Metallothionein gene deficiency facilitates the differentiation of C2C12 myoblasts into slow-twitch myotubes

Abstract

Metallothionein (MT) 1 and 2 are ubiquitously expressed cysteine-rich, low molecular weight proteins. MT expression is upregulated in skeletal muscle during aging. MTs also play role in multiple types of skeletal muscle atrophy. Meanwhile, it has been reported that MT1 and MT2 gene deficiency increases myogenesis in MT knockout (MTKO) mice. However, little is known about the effect of MTs on muscle formation and atrophy. In this study, we investigated the effect of MT1 and MT2 gene knock-out using the CRISPR-Cas9 system in an in vitro skeletal muscle differentiation model (C2C12 cell line). Although the number of myotubes in C2C12 MTKO cells was comparable to that of mock-transfected cells, MT deficiency promoted myocyte fusion. Muscle-specific transcription factors myoD and myogenin were found to be upregulated at the late stage of myotube differentiation. Furthermore, the fast-twitch myosin heavy chain (MyHC) protein expression was similar in MTKO and mock-transfected myotubes, but slow-MyHC expression was higher in MTKO cells than in mock cells. The MT gene deletion did not affect the number of fast MyHC-positive myotubes but increased the number of slow MyHC-positive myotubes. Treatment with the antioxidant N-acetylcysteine (NAC) inhibited the increase in the number of slow MyHC-positive myotubes as well as slow-MyHC expression in MTKO cells. In contrast, NAC treatment did not alter the number of fast MyHC-positive myotubes or the expression of fast-MyHC in MTKO cells. These results suggest that the antioxidant effects of MTs may be involved in slow-twitch myofiber formation in skeletal muscle.

Cysteine Rich Intestinal Protein 2 is a copper-responsive regulator of skeletal muscle differentiation

“…Furthermore, while the expression of fast-twitch MHC protein expression remained stable, the expression of slow-MyHC expression was higher in KO cells than controls (183). This effect was reverted by the antioxidant N-acetylcysteine, suggesting that the antioxidant effects of Mts may be involved in slow-twitch myofiber formation in skeletal muscle (183).…”

“…Furthermore, while the expression of fast-twitch MHC protein expression remained stable, the expression of slow-MyHC expression was higher in KO cells than controls (183). This effect was reverted by the antioxidant N-acetylcysteine, suggesting that the antioxidant effects of Mts may be involved in slow-twitch myofiber formation in skeletal muscle (183). Although an important function for mCrip2 as a nuclear protein has emerged as a transcription co-adaptor protein (184), further research into the nuclear function of mCrip2 in response to Cu-supplementation during skeletal muscle differentiation is needed to fully understand the multifaceted role of this factor during myogenesis.…”

“…In fact, a recent study explored the role of ubiquitously expressing Mt on muscle differentiation. CRISPR-Cas9 mediated deletion of Mt1 and Mt2 genes in C2C12 myoblasts showed that deficiency of these genes promotes myocyte fusion, with MyoD1 and myogenin upregulated at the late stage of myotube differentiation (183).…”

“…In fact, a recent study explored the role of ubiquitously expressing Mt on muscle differentiation. CRISPR-Cas9 mediated deletion of Mt1 and Mt2 genes in C2C12 myoblasts showed that deficiency of these genes promotes myocyte fusion, with MyoD1 and myogenin upregulated at the late stage of myotube differentiation (183). Furthermore, while the expression of fast-twitch MHC protein expression remained stable, the expression of slow-MyHC expression was higher in KO cells than controls (183).…”

“…CRISPR-Cas9 mediated deletion of Mt1 and Mt2 genes in C2C12 myoblasts showed that deficiency of these genes promotes myocyte fusion, with MyoD1 and myogenin upregulated at the late stage of myotube differentiation (183). Furthermore, while the expression of fast-twitch MHC protein expression remained stable, the expression of slow-MyHC expression was higher in KO cells than controls (183). This effect was reverted by the antioxidant N-acetylcysteine, suggesting that the antioxidant effects of Mts may be involved in slow-twitch myofiber formation in skeletal muscle (183).…”