Global gene expression during muscle hypertrophy induced by myostatin suppression and/or beta-adrenergic agonist

Kim, Kyung Ho
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[Honolulu] : [University of Hawaii at Manoa], [December 2011]
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Skeletal muscle growth is important for animal agriculture, particularly for meat-producing animals, and for human health as well. A better understanding of the mechanisms regulating skeletal muscle growth is expected to contribute to improving the efficiency of meat animal production and alleviating human suffereings caused by muscle atrorphic conditions. Currently, two molecules have been shown to have dramatic effects on skeletal muscle mass: myostatin and β-adrenergic agonists. Myostatin (Mstn), a member of the TGF-β superfamily proteins, acts as a potent negative regulator of skeletal muscle growth. Suppression of Mstn by varying means has shown to increase skeletal muscle mass of animals. Administration of β-adrenergic agonist (BAA) such as clenbuterol (CL) induces dramatic increase in skeletal muscle mass or inhibits muscle atrophy. Although there have been numerous demonstrations of the muscle growth--promoting effect of BAA administration or Mstn suppression, the effect of a combination of BAA administration and Mstn inhibition on skeletal muscle growth has not been investigated. Similarly, very little is known about the molecular signaling pathways leading to muscle hypertrophy induced by the two stimuli and the genes that are commonly regulated by both Mstn and BAA administration. Therefore, the objectives of this study were 1) to investigate the combined effect of Mstn-suppression and BAA administration on skeletal muscle growth, 2) to examine the role of the Akt/mTOR pathway in the two muscle hypertrophic models, 3) to examine global changes in gene expression in skeletal muscle undergoing hypertrophy induced by chronic suppression of Mstn or BAA administration , and 4) to compare the changes in gene expression between these two muscle hypertrophic models. We used a transgenic mouse strain that overexpresses the Mstn-prodomain (Mstn-pro) and exhibits a significant increase in skeletal muscle mass regardless of age and sex. Clenbuterol (CL) was used as a BAA compound. Heterozygous Mstn-pro and wild-type littermates were produced and were given 0 or 20 ppm of CL in their drinking water. Phosphorylation of molecules involved in the Akt/mTOR pathway was examined by using the Western blot analysis. RNA samples of the gastrocnemius muscle in each group were subjected to microarray analysis using the Affymetrix GeneChip Mouse 430-2.0 platform. CL increased body and muscle mass of male and female mice in both genotypes, indincating that the muscle-hypertrophic effect of CL is additive to the effect of Mstn suppression. Levels of phosphorylated muscle 4E-BP1 and p70S6k, two downstream effectors of the mTOR pathway, were higher in Mstn-pro mice than in wild type mice. Levels of phosphorylated muscle Akt, an upstream effector of the mTOR pathway, were also higher in Mstn-pro mice than in wild type mice, indicating that the Akt/mTOR anabolic pathway is involved in the regulation of muscle mass by Mstn. CL increased the phosphorylation of Akt, 4E-BP1 and p70S6k in both genotypes, resulting in the highest phosphorylation levels of Akt, 4E-BP1 and p70S6k in CL-fed Mstn-pro mice. This result suggests that like Mstn, BAA also regulates muscle hypertrophy through the Akt/mTOR pathway, and the pathways of Mstn and CL signaling converge to the Akt/mTOR anabolic pathway to regulate skeletal muscle hypertrophy. Microarray analysis of global gene expression showed that Mstn suppression and CL administration induced significant changes in the mRNA abundance of various genes associated with muscle contraction, initiation of translation, transcription, and muscle hypertrophic signal pathway, suggesting that increased protein synthesis is partly responsible for the hypetrophy induced by Mstn and CL. Additionally, the alteration of Igf2 obsderved in Mstn suppressed mice, and the alterations of eIF4e, Acvr2b, FoxO and PTEN observed in mice treated with CL indicate that the pathways of Mstn and CL signaling converge to the Akt/mTOR anabolic pathway to regulate skeletal muscle hypertrophy.
Ph.D. University of Hawaii at Manoa 2011.
Includes bibliographical references.
myostatin suppression
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Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Molecular Biosciences and Bioengineering.
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