Knockdown of CNN3 Impairs Myoblast Proliferation, Differentiation, and Protein Synthesis via the mTOR Pathway

Abstract

BackgroundMyogenesis is a complex process that requires optimal outside–in substrate–cell signaling. Calponin 3 (CNN3) plays an important role in regulating myogenic differentiation and muscle regeneration; however, the precise function of CNN3 in myogenesis regulation remains poorly understood. Here, we investigated the role of CNN3 in a knockdown model in the mouse muscle cell line C2C12.MethodsMyoblast proliferation, migration, differentiation, fusion, and protein synthesis were examined in CNN3 knockdown C2C12 mouse muscle cells. Involvement of the mTOR pathway in CNN3 signaling was explored by treating cells with the mTOR activator MHY1485. The regulatory mechanisms of CNN3 in myogenesis were further examined by RNA sequencing and subsequent gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA).ResultsDuring proliferation, CNN3 knockdown caused a decrease in cell proliferation and migration. During differentiation, CNN3 knockdown inhibited myogenic differentiation, fusion, and protein synthesis in C2C12 cells via the AKT/mTOR and AMPK/mTOR pathways; this effect was reversed by MHY1485 treatment. Finally, KEGG and GSEA indicated that the NOD-like receptor signaling pathway is affected in CNN3 knockdown cell lines.ConclusionCNN3 may promote C2C12 cell growth by regulating AKT/mTOR and AMPK/mTOR signaling. The KEGG and GSEA indicated that inhibiting CNN3 may activate several pathways, including the NOD-like receptor pathway and pathways involved in necroptosis, apoptosis, and inflammation.

Promoting effects of calponin 3 on the growth of diffuse large B‑cell lymphoma cells

“…Its effects on proliferation have been reported in recent years. For instance, CNN3 knockdown impairs the proliferation of myoblasts and fibroblasts (33,34). Increased expression of CNN3 promoted the proliferation of osteosarcoma and cervical cancer cells (11,35).…”

Role of Actin-Binding Proteins in Skeletal Myogenesis

“…Mutations in CNN3 have been linked to neonatal lethality and extensive malformations in mice embryos due to induced actin cytoskeletal reorganization [210]. In addition, CNN3 has been observed to be upregulated during muscle development and regeneration in various species, including pigs and mice, hinting at a potential association with muscle development [211,212]. Interestingly, silencing CNN3 using siRNA dramatically repressed the expression of proliferationrelated genes, such as cyclin D, CDK2, CDK4/6, Ki67, and MyoD [211,212].…”

“…In addition, CNN3 has been observed to be upregulated during muscle development and regeneration in various species, including pigs and mice, hinting at a potential association with muscle development [211,212]. Interestingly, silencing CNN3 using siRNA dramatically repressed the expression of proliferationrelated genes, such as cyclin D, CDK2, CDK4/6, Ki67, and MyoD [211,212]. Besides, the knockdown of CNN3 dramatically suppresses the expression of differentiation marker proteins (MEF2A, MyoG) and myotube formation [211,212].…”

“…Interestingly, silencing CNN3 using siRNA dramatically repressed the expression of proliferationrelated genes, such as cyclin D, CDK2, CDK4/6, Ki67, and MyoD [211,212]. Besides, the knockdown of CNN3 dramatically suppresses the expression of differentiation marker proteins (MEF2A, MyoG) and myotube formation [211,212]. Studies by She et al revealed that the inhibition of CNN3 protein synthesis is associated with a negative correlation with AMPK/mTOR and Akt/mTOR signaling pathways impacting the proliferation and differentiation of C2C12 myoblasts [211].…”

Evolution and function of calponin and transgelin

“…The inhibitory effects of calponin 3 on myogenesis and myocyte fusion are regulated by the Rho-associated protein kinase (ROCK) signaling pathway ( Shibukawa et al, 2013 ). Another study showed that calponin 3 regulates myoblast proliferation, differentiation and protein synthesis through mTOR pathway ( She et al, 2021 ). Calponin 3 is also found in chondrocytes as a Smad-binding regulator to inhibit bone morphogenic protein (BMP)-mediated transcription ( Haag and Aigner, 2007 ).…”