GLOBAL GENE EXPRESSION DURING MUSCLE HYPERTROPHY INDUCED BY MYOSTATIN SUPPRESSION AND/OR BETA-ADRENERGIC AGONIST A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII AT MANOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR BIOSCIENCES AND BIOENGINEERING DECEMBER 2011 BY KYUNGHO KIM Dissertation Committee: Yong Soo Kim, Chairperson Dulal Borthakur Jinzeng Yang Michael A. Dunn Yong Li ACKNOWLEDGMENTS Having received help from so many people, it is hard to include everyone, but there are several individuals without whom, I could not have made it this far. First, I would like to thank my advisor, Dr. Yong Soo Kim, for his helpful advice, for his financial support, for his patient guidance and critical comments on my research, for sharing his expertise, knowledge, thoughts and perspectives in the field of animal biotechnology and growth biology, and for his great encouragement, help and care during all my studies at University of Hawaii. I would also like to thank my dissertation committee members, Dr. Jinzeng Yand, Dr. Michael A. Dunn, Dr. Dulal Borthakur and Dr, Yong Li, for their keen review, advice and constructive comments. They also played crucial roles in my learning process as they provided their academic expertise and laboratory resources. I especially thank Dr. Hyun Wong Shin, my Master’s advisor, a professor at South Korea Soonchunhyang University, for his great support to my graduate admission application to University of Hawaii, his continuous encouragement, enlightening advice during all my studies. I thank Mrs. Cory S. Tauyan, for training and help in carrying out mice project. Working with my colleagues, especially Rocky, Mandy and Arthor had always been fun for they made the circumstances in the lab pleasant to work in. I would like to thank my family (my father Joo-Bok Kim, my mother Kyung- Sook Lee, my wife Na-Yeong Oh, my son Brian T. Kim, my brother Soo-Ho Kim, my father-in-law Se-Hwa Oh, my mother-in-law Sung-Re Yoo), for their love, concern, prayers, encouragement and support during all my studies at University of Hawaii. It is acknowledged that the United State Department of Agriculture (USDA) T-STAR program supported this project. i ABSTRACT 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 ii (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, iii 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. iv TABLE OF CONTENTS Acknowledgements……………………………………………………………………...i Abstract………………………………………………………………………………....ii List of tables……….………………………………………………………………….....x List of figures………………………………………………………………………….xiii CHAPTER 1: LITERATURE REVIEW 1.1. SKELETAL MUSCLE GROWTH AND DVELOPMENT.......................................1 1.1.1. Embryonic development of skeletal muscle..…………………………………..1 1.1.2. Myogenic regulatory factors (MRFs)....………………………………………..2 1.1.3. Postnatal development of skeletal muscle.....…………………………………..3 1.1.4. Muscle hypertrophy and satellite cell....………………………………………..5 1.2. SIGNALING PATHWAYS IN SKELETAL MUSCLE HYPERTROPHY AND ATROPHY………………………………………………………………………….6 1.2.1. Akt/mTOR signaling pathway ……....................................................................7 1.2.2. FoxO transcription factors ……………………………………………………..9 1.2.3. NF-kB signaling pathway..................................................................................10 1.2.4. MAPK/ERK signaling pathway.........................................................................11 1.3. REGULATION OF MUSCLE GROWTH BY MYOSTATIN ...………………….12 1.3.1. Role of myostatin in the control of myoblast proliferation and differentiation…………………………………………………………………13 1.3.2. Role of myostatin in the control of post-natal muscle growth…………….15 1.3.3. Role of myostatin on muscle fiber type……………………………….………18 1.3.4. Myostatin synthesis, processing, activation and inhibition…………………...20 1.3.5. Myostatin signal transduction…...….....………………………………………23 v 1.4. REGULATION OF MUSCLE GROWTH BY β-ADRENERGIC AGONISTS…..25 1.4.1. Function of β-adrenergic agonists...............................................................26 1.4.2. Skeletal muscle hypertrophic effect of β-adrenergic agonists……………26 1.4.3. β-adrenergic receptors...………………………………………………………27 1.4.4. Signaling pathways of β2-adrenergic receptors leading to muscle hypertrophy…………………………………………………………………28 1.4.5. Adaptation of animals to growth promoting effects of β-adrenergic agonists………………………………………………………………………29 1.5. MICROARRAY TECHNOLOGY..………………………………………………..30 1.5.1. Principles of DNA microarray…..............................…………….……………30 1.5.2. Two-channel and one-channel microarray detection ..…………..……………31 1.5.3. Use of microarray analysis to examine global gene expression during skeletal muscle growth …………………………………………………..…32 1.6. POTENTIALS OF MYOSTATIN AND BETA-ADRENERGIC AGONISTS IN ANIMAL PRODCUTION AND HUMAN HELATH……………………………35 CHAPTER 2: THE MUSCLE-HYPERTROPHIC EFFECT OF CLENBUTEROL IS ADDITIVE TO THE HYPERTROPHIC EFFECT OF MYOSTATIN SUPPRESSION 2.1. Abstract……………..……………………………………………………………...40 2.2. Introduction………………………………………………………………………..40 2.3. Materials and methods..……………………………………………………………43 2.3.1. Animals and sample collection ……………………………………….………43 2.3.2. Genotyping …………………………………………………………….……..44 2.3.3. Measurement of skeletal muscle DNA and RNA concentration……………...44 vi 2.3.4. Western blot analysis of the phosphorylation of 4E-BP1 and p70s6k …….....45 2.3.5. Statistical analysis ……………………………………………………………46 2.4. Results……………………………………………………………………………..46 2.4.1. Effects of CL on body and muscle weights of wild type and Mstn-pro mice...46 2.4.2. Effects of CL on muscle DNA and RNA concentrations of wild