The critical role of myostatin in differentiation of sheep myoblasts

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Abstract

Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

Highlights

► Identification of the effective and specific shRNA to knockdown MSTN. ► Overexpression of MSTN reversibly suppressed myogenic differentiation. ► shRNA knockdown of endogenous MSTN promoted ovine myoblast differentiation. ► MSTN inhibits myogenic differentiation through down-regulation of MyoD and Myogenin and up-regulation of Smad3. ► Provides a promise for the generation of transgenic sheep to improve meat productivity.

Introduction

Myostatin (MSTN), also known as growth differentiation factor 8 (GDF8), is a member of the TGFβ superfamily. Previous studies reported that disruptive mutations in the coding region of bovine MSTN gene were associated with dramatic and widespread increase of skeletal muscle mass, which was characterized as “double muscling” [1], [2]. Similar double muscling trait was also observed in human, accompanied by naturally occurred loss-of-function mutations in the MSTN gene [3]. In line with these findings, knockout of MSTN in mice resulted in significant hyperplasia (increase in number of muscle fibers) and hypertrophy (increase in size of muscle fibers) [4]. In contrast, over expression of MSTN in mice showed decreased skeletal muscle mass [5] and the development of cachectic-like muscle wasting [6]. These studies demonstrate that MSTN plays a critical role in muscle growth and development.

Based on accumulating evidence, it is conceivable that inhibition of MSTN function in farm animal myoblasts could effectively increase muscle mass so as to increase meat productivity. This has been an interest to many investigators. Several studies revealed that dominant negative proteins, such as MSTN prepropeptide and follistatin, can suppress MSTN activity and result in increased muscle mass [7], [8]. However, these dominant negative proteins may cause undesirable immune response and result in perturbation of their inhibitory functions. Though MSTN knockout mice have been successfully obtained [9], gene knockout in farm animals has still been time consuming and labor intensive. RNA interference (RNAi) is a promising method to specifically and efficiently silence gene expression [10]. It has been widely used in gene function analysis and potential therapeutics of diseases [11]. A recent study suggested that using RNAi to silence the MSTN gene could be an effective approach to improve meat productivity [12].

In this study, we used both ectopic overexpression and shRNA knockdown approaches to investigate the role of MSTN in the differentiation of primary ovine myoblasts. The results of our study document that MSTN inhibits sheep myoblast differentiation. Further, our study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

Section snippets

Cell culture

TLA-HEK293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Ovine myoblasts were derived from vastus lateralis of 60-day fetus of Xinjiang Merino Sheep as previously described [13]. Briefly, ovine myoblasts were liberated with at least 90% myoblast purity from fresh vastus lateralis by mincing the muscle tissue. Muscle scrap was digested with 0.02% Type 1-A collagenase (Sigma, St Louis, MO) for 45 min at 37 °C, and cultured in DMEM with 1 × 105 IU/liter penicillin,

Identification of the most effective and specific shRNA to knockdown MSTN

Six shRNAs were designed by using the Dharmacon siDESIGN Center database and cloned into the pLL3.7 lentiviral vector (Table 1 and Fig. 1A). To determine the efficacies of these shRNA of knocking down MSTN, the MSTN-shRNA constructs were co-transfected with pLex-MSTN into 293T cells. Western blotting was performed to measure MSTN protein levels in the transfected cells by using an anti-HA antibody. Interestingly, all the shRNAs except for shR539, caused substantial down-regulation of MSTN

Discussion

Increase of muscle mass is a consequence of hyperplasia and hypertrophy. This process includes both myoblast proliferation and differentiation. When early muscle progenitors are specified, they can withdraw from the cell cycle to terminally differentiate into functional muscle fibers [19]. Identifying critical regulators is essential to enhance the understanding of the mechanisms of muscle growth and development, and to improve meat productivity of farm animals. MSTN has been considered as a

Acknowledgments

We thank Dr. Wenlai Zhou for kindly providing the lentivirus vector. This study was funded by grants of National High Technology Research and Development Program of China (863 Program), 2009AA10Z110 and 2011AA100307.

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