Limits...
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.


Mss51-disrupted myoblasts proliferate and differentiate normally. (A) Proliferating control and Mss51-disrupted myoblasts labeled with EdU (red) and Hoeschst 33342 (blue). (B) Quantification of EdU staining (n = 6 wells imaged per sample). (C) Representative myosin heavy chain (MF20, green) staining of control and Mss51-disrupted myotubes 2 days after induction of differentiation with nuclei stained by DAPI (blue). (D) Quantification of fusion index (percentage of nuclei found in MF20+ myotubes, n = 6). (E) Creatine kinase (CK) activity in myotubes after 6 days of differentiation. (F) Protein synthesis rates as measured by puromycin incorporation on the left and total protein stained by SYPRO Ruby on the right after 6 days of differentiation. (G) Densitometric analysis of SUnSET assay as shown in F, normalized to SYPRO Ruby-stained total protein (n = 3). Differences between groups were not statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664537&req=5

jnd-2-4-jnd150119-g005: Mss51-disrupted myoblasts proliferate and differentiate normally. (A) Proliferating control and Mss51-disrupted myoblasts labeled with EdU (red) and Hoeschst 33342 (blue). (B) Quantification of EdU staining (n = 6 wells imaged per sample). (C) Representative myosin heavy chain (MF20, green) staining of control and Mss51-disrupted myotubes 2 days after induction of differentiation with nuclei stained by DAPI (blue). (D) Quantification of fusion index (percentage of nuclei found in MF20+ myotubes, n = 6). (E) Creatine kinase (CK) activity in myotubes after 6 days of differentiation. (F) Protein synthesis rates as measured by puromycin incorporation on the left and total protein stained by SYPRO Ruby on the right after 6 days of differentiation. (G) Densitometric analysis of SUnSET assay as shown in F, normalized to SYPRO Ruby-stained total protein (n = 3). Differences between groups were not statistically significant.

Mentions: Myostatin, TGF-β, and IGF-1 all modulate myoblast proliferation and differentiation and, as shown in Fig. 1B, also are associated with altered Mss51 gene expression. For this reason, we examined cell proliferation and differentiation in Mss51-disrupted cells. Proliferation was unchanged compared to control cells, as measured by EdU incorporation (Fig. 5A, B) and alamarBlue assay (Supplementary Figure 1). Differentiation was also equivalent in Mss51-disrupted and control cell populations by myofusion index, the proportion of nuclei found in myosin heavy chain-positive fibers at day 5 post differentiation media (Fig. 5C, D). Creatine kinase enzyme activity, a marker of differentiation, did not differ significantly between control and Mss51-disrupted myotubes after six days of differentiation (Fig. 5E). Myotubes were differentiated for six days to ensure that stable levels of Mss51 transcript and protein were present in control cells, corresponding to the greatest difference between control and disrupted populations. We also looked at expression of myogenic regulatory factors by qPCR and did not see meaningful differences between control and Mss51-disrupted cells (data not shown). Protein synthesis, as measured by puromycin incorporation using the SUnSET assay, was not altered in Mss51-disrupted myotubes after 6 days of differentiation (Fig. 5F, G).


Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism
Mss51-disrupted myoblasts proliferate and differentiate normally. (A) Proliferating control and Mss51-disrupted myoblasts labeled with EdU (red) and Hoeschst 33342 (blue). (B) Quantification of EdU staining (n = 6 wells imaged per sample). (C) Representative myosin heavy chain (MF20, green) staining of control and Mss51-disrupted myotubes 2 days after induction of differentiation with nuclei stained by DAPI (blue). (D) Quantification of fusion index (percentage of nuclei found in MF20+ myotubes, n = 6). (E) Creatine kinase (CK) activity in myotubes after 6 days of differentiation. (F) Protein synthesis rates as measured by puromycin incorporation on the left and total protein stained by SYPRO Ruby on the right after 6 days of differentiation. (G) Densitometric analysis of SUnSET assay as shown in F, normalized to SYPRO Ruby-stained total protein (n = 3). Differences between groups were not statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

jnd-2-4-jnd150119-g005: Mss51-disrupted myoblasts proliferate and differentiate normally. (A) Proliferating control and Mss51-disrupted myoblasts labeled with EdU (red) and Hoeschst 33342 (blue). (B) Quantification of EdU staining (n = 6 wells imaged per sample). (C) Representative myosin heavy chain (MF20, green) staining of control and Mss51-disrupted myotubes 2 days after induction of differentiation with nuclei stained by DAPI (blue). (D) Quantification of fusion index (percentage of nuclei found in MF20+ myotubes, n = 6). (E) Creatine kinase (CK) activity in myotubes after 6 days of differentiation. (F) Protein synthesis rates as measured by puromycin incorporation on the left and total protein stained by SYPRO Ruby on the right after 6 days of differentiation. (G) Densitometric analysis of SUnSET assay as shown in F, normalized to SYPRO Ruby-stained total protein (n = 3). Differences between groups were not statistically significant.
Mentions: Myostatin, TGF-β, and IGF-1 all modulate myoblast proliferation and differentiation and, as shown in Fig. 1B, also are associated with altered Mss51 gene expression. For this reason, we examined cell proliferation and differentiation in Mss51-disrupted cells. Proliferation was unchanged compared to control cells, as measured by EdU incorporation (Fig. 5A, B) and alamarBlue assay (Supplementary Figure 1). Differentiation was also equivalent in Mss51-disrupted and control cell populations by myofusion index, the proportion of nuclei found in myosin heavy chain-positive fibers at day 5 post differentiation media (Fig. 5C, D). Creatine kinase enzyme activity, a marker of differentiation, did not differ significantly between control and Mss51-disrupted myotubes after six days of differentiation (Fig. 5E). Myotubes were differentiated for six days to ensure that stable levels of Mss51 transcript and protein were present in control cells, corresponding to the greatest difference between control and disrupted populations. We also looked at expression of myogenic regulatory factors by qPCR and did not see meaningful differences between control and Mss51-disrupted cells (data not shown). Protein synthesis, as measured by puromycin incorporation using the SUnSET assay, was not altered in Mss51-disrupted myotubes after 6 days of differentiation (Fig. 5F, G).

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.