Leucyl-tRNA synthetase is required for the myogenic differentiation of C2C12 myoblasts, but not for hypertrophy or metabolic alteration of myotubes

Feb 10, 2018Experimental cell research

Leucyl-tRNA synthetase is needed for muscle cell formation but not for muscle growth or metabolism changes

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Abstract

siRNA-mediated knockdown of leucyl-tRNA synthetase (Lars) decreased phosphorylated p70 S6 kinase levels in skeletal muscle cells.

Lars knockdown inhibited the differentiation of C2C12 mouse myoblasts into myotubes, as shown by a decreased fusion index.
Reduced mRNA and protein expression levels of myogenic markers were observed following Lars knockdown.
Knockdown of Lars led to decreased levels of Insulin-like growth factor 2 (Igf2) mRNA early in differentiation.
Lars knockdown did not affect phosphorylated mTOR levels in differentiated myotubes.
There was no impact on the hypertrophy of myotubes measured by their diameters or hypertrophy marker expression.
Metabolic functions, including glycolysis and mitochondrial respiration, were not altered by Lars knockdown.

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Mammalian target of rapamycin (mTOR) signaling controls skeletal muscle cell differentiation, growth, and metabolism by sensing the intracellular energy status and nutrients. Recently, leucyl-tRNA synthetase (Lars) was identified as an intracellular sensor of leucine involved in the activation of mTOR signaling. However, there is still no evidence for the activation of mTOR signaling by Lars and its physiological roles in skeletal muscle cells. In this study, we determined the potential roles of Lars for the activation of mTOR signaling, skeletal muscle cell differentiation, hypertrophy, and metabolism using small interfering (si)-RNA knockdown. siRNA-mediated knockdown of Lars decreased phosphorylated p70 S6 kinase and inhibited the differentiation of C2C12 mouse myoblasts into myotubes, as evidenced by a decreased fusion index and decreased mRNA and protein expression levels of myogenic markers. Importantly, si-Lars decreased the level of Insulin-like growth factor 2 (Igf2) mRNA expression from the early stages of differentiation, indicating the possibility of an association between the mTOR-IGF2 axis and Lars. However, Lars knockdown did not decrease phosphorylated mTOR in differentiated myotubes, nor did it affect the hypertrophy of myotubes as evidenced by measuring their diameters and detecting the mRNA and protein expression of hypertrophy markers. Similarly, an extracellular flux analyzer showed that Lars knockdown did not affect the metabolism (glycolysis and mitochondrial respiration) of myotubes. These results demonstrate that Lars is required for skeletal muscle differentiation through the activation of mTOR signaling, but not for hypertrophy or metabolic alteration of myotubes.

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Leucyl-tRNA synthetase is required for the myogenic differentiation of C2C12 myoblasts, but not for hypertrophy or metabolic alteration of myotubes

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