Beta-agonist drugs modulate the proliferation and differentiation of skeletal muscle cells in vitro

Abstract

Essentially employed for the treatment of airway obstructions in humans, β-agonists are also known to have an anabolic effect in animals' skeletal muscle. In vivo and in vitro studies have attested the increase in animal body mass and the hypertrophy of muscle cells following the administration of specific β-agonists. However, the contribution of β-agonists to C2C12 myoblasts growth remains obscure. We therefore aimed to investigate the impact of β1-and β2-agonist drugs on the proliferation and differentiation of skeletal muscle cells. Direct observations and cytotoxicity assay showed that clenbuterol, salbutamol, cimaterol and ractopamine enhanced muscle cell growth and viability during the proliferation stage. Structural examinations coupled to Western blot analysis indicated that salbutamol and cimaterol triggered a decrease in myotube formation. A better comprehension of the effect of β-agonists on myogenic regulatory genes in the muscle cells is crucial to establish a specific role of β-agonists in muscle development, growth, and regeneration.

Keywords: Agonist drugs; C2C12; DM, Differentiation Medium; Differentiation; GM, Growth medium; MyHC, Myosin heavy chain; MyoD, Myoblast determination protein 1; Proliferation.

Fig. 1

Beta-agonists effect on cAMP production and proliferation of C2C12. C2C12 cells treated with clenbuterol, salbutamol, cimaterol, and ractopamine at the final concentration of (A) 10⁻⁶ M and (B) 10⁻⁷ M for 30 min. Data are presented as mean ± SEM of three independent replicates and comparison between treated and untreated cells were assessed by using a Student's t-test. (C) MTT assay performed in triplicate at specific time points, from day 1 to day 8. Data are presented as mean ± SEM of four independent experiments with Two-way ANOVA analysis. (*) p < 0.05.

Fig. 2

C2C12 cells culture protocol and bright field observation. Photographs of myoblast cells treated with 10⁻⁶ M clenbuterol, salbutamol, cimaterol, ractopamine and control cells were taken with Nikon eclipse TS 100 microscope at 10x magnification. Yellow stars show the specific time when the pictures were taken following the culture protocol. Scale bar: 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Morphological changes induced by β-agonist drugs. Hematoxylin and eosin staining of (A) myoblasts at differentiation day 0 and myotubes at differentiation day 7. Myotubes with darker pink cytoplasm contained multiple nuclei indicated in black arrows. Scale bar: 100 μm. (B) Myotubes fusion index represented the ratio of average nuclei inside myotubes to the total nuclei for six random fields/coverslip. (C) Average number of myotubes formed per field for each group of drug treatment. (D) Average number of nuclei within the myotubes. Data are presented as mean ± SEM of three independent experiments. Student's t-test was performed for statistical analysis. (*) p < 0.05, n. s: non-significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Effect of β-agonists on myogenic proteins expression during cell differentiation. (A) Western blot imaging of proteins extracted at differentiation days 0, 1, 4, and 7 from cells exposed to drugs at seeding. Densitometry analysis of (B) MyHC, (C) α-actinin-2, (D) MyoD, and (E) myogenin proteins normalized against GAPDH. Data are presented as mean ± SEM of three independent biological replicates. Ct: control, Cl: clenbuterol, Sa: salbutamol, Ci: cimaterol, Ra: ractopamine. (*) p < 0.05.