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Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism

View Article: PubMed Central - PubMed

ABSTRACT

Background:: The transforming growth factor β (TGF-β) signaling pathways modulate skeletal muscle growth, regeneration, and cellular metabolism. Several recent gene expression studies have shown that inhibition of myostatin and TGF-β1 signaling consistently leads to a significant reduction in expression of Mss51, also named Zmynd17. The function of mammalian Mss51 is unknown although a putative homolog in yeast is a mitochondrial translational activator.

Objective:: The objective of this work was to characterize mammalian MSS51.

Methods:: Quantitative RT-PCR and immunoblot of subcellular fractionation were used to determine expression patterns and localization of Mss51. The CRISPR/Cas9 system was used to reduce expression of Mss51 in C2C12 myoblasts and the function of Mss51 was evaluated in assays of proliferation, differentiation and cellular metabolism.

Results:: Mss51 was predominantly expressed in skeletal muscle and in those muscles dominated by fast-twitch fibers. In vitro, its expression was upregulated upon differentiation of C2C12 myoblasts into myotubes. Expression of Mss51 was modulated in response to altered TGF-β family signaling. In human muscle, MSS51 localized to the mitochondria. Its genetic disruption resulted in increased levels of cellular ATP, β-oxidation, glycolysis, and oxidative phosphorylation.

Conclusions:: Mss51 is a novel, skeletal muscle-specific gene and a key target of myostatin and TGF-β1 signaling. Unlike myostatin, TGF-β1 and IGF-1, Mss51 does not regulate myoblast proliferation or differentiation. Rather, Mss51 appears to be one of the effectors of these growth factors on metabolic processes including fatty acid oxidation, glycolysis and oxidative phosphorylation.

No MeSH data available.


Gene and protein expression in Mss51-disrupted differentiated myotubes (A) Myosin heavy chain expression measured by qRT-PCR in control and Mss51-disrupted C2C12 myotubes, normalized to expression levels in control myotubes using reference genes Tfam and TBP (n = 3). (B) Expression of metabolic genes in control and Mss51-disrupted C2C12 myotubes, normalized to expression levels in control myotubes using reference genes Tfam and TBP (n = 3). (C) Western blots of control andMss51-disrupted C2C12 myotubes examining expression and phosphorylation of AMPKα. (D) Densitometric analysis of AMPKα expression normalized to GAPDH and of AMPKα phosphorylation normalized to both GAPDH and total AMPKα (n = 3). *p <  0.05, **p <  0.01.
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jnd-2-4-jnd150119-g006: Gene and protein expression in Mss51-disrupted differentiated myotubes (A) Myosin heavy chain expression measured by qRT-PCR in control and Mss51-disrupted C2C12 myotubes, normalized to expression levels in control myotubes using reference genes Tfam and TBP (n = 3). (B) Expression of metabolic genes in control and Mss51-disrupted C2C12 myotubes, normalized to expression levels in control myotubes using reference genes Tfam and TBP (n = 3). (C) Western blots of control andMss51-disrupted C2C12 myotubes examining expression and phosphorylation of AMPKα. (D) Densitometric analysis of AMPKα expression normalized to GAPDH and of AMPKα phosphorylation normalized to both GAPDH and total AMPKα (n = 3). *p <  0.05, **p <  0.01.

Mentions: Myostatin knockout and IGF-1 transgene expression lead to an alteration in fiber-type composition with a decrease in type 1 slow oxidative fibers and an increase in type IIB fast glycolytic fibers [9, 10, 19, 39– 42]. We therefore examined whether CRISPR/Cas9-mediated disruption of the Mss51 locus altered expression of the various myosin heavy chain isoforms (MyHCs) expressed by C2C12 myotubes. In Mss51-disrupted C2C12 myotubes, embryonic MyHC (Myh3), neonatal MyHC (Myh8), and MyHC IIX (Myh1) were significantly decreased, MyHC IIB (Myh4) was significantly increased, and MyHC IIA (Myh2) and MyHC I (Myh7) were unchanged (Fig. 6A). The changes exhibited in Mss51-disrupted myotubes indicated a shift towards more fast-twitch MyHC expression.


Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism
Gene and protein expression in Mss51-disrupted differentiated myotubes (A) Myosin heavy chain expression measured by qRT-PCR in control and Mss51-disrupted C2C12 myotubes, normalized to expression levels in control myotubes using reference genes Tfam and TBP (n = 3). (B) Expression of metabolic genes in control and Mss51-disrupted C2C12 myotubes, normalized to expression levels in control myotubes using reference genes Tfam and TBP (n = 3). (C) Western blots of control andMss51-disrupted C2C12 myotubes examining expression and phosphorylation of AMPKα. (D) Densitometric analysis of AMPKα expression normalized to GAPDH and of AMPKα phosphorylation normalized to both GAPDH and total AMPKα (n = 3). *p <  0.05, **p <  0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664537&req=5

jnd-2-4-jnd150119-g006: Gene and protein expression in Mss51-disrupted differentiated myotubes (A) Myosin heavy chain expression measured by qRT-PCR in control and Mss51-disrupted C2C12 myotubes, normalized to expression levels in control myotubes using reference genes Tfam and TBP (n = 3). (B) Expression of metabolic genes in control and Mss51-disrupted C2C12 myotubes, normalized to expression levels in control myotubes using reference genes Tfam and TBP (n = 3). (C) Western blots of control andMss51-disrupted C2C12 myotubes examining expression and phosphorylation of AMPKα. (D) Densitometric analysis of AMPKα expression normalized to GAPDH and of AMPKα phosphorylation normalized to both GAPDH and total AMPKα (n = 3). *p <  0.05, **p <  0.01.
Mentions: Myostatin knockout and IGF-1 transgene expression lead to an alteration in fiber-type composition with a decrease in type 1 slow oxidative fibers and an increase in type IIB fast glycolytic fibers [9, 10, 19, 39– 42]. We therefore examined whether CRISPR/Cas9-mediated disruption of the Mss51 locus altered expression of the various myosin heavy chain isoforms (MyHCs) expressed by C2C12 myotubes. In Mss51-disrupted C2C12 myotubes, embryonic MyHC (Myh3), neonatal MyHC (Myh8), and MyHC IIX (Myh1) were significantly decreased, MyHC IIB (Myh4) was significantly increased, and MyHC IIA (Myh2) and MyHC I (Myh7) were unchanged (Fig. 6A). The changes exhibited in Mss51-disrupted myotubes indicated a shift towards more fast-twitch MyHC expression.

View Article: PubMed Central - PubMed

ABSTRACT

Background:: The transforming growth factor &beta; (TGF-&beta;) signaling pathways modulate skeletal muscle growth, regeneration, and cellular metabolism. Several recent gene expression studies have shown that inhibition of myostatin and TGF-&beta;1 signaling consistently leads to a significant reduction in expression of Mss51, also named Zmynd17. The function of mammalian Mss51 is unknown although a putative homolog in yeast is a mitochondrial translational activator.

Objective:: The objective of this work was to characterize mammalian MSS51.

Methods:: Quantitative RT-PCR and immunoblot of subcellular fractionation were used to determine expression patterns and localization of Mss51. The CRISPR/Cas9 system was used to reduce expression of Mss51 in C2C12 myoblasts and the function of Mss51 was evaluated in assays of proliferation, differentiation and cellular metabolism.

Results:: Mss51 was predominantly expressed in skeletal muscle and in those muscles dominated by fast-twitch fibers. In vitro, its expression was upregulated upon differentiation of C2C12 myoblasts into myotubes. Expression of Mss51 was modulated in response to altered TGF-&beta; family signaling. In human muscle, MSS51 localized to the mitochondria. Its genetic disruption resulted in increased levels of cellular ATP, &beta;-oxidation, glycolysis, and oxidative phosphorylation.

Conclusions:: Mss51 is a novel, skeletal muscle-specific gene and a key target of myostatin and TGF-&beta;1 signaling. Unlike myostatin, TGF-&beta;1 and IGF-1, Mss51 does not regulate myoblast proliferation or differentiation. Rather, Mss51 appears to be one of the effectors of these growth factors on metabolic processes including fatty acid oxidation, glycolysis and oxidative phosphorylation.

No MeSH data available.