrated that the activation of AMPK was
prior to SIRT1. PGC-1, the only target of SIRT1, was reported to promote the formation of slow-twitch fiber[19]. In our study, we showed that resveratrol affected AMPK/SIRT1/PGC-1 pathway and Compound C attenuated the effect of resveratrol on increasing slow MyHC protein and decreasing fast MyHC protein, suggesting that resveratrol regulated muscle fiber transformation though the AMPK/SIRT1/PGC-1 pathway in C2C12 myotubes.
MicroRNAs, a kind of single chain noncoding RNAs, are involved in a variety of biological processes. It has been reported that miR-22 was able to inhibit muscle cell

proliferation and promote differentiation[25]. In this study, we showed that miR-22-3p was abundant in fast-twitch muscles. We also showed that overexpression of miR-22-3p promoted MyHC IIb protein level and suppressed MyHC I and MyHC IIa protein levels, while knockdown of miR-22-3p obtained a reverse result. Immunofluorescent assay further confirmed that overexpression of miR-22-3p caused a decrease in the number of slow MyHC-positive myotubes, whereas inhibition of miR-22-3p got the opposite result. Taken together, these findings suggested that miR-22-3p promoted muscle fiber type conversion from slow-twitch to fast-twitch in vitro. It is well known that nutrients could regulate the expression of microRNAs. In this study, we showed that resveratrol inhibited miR-22-3p expression. Interestingly, we also found that overexpression of miR-22-3p repressed muscle fiber type conversion from fast-twitch to slow-twitch cause by resveratrol, indicating that miR-22-3p contributed to resveratrol-induced muscle fiber type conversion in C2C12 myotubes.
In conclusion, we provided the first SRT501 evidence that resveratrol promotes muscle fiber type conversion from fast-twitch to slow-twitch via miR-22-3p and AMPK/SIRT1/PGC-1 pathway in C2C12 myotubes.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the National Key R&D Program of China (No.

2018YFD0500403).

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Fig. 1. Resveratrol regulates muscle fiber type conversion in C2C12 myotubes. C2C12 myotubes cultured in differentiated medium were treated with resveratrol for 3 days. (A) Western blotting measured the expression levels of slow MyHC and fast

MyHC. (B) Immunofluorescence analyzed slow MyHC and fast MyHC expression. Magnification:  200. (C) Real-time quantitative PCR measured MyHC I, MyHC IIa, MyHC IIx, MyHC IIb, TNNI1, TNNC1 and TNNT1 mRNA levels. Data were means ±
SE from three independent experiments. *P＜.05, **P＜.01 and ***P＜.001 as
compared with control group.

Fig. 2. Effect of resveratrol on metabolic enzyme activities. C2C12 myotubes cultured in differentiated medium were treated with resveratrol for 3 days. SDH, MDH and LDH activities were measured by commercial kits. Data were means ± SE from three

independent experiments. *P＜.05, **P＜.01 and ***P＜.001 as compared with
control group

Fig. 3. Resveratrol regulates muscle fiber type conversion though

(E)IRT1/PGC-1 pathway in C2C12 myotubes. (A) C2C12 myotubes culturedin differentiated medium were treated with resveratrol for 3 days. Western blotting measured the expression levels of p-AMPK, SIRT1 and PGC-1. (B) C2C12 myotubes cultured in differentiated medium were treated with resveratrol for 3 days. Real-time quantitative PCR measured PGC-1, SIRT1, AMPK1 and AMPK2 mRNA levels. (C) C2C12 myotubes cultured in differentiated medium were treated with resveratrol for 3 days. ELISA measured AMPK enzyme activity. (D) C2C12

myotubes cultured in differentiated medium were pretreated with Compound C (50 µM) for 1 h and then treated with resveratrol (50 µM) for 3 days. Western blotting measured the expression levels of AMPK, p-AMPK, slow MyHC and fast MyHC. (E) C2C12 myotubes cultured in differentiated medium were pretreated with Compound

C (50 µM) for 1 h and then treated with resveratrol (50 µM) for 3 days.

Immunofluorescence analyzed slow MyHC expression. Magnification:  200. Data

were means ± SE from three independent experiments. **P＜.01 and ***P＜.001 as compared with control group.

Fig. 4. Relative miR-22-3p expression in mouse different tissues and different type of skeletal muscles. Real-time quantitative PCR measured miR-22-3p expression. Data

were means ± SE from three mice. **P＜.01 and ***P＜.001.
Fig. 5. miR-22-3p regulates skeletal muscle fiber type conversion in C2C12 myotubes. C2C12 myotubes cultured in differentiation medium were transfected with 200 nM miRNA mimics Neg. control, 200 nM miR-22-3p mimics, 400 nM miRNA inhibitor Neg. control or 400 nM miR-22-3p inhibitor for 3 d. (A) Real-time quantitative PCR measured the miR-22-3p level. (B) Western blotting measured the expression levels of MyHC I (slow MyHC), MyHC IIa and MyHC IIb. (C) Immunofluorescence analyzed slow MyHC expression. Magnification:  200. Data were means ± SE from three

independent experiments. *P＜.05, **P＜.01 and ***P＜.001 as compared with
control group.

Fig. 6. Effect of resveratrol on miR-22-3p expression level. C2C12 myotubes cultured in differentiated medium were treated with resveratrol for 3 days. Real-time quantitative PCR measured the miR-22-3p level. Data were means ± SE from three

independent experiments. **P＜.01 and ***P＜.001 as compared with control group.

Fig. 7. miR-22-3p contributes to resveratrol-induced skeletal muscle fiber type conversion. C2C12 myotubes cultured in differentiation medium were transfected with 200 nM miRNA mimics Neg. control or 200 nM miR-22-3p mimics. After 24 h of transfection, 50 µM resveratrol was added to differentiation medium simultaneously for another 3 d. Western blotting analyzed MyHC I (slow MyHC), MyHC IIa and MyHC IIb protein levels. Data were means ± SE from three

independent experiments. *P＜.05, **P＜.01 and ***P＜.001 as compared with control group.

Table 1. List of genes, primer sequences, GenBank accession numbers, and product sizes in this study