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An Inbred Rat Model of Exercise Capacity: the Path to Identifying Alleles Regulating Variation in Treadmill Running Performance and Associated Phenotypes

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An Inbred Rat Model of Exercise Capacity: the Path to Identifying Alleles Regulating Variation in Treadmill Running Performance and Associated Phenotypes Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences An Inbred Rat Model of Exercise Capacity: The Path to Identifying Alleles Regulating Variation in Treadmill Running Performance and Associated Phenotypes Submitted by: Justin A. Ways In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences Examination Committee Major Advisor: George Cicila, Ph.D. Academic Abraham Lee, Ph.D., PT Advisory Committee: Yasser Saad, Ph.D. Joana Chakraborty, Ph.D. John C. Barbato, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: June 22, 2007 An Inbred Rat Model of Exercise Capacity: The Path to Identifying Alleles Regulating Variation in Treadmill Running Performance and Associated Phenotypes Justin Andrew Ways The University of Toledo College of Medicine 2007 Copyright 2007, Justin Andrew Ways ii DEDICATION I am dedicating this dissertation to: all the people in my life who believe in me, especially when I don’t believe in myself; all others who struggle with who they are and what they want to do in life, it is never too late to find your path; all the teachers who had a profound impact in my life, not only educational, but spiritual and moral as well - I can only hope to give to others as much as you have given to me, my nephews, Nathan and Ethen, who I hope and pray achieve all their dreams in life. iii ACKNOWLEDGEMENTS I am deeply grateful to the following: God, for giving me the ability to achieve the pursuit of higher education and follow my dreams; my family, for understanding the commitment it takes to pursue higher education and for their support, even if they can’t remember what it is I do; Drs. George Cicila and Soon Jin Lee for providing me the opportunity to pursue my doctoral degree in their lab and for allowing me to embark on a teaching career while performing my graduate studies; Dr. John Barbato, for being a trusted mentor, friend, and brother; Dr. Abraham Lee, for his guidance, both academic and spiritual; Dr. Joana Chakraborty, for her outstanding support of students, personal guidance, and providing me with the opportunity to utilize and practice physiological education; Dr. Yasser Saad, for challenging discussions that forced intellectual growth; Dr. Brian Smith, an excellent rat, and probably human, surgeon; Dr. Eric Morgan, for being a trusted friend and excellent cardiovascular resource; Sarah, Kris, and Ramona, without whom none of the work in the past five years would have been possible; my mentors and friends at Mercy College of Northwest Ohio; and last, but not least, Mom (a.k.a. Marianne), without whom I would not be where I am or who I am today. You will always be the one I hold most dear. iv TABLE OF CONTENTS Introduction…………………………………………………………………….1 Literature Review………………………………………………………..…….6 Materials and Methods……………………………….……………..….…...53 Results…...…………………………………………………………….……..85 Discussion...…..……..……………………………………………….…….113 Conclusions...………………………………………………………………138 Summary……………………………………………………………………141 References……………………………………………………………….…142 Appendix A……………………………………………………………….…184 Appendix B……………………………………………………………….…185 Abstract..………………………………………………………………….…186 v A LONG TIME AGO, WISE MEN ONCE SAID… ‘‘In a word, all parts of the body which were made for active use, if moderately used and exercised at the labor to which they are habituated, become healthy, increase in bulk, and bear their age well, but when not used, and when left without exercise, they become diseased, their growth is arrested, and they soon become old.” --Hippocrates Socrates: "And is not bodily habit spoiled by rest and illness, but preserved for a long time by motion and exercise?" Theaetetus: "True." --Plato, approximately 400 B.C. (Plato: Theaetetus, Great Books, Vol 7, 1952, p. 518.) vi INTRODUCTION Aerobic exercise tests measure the integrative ability of multiple physiological systems to adapt to acute aerobic exercise and are often used to determine physical fitness, assess overall health, and predict mortality (Hammond and Froelicher, 1984; Lee et al., 1999; Myers et al., 2002). The functional capacity of each system determines the overall efficiency of adaptation, which in turn determines the quality of performance on an exercise test. Low performance, then, reflects a compromise in the functional capacity of one or more physiological systems, denotes substandard physical fitness, and may indicate an elevated risk for disease development. While the associations between aerobic performance, physical fitness, and overall health are well known, the underlying factors