Myostatin Exhibits an Evolutionarily Conserved Circadian Pattern in Skeletal Muscles

Introduction: Myostatin (MSTN), a transforming growth factor-beta (TGF-β) superfamily member, is an evolutionarily conserved negative regulator of skeletal muscle mass. Loss of MSTN commonly promotes augmentation in skeletal muscle mass in all animal species examined. Recent studies have demonstrated that circadian clock proteins play a critical role in the regulation of muscle mass and function, in part by modulating the expression of key muscle-related genes. While myostatin has an important role in sustaining skeletal muscle protein turnover, it is unknown if circadian clock proteins regulate myostatin in a circadian pattern. Methods: We analysed time-course muscle samples from 16 animal species ranging from Caenorhabditis elegans to humans and examined the rhythmic expression pattern of Mstn. We also used various circadian clock deficient models such as muscle-specific Bmal1 knockout, Per1/Per2 double knockout, genetic knockout of per0 and tim0 genes in fruit flies, clocka gene in zebrafish and environmental perturbation. Results: Both mRNA and protein of MSTN exhibit rhythmic expression patterns in a variety of animal species ranging from Caenorhabditis elegans (C. elegans) to humans. The rhythmicity of Mstn orthologs in muscle is evolutionarily conserved along with their sequence evolution in C. elegans, Drosophila melanogaster, Crustacea, fish and mammals including mice (mRNA: amplitude = 0.188, p < 0.0001; protein: amplitude = 0.255, p < 0.05), goats, pigs and humans. In murine skeletal muscle, rhythmic expression of Mstn is synchronized with the core circadian genes, Per2. We then constructed a muscle-specific Bmal1 knockout mouse model (Bmal1^MKO). Notably, Bmal1^MKO mice had increased body weight (29.30 ± 0.85 vs. 32.16 ± 0.79, p < 0.05) and lean mass (WT 23.33 ± 0.35 vs. 25.35 ± 0.45, p < 0.01), while the difference in lean mass at 12 weeks of age (~1.996 g) closely matches the difference in total body weight (~ 2.000 g). Muscle-specific Bmal1 knockout reduced the mRNA and protein levels of mstn/MSTN by ~ 50%. In addition, disruption of the circadian clock by constant light or Per1/Per2 double knockout also abolishes the rhythmicity of Mstn. Similarly, genetic knockout of per0 and tim0 genes in fruit flies, clocka gene in zebrafish (mstna: p < 0.01 vs. p = 0.6397) and environmental perturbation (Aplodinotus grunniens, mstn1: p < 0.0001 vs. p = 0.04; mstn2: p < 0.05 vs. p = 0.06) all alter Mstn oscillation profoundly. Conclusions: These findings reveal an evolutionarily conserved rhythmic expression pattern of Mstn in skeletal muscles.