Changes in expression of serine biosynthesis and integrated stress response genes during myogenic differentiation of C2C12 cells

Abstract

Skeletal muscle is a highly metabolic and dynamic tissue that is formed through the complex and well-organised process of myogenesis. Although there is a good understanding about the role of the Muscle Regulatory Factors during myogenesis, little is known about the potential interplay of other metabolic proteins. The aim of this study was to determine the endogenous mRNA expression profile for a novel group of genes, recently associated with β2-adrenergic agonist (BA) induced muscle hypertrophy in pigs [1], during myogenic differentiation in C2C12 cells and their response to dibutyryl cyclic-AMP (dbcAMP). These genes included mitochondrial phosphoenolpyruvate carboxykinase (PCK2/PEPCK-M), genes involved in serine biosynthesis (Phosphoglycerate dehydrogenase, PHGDH; Phosphoserine aminotransferase-1, PSAT1; Phosphoserine phosphatase, PSPH) and those involved in an integrated stress response (Asparagine synthetase, ASNS; Sestrin-2, SESN2; and Activating transcription factor-5, ATF5).A coordinated peak in endogenous PCK2, PHGDH, PSAT1, PSPH, ASNS, ATF5 and SESN2 mRNA expression was observed at day 2 of differentiation (P < 0.001) in C2C12 cells, which coincided with the peak in myogenin mRNA. Myotube hypertrophy was induced with dbcAMP (1 mM) treatment from day 0, thereby mimicking the in vivo BA response. Although dbcAMP treatment from day 0 induced larger myotubes and increased both myosin heavy chain-IIB (MyHC-IIB) and pyruvate carboxylase (PC) mRNA, the expression of PCK2, PHGDH, PSAT1 and ASNS mRNA were all unaffected. Treatment with dbcAMP from day 4 increased MyHC-IIB mRNA, however this was less dramatic compared to the response observed following treatment from day 0, but there was no effect on PC mRNA. There was also no effect of dbcAMP treatment from day 4 on PCK2, PHGDH, PSAT1 and ASNS mRNA.To conclude, the coordinated day 2 peak in endogenous expression of PCK2, PHGDH, PSAT1, PSPH, ASNS, ATF5 and SESN2 mRNA may relate to a shift in biosynthetic demand required to initiate myogenic differentiation. However, dbcAMP had no effect on the expression of these genes in vitro suggesting that the effects observed in BA-treated pigs might be via other signalling pathways from the activation of the β2-adrenergic receptor, but independent of cAMP, or that there are species differences in the response. Keywords: C2C12, dbcAMP, Hypertrophy, Myogenesis, PCK2/PEPCK-M, PHGDH