Effects of polygalacin D extracted from Platycodon grandiflorum on myoblast differentiation and muscle atrophy

Purpose: The balance between synthesis and degradation of proteins plays a critical role in the maintenance of skeletal muscle mass.Mitochondrial dysfunction has been closely associated with skeletal muscle atrophy caused by aging, cancer, and chemotherapy.Polygalacin D is a saponin derivative isolated from Platycodon grandiflorum (Jacq.) A. DC.This study aimed to investigate the effects of polygalacin D on myoblast differentiation and muscle atrophy in association with mitochondrial function in in vitro and in zebrafish models in vivo.Methods: C2C12 myoblasts were cultured in differentiation media containing different concentrations of polygalacin D, followed by the immunostaining of the myotubes with myosin heavy chain (MHC).The mRNA expression of markers related to myogenesis, muscle atrophy, and mitochondrial function was determined by real-time quantitative reverse transcription polymerase chain reaction.Wild type AB* zebrafish (Danio rerio) embryos were treated with 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI) with or without polygalacin D, and immunostained to detect slow and fast types of muscle fibers.The Tg(Xla.Eef1a1:mito-EGFP) zebrafish expressing mitochondria-targeted green fluorescent protein was used to monitor mitochondrial morphology.Results: The exposure of C2C12 myotubes to 0.1 ng/mL of polygalacin D increased the formation of MHC-positive multinucleated myotubes (≥ 8 nuclei) compared with the control.Polygalacin D significantly increased the expression of MHC isoforms (Myh1, Myh2, Myh4, and Myh7) involved in myoblast differentiation while it decreased the expression of atrophic markers including muscle RING-finger protein-1 (MuRF1), mothers against decapentaplegic homolog (Smad)2, and Smad3.In addition, polygalacin D promoted peroxisome proliferatoractivated receptor-gamma coactivator (Pgc1α) expression and reduced the level of mitochondrial fission regulators such as dynamin-1-like protein (Drp1) and mitochondrial