involved are poorly understood (Blair et al., 2001). Identifying these underlying factors is therefore an essential step toward a more throrough understanding of the relationship between physical fitness and health. Aerobic exercise capacity is a decidedly complex trait for which measures of performance display continuous variation from low to high values in populations (Britton and Koch, 2001; Koch and Britton, 2005). The additive effect of multiple genetic and environmental factors influencing the physiological systems involved in the adaptive response to exercise gives rise to the observed variation in performance. Genetic factors alone may account for a significant portion of this variation as human and rodent studies estimate the heritability of exercise performance to range from 39% to 73% (Bouchard et al., 1998; Koch et al., 1998; 1 Bouchard et al., 1999; Lightfoot et al., 2001; Lightfoot et al., 2007). These genetic factors are likely a complex mixture of multiple genes, or rather their allelic variants, each exerting relatively minor effects on performance (Jennings et al., 1989; Blair et al., 1995; Bouchard et al., 1999; Koch et al., 1999; Lightfoot et al., 2001; Barton and Keightley, 2002; Lerman et al., 2002). As numerous physiological systems are involved in determining the response to exercise, subsets of the aforementioned genes may be operating in only one or a few of these systems and behaving as discrete units. The sum effect of the response of each allelic variant in each organ system determines the overall response to exercise, and therefore overall performance capacity. Complex systems such as those that determine aerobic exercise performance are difficult to study without the use of appropriate models. Such models help reduce the complexity of the system by providing the means to dissect it into simpler components. Rats have historically been used as models for physiological processes (James and Lindpaintner, 1997), but in more recent years they have been used to study the genetic component of complex traits such as hypertension, diabetes, and aerobic running capacity (Dahl et al., 1962b; Dahl et al., 1962a; Colle et al., 1983; Barbato et al., 1998; Koch et al., 1998). A vast array of genetic tools has been developed to help resolve the molecular mechanisms underlying the complex physiology of the rat. However, congenic strains have been particularly useful because they isolate individual genetic factors and allow their effects on trait variation to be investigated independent of 2 other causative genetic factors (Joe, 2005; Lazar et al., 2005). Furthermore, congenic strains facilitate the study of natural variation in complex traits rather than using physical or chemical interventions that attempt to mimic trait variation or induce disease (Goldblatt et al., 1934; Rossini et al., 1977; Zolotareva and Kogan, 1978; Ahn et al., 2004; Pinet et al., 2004). Combined with the knowledge extracted from the relatively recent sequencing of the rat genome (Gibbs et al., 2004), congenic rat strains provide a powerful tool for understanding factors that contribute to the natural physiological variation for which so much data exists, including exercise performance. Work in our laboratory has focused on studying the relationship between the complex physiological nature of aerobic exercise performance and the underlying allelic variants that determine variation in performance by developing rat models of aerobic running capacity (ARC). A treadmill running test similar to that of the Bruce test used for human cardiovascular performance evaluations (Bruce et al., 1963) has been used to selectively breed for high and low ARC in rats (Koch et al., 1998; Koch and Britton, 2001) and to survey the ARC of commercially available inbred rat strains, which identified the Copenhagen (COP) and DA strains as being the most divergent among eleven inbred strains evaluated (Barbato et al., 1998). While the use of selectively bred rat strains is superior to available inbred strains with respect to being more highly divergent for ARC specifically, they are still genetically heterogeneous, which makes identifying the genetic factors responsible for the divergence difficult (Flint and Mott, 2001). The 3 COP and DA rats, on the other hand, are genetically homogeneous, naturally divergent for ARC, and readily available for the application of existing genetic tools that can be used to identify the underlying genetic factors responsible for variation in ARC. Using a segregating F2 population bred from COP and DA rats we performed a genome scan to identify chromosomal regions, or quantitative trait loci (QTLs), linked to the strain variation in ARC. We detected two such regions on rat chromosome 16 (RNO16) and one region on rat chromosome 3 (RNO3) (Ways et al., 2002). We also observed an interaction between loci in two intervals carrying ARC QTLs,
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