Proteomic Studies on the Mechanism of Myostatin Regulating Cattle Skeletal Muscle Development.

Hui Sheng,Manning Miao,Xin Li,Yiwen Guo,Guangpeng Li,Haoyun Tan,Junxing Zhang,Xiangbin Ding,Debao Hu,Linlin Zhang,Hong Guo

doi:10.3389/fgene.2021.752129

Hui Sheng, Manning Miao + Show 9 more

Open Access

https://doi.org/10.3389/fgene.2021.752129

Copy DOI

Abstract

Myostatin (MSTN) is an important negative regulator of muscle growth and development. In this study, we performed comparatively the proteomics analyses of gluteus tissues from MSTN+/− Mongolian cattle (MG.MSTN+/−) and wild type Mongolian cattle (MG.WT) using a shotgun-based tandem mass tag (TMT) 6-plex labeling method to investigate the regulation mechanism of MSTN on the growth and development of bovine skeletal muscle. A total of 1,950 proteins were identified in MG.MSTN+/− and MG.WT. Compared with MG.WT cattle, a total of 320 differentially expressed proteins were identified in MG.MSTN cattle, including 245 up-regulated differentially expressed proteins and 75 down-regulated differentially expressed proteins. Bioinformatics analysis showed that knockdown of the MSTN gene increased the expression of extracellular matrix and ribosome-related proteins, induced activation of focal adhesion, PI3K-AKT, and Ribosomal pathways. The results of proteomic analysis were verified by muscle tissue Western blot test and in vitro MSTN gene knockdown test, and it was found that knockdown MSTN gene expression could promote the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells (BSMSCs). At the same time, Co-Immunoprecipitation (CO-IP) assay showed that MSTN gene interacted with extracellular matrix related protein type I collagen α 1 (COL1A1), and knocking down the expression of COL1A1 could inhibit the activity of adhesion, PI3K-AKT and ribosome pathway, thus inhibit BSMSCs proliferation. These results suggest that the MSTN gene regulates focal adhesion, PI3K-AKT, and Ribosomal pathway through the COL1A1 gene. In general, this study provides new insights into the regulatory mechanism of MSTN involved in muscle growth and development.

Highlights

Myostatin (MSTN), known as growth differentiation factor-8 (GDF-8), is a highly conservative member of the transforming growth factor β (TGF-β) superfamily (McPherron et al, 1997)
We found that compared with MG.WT, the expression of a large number of extracellular matrix-related proteins in the gluteal muscle tissues of MG.MSTN+/− was up-regulated, among which the expression of COL1A1 The levels were raised by 2.3 times
The results showed that in bovine skeletal muscle satellite cells (BSMSCs) transfected with si-COL1A1, the mRNA expression levels of majority differentially expressed proteins involved in focal adhesion, PI3K-AKT, and ribosomal pathways did not change significantly (Figure 10A), but the protein expression levels decreased significantly, especially the protein expression levels of key genes p-FAK, p-AKT1 and p-RPS6 in the pathway (Figures 10B,C)

Summary

Introduction

Myostatin (MSTN), known as growth differentiation factor-8 (GDF-8), is a highly conservative member of the transforming growth factor β (TGF-β) superfamily (McPherron et al, 1997). MSTN has been confirmed to be a secreted growth factor expressed predominantly in skeletal muscle (McPherron, 1997; Kollias and McDermott, 2008) and plays a key role in the negative regulation of muscle development (Tsuchida, 2008). Overexpression of MSTN or systemic administration can lead to muscle atrophy (Zimmers et al, 2002; Durieux et al, 2007). All these effects are mainly achieved by regulating the proliferation and differentiation of myoblasts (Thomas et al, 2000; Rios et al, 2001; Langley et al, 2002; Rios et al, 2002). MSTN signal cascade plays a central role in regulating muscle weight, the mechanism of this signal cascade is still unclear (Elkina et al, 2011)

Methods

Results

Conclusion