BIO101 stimulates myoblast differentiation and improves muscle function in adult and old mice

Abstract

Background: Muscle aging is associated with a consistent decrease in the ability of muscle tissue to regenerate following intrinsic muscle degradation, injury or overuse. Age-related imbalance of protein synthesis and degradation, mainly regulated by AKT/mTOR pathway, leads to progressive loss of muscle mass. Maintenance of anabolic and regenerative capacities of skeletal muscles may be regarded as a therapeutic option for sarcopenia and other muscle wasting diseases. Our previous studies have demonstrated that BIO101, a pharmaceutical grade 20-hydroxyecdysone, increases protein synthesis through the activation of MAS receptor involved in the protective arm of renin-angiotensin-aldosterone system. The purpose of the present study was to assess the anabolic and pro-differentiating properties of BIO101 on C2C12 muscle cells in vitro and to investigate its effects on adult and old mice models in vivo.

Methods: The effects of BIO101 on C2C12 differentiation were assessed using myogenic transcription factors and protein expression of major kinases of AKT/mTOR pathway by Western blot. The in vivo effects of BIO101 have been investigated in BIO101 orally-treated (50 mg/kg/day) adult mice (3 months) for 28 days. To demonstrate potential beneficial effect of BIO101 treatment in a sarcopenic mouse model, we use orally treated 22-month-old C57Bl6/J mice, for 14 weeks with vehicle or BIO101. Mice body and muscle weight were recorded. Physical performances were assessed using running capacity and muscle contractility tests.

Results: Anabolic properties of BIO101 were confirmed by the rapid activation of AKT/mTOR, leading to an increase of C2C12 myotubes diameters (+26%, P < 0.001). Pro-differentiating effects of BIO101 on C2C12 myoblasts were revealed by increased expression of muscle-specific differentiation transcription factors (MyoD, myogenin), resulting in increased fusion index and number of nuclei per myotube (+39% and +53%, respectively, at day 6). These effects of BIO101 were like those of angiotensin (1-7) and were abolished with the use of A779, a MAS receptor specific antagonist. Chronic BIO101 oral treatment induced AKT/mTOR activation and anabolic effects accompanied with improved physical performances in adult and old animals (maximal running distance and maximal running velocity).

Conclusions: Our data suggest beneficial anabolic and pro-differentiating effects of BIO101 rendering BIO101 a potent drug candidate for treating sarcopenia and possibly other muscle wasting disorders.

Keywords: 20-hydroxyecdysone (20E); Ecdysteroids; MAS receptor; Muscle cell differentiation; Renin-angiotensin-aldosterone system (RAAS); Sarcopenia.

Figure 1

Anabolic and metabolic effects of BIO101 on C2C12 myotubes. (A, left panels): MHC staining of 8 day‐differentiated myotubes without drug and in the presence of 5 μM BIO101 for last 2 days, scale bar represents 50 μm. (A, right panel): Myotube diameters after 2 days of treatment with 5 μM BIO101. N = 9 independent experiments. (B) Effects of BIO101 on AKT/mTOR pathway in C2C12 myotubes detected by Western blot. (B, left panel): Representative examples of six independent experiments, of AKT and P70S6K phosphorylation in response to BIO101 exposure (10 μM) in C2C12 myotubes. (B, right panels): Densitometric analysis of AKT and P70S6K phosphorylation relative to non‐phosphorylated protein, vinculin housekeeping protein and untreated control for each timepoint. (C) Effects of BIO101 on AMPK pathway in C2C12 myotubes detected by Western blot. (C, left panel): Representative examples of three independent experiments, of AMPKα and ACC phosphorylation in response to BIO101 exposure (10 μM) in C2C12 myotubes. (C, right panels): Densitometry analysis of AMPKα and ACC phosphorylation relative to non‐phosphorylated protein, vinculin housekeeping protein and untreated control for each timepoint. Data are presented as mean ± SEM. *P < 0.05 and ***P < 0.001.

Figure 2

BIO101 activates AKT/mTOR and AMPK pathways at early stage of differentiation process. (A) Effects of BIO101 on AKT/mTOR pathway in C2C12 myoblasts initiated for differentiation detected by Western blot. (A, left panel): Representative examples of five independent experiments, of AKT and S6 phosphorylation in response to BIO101 exposure (10 μM). (A, right panels): Densitometric analysis of kinase phosphorylation relative to non‐phosphorylated protein, vinculin housekeeping protein and untreated control for each timepoint. (B) Effects of BIO101 on AMPK pathway in C2C12 myoblasts initiated for differentiation detected by Western blot. (B, left panel): Representative examples of three individual Western blot experiments. (B, right panels): Densitometric analysis of AMPKα and ACC kinase phosphorylation. Data are presented as mean ± SEM. *P < 0.05.

Figure 3

Effects of BIO101 on C2C12 cells differentiation. (A) MHC staining of C2C12‐derived differentiated myotubes without drug or treated with 5 μM BIO101 for 6 days. Scale bars represents 50 μM and quantification of myotubes diameters, fusion index and number of nuclei per myotube after 6 days of differentiation in the presence of 0.1–5 μM BIO101. N = at least five independent experiments. (B) Distribution of plurinucleate myotubes after 5 μM BIO101 treatment. (C) Kinetics of MyoD, Myogenin and MHC expression by Western blot. (D, E) Kinetics of MyoD (n = 3) and myogenin (n = 5) by immunofluorescence in C2C12 during differentiation. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 4

Effects of MAS inhibition with A779 on BIO101‐ and Ang1–7‐stimulated C2C12 differentiation. A‐C ‐ fusion index (A), number of nuclei per myotubes (B) and myotube diameters (C) were measured after 6‐day differentiation in the presence of the drugs. (D–F) Biological response versus target engagement ratio or TER (ratio between drug concentration and EC₅₀) for fusion index (D), number of nuclei per myotube (E) and diameters (F) measured after 6‐day differentiation. Each bar corresponds to the mean ± SEM of at least three independent experiments. Data are presented as mean ± SEM. *P < 0.05, and ***P < 0.001 compared with control, ^# P < 0.05 compared with BIO101 alone.

Figure 5

Effects of BIO101 in adult and aged mice. Old C57Bl6/J mice (22 months) were orally treated for 14 weeks with either vehicle (n = 7) or 50 mg/kg/day of BIO101 (n = 9). Adult C57Bl6/J mice (12 months; n = 10) has been used as control and were orally treated with vehicle. (A) Delta of weight gain of adult mice (n = 9), and vehicle (n = 9) or BIO101‐treated old mice (n = 10) orally during 14 weeks. (B) Gastrocnemius muscle weight of adult mice (n = 9), old vehicle (n = 9) or BIO101‐treated (n = 10) mice orally during 14 weeks. (C) Maximal running velocity. (D) Western blot and densitometry analysis of protein extracts from gastrocnemius muscles for (p)‐AKT, (p)‐P70S6K, and MyoD expression. (E) Western blot and densitometry analysis of protein extracts from gastrocnemius muscles for (p)‐AMPKα and (p)‐ACC expression. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.