University of Kentucky UKnowledge Veterinary Science Faculty Publications Veterinary Science 12-30-2009 Transcriptional adaptations following exercise in thoroughbred horse skeletal muscle highlights molecular mechanisms that lead to muscle hypertrophy Beatrice A. McGivney University College Dublin, Ireland Suzanne S. Eivers University College Dublin, Ireland David E. MacHugh University College Dublin, Ireland James N. MacLeod University of Kentucky, [email protected] Grace M. O'Gorman University College Dublin, Ireland Follow this and additional works at: https://uknowledge.uky.edu/gluck_facpub See next page for additional authors Part of the Veterinary Medicine Commons Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Repository Citation McGivney, Beatrice A.; Eivers, Suzanne S.; MacHugh, David E.; MacLeod, James N.; O'Gorman, Grace M.; Park, Stephen D.E.; Katz, Lisa M.; and Hill, Emmeline W., "Transcriptional adaptations following exercise in thoroughbred horse skeletal muscle highlights molecular mechanisms that lead to muscle hypertrophy" (2009). Veterinary Science Faculty Publications. 13. https://uknowledge.uky.edu/gluck_facpub/13 This Article is brought to you for free and open access by the Veterinary Science at UKnowledge. It has been accepted for inclusion in Veterinary Science Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact [email protected]. Authors Beatrice A. McGivney, Suzanne S. Eivers, David E. MacHugh, James N. MacLeod, Grace M. O'Gorman, Stephen D.E. Park, Lisa M. Katz, and Emmeline W. Hill Transcriptional adaptations following exercise in thoroughbred horse skeletal muscle highlights molecular mechanisms that lead to muscle hypertrophy Notes/Citation Information Published in BMC Genomics, v. 10, 638. © 2009 McGivney et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Digital Object Identifier (DOI) http://dx.doi.org/10.1186/1471-2164-10-638 This article is available at UKnowledge: https://uknowledge.uky.edu/gluck_facpub/13 BMC Genomics BioMed Central Research article Open Access Transcriptional adaptations following exercise in Thoroughbred horse skeletal muscle highlights molecular mechanisms that lead to muscle hypertrophy Beatrice A McGivney1, Suzanne S Eivers1, David E MacHugh1,2, James N MacLeod3, Grace M O'Gorman1, Stephen DE Park1, LisaMKatz4 and Emmeline W Hill*1 Address: 1Animal Genomics Laboratory, UCD School of Agriculture, Food Science and Veterinary Medicine, UCD College of Life Sciences, University College Dublin, Belfield, Dublin 4, Ireland, 2UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland, 3Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546-0099, USA and 4University Veterinary Hospital, UCD School of Agriculture, Food Science and Veterinary Medicine, UCD College of Life Sciences, University College Dublin, Belfield, Dublin 4, Ireland Email: Beatrice A McGivney - [email protected]; Suzanne S Eivers - [email protected]; David E MacHugh - [email protected]; James N MacLeod - [email protected]; Grace M O'Gorman - [email protected]; Stephen DE Park - [email protected]; Lisa M Katz - [email protected]; Emmeline W Hill* - [email protected] * Corresponding author Published: 30 December 2009 Received: 25 June 2009 Accepted: 30 December 2009 BMC Genomics 2009, 10:638 doi:10.1186/1471-2164-10-638 This article is available from: http://www.biomedcentral.com/1471-2164/10/638 © 2009 McGivney et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Selection for exercise-adapted phenotypes in the Thoroughbred racehorse has provided a valuable model system to understand molecular responses to exercise in skeletal muscle. Exercise stimulates immediate early molecular responses as well as delayed responses during recovery, resulting in a return to homeostasis and enabling long term adaptation. Global mRNA expression during the immediate-response period has not previously been reported in skeletal muscle following exercise in any species. Also, global gene expression changes in equine skeletal muscle following exercise have not been reported. Therefore, to identify novel genes and key regulatory pathways responsible for exercise adaptation we have used equine-specific cDNA microarrays to examine global mRNA expression in skeletal muscle from a cohort of Thoroughbred horses (n = 8) at three time points (before exercise, immediately post-exercise, and four hours post-exercise) following a single bout of treadmill exercise. Results: Skeletal muscle biopsies were taken from the gluteus medius before (T0), immediately after (T1) and four hours after (T2) exercise. Statistically significant differences in mRNA abundance between time points (T0 vs T1 and T0 vs T2) were determined using the empirical Bayes moderated t-test in the Bioconductor package Linear Models for Microarray Data (LIMMA) and the expression of a select panel of genes was validated using real time quantitative reverse transcription PCR (qRT- PCR). While only two genes had increased expression at T1 (P < 0.05), by T2 932 genes had increased (P < 0.05) and 562 genes had decreased expression (P < 0.05). Functional analysis of genes differentially expressed during the recovery phase (T2) revealed an over-representation of genes localized to the actin cytoskeleton and with functions in the MAPK signalling, focal adhesion, insulin signalling, mTOR signaling, p53 signaling and Type II diabetes mellitus pathways. At T1, using a less stringent statistical approach, we observed an over-representation of genes involved in the stress Page 1 of 18 (page number not for citation purposes) BMC Genomics 2009, 10:638 http://www.biomedcentral.com/1471-2164/10/638 response, metabolism and intracellular signaling. These findings suggest that protein synthesis, mechanosensation and muscle remodeling contribute to skeletal muscle adaptation towards improved integrity and hypertrophy. Conclusions: This is the first study to characterize global mRNA expression profiles in equine skeletal muscle using an equine-specific microarray platform. Here we reveal novel genes and mechanisms that are temporally expressed following exercise providing new knowledge about the early and late molecular responses to exercise in the equine skeletal muscle transcriptome. Background ies have shown that a large number of genes are differen- The Thoroughbred racehorse is an elite athlete, that for tially expressed in skeletal muscle following exercise [19]. four hundred years has been selected for physiological A single bout of exercise has been shown to increase traits enabling exceptional speed and stamina. As a highly mRNA expression particularly in genes involved in mito- adapted athlete the Thoroughbred is a suitable model for chondrial biogenesis and metabolism [20]. understanding the physiology of exercise [1]. Thorough- breds have a very high aerobic capacity or maximal oxy- While protein changes and mRNA quantified in small gen uptake (VO2max) [2] relative to their body mass. A panels of genes by Western blotting and real time qRT- bout of intense exercise requires both aerobic and anaero- PCR [21-24] have been investigated, global mRNA expres- bic energy production and a Thoroughbred may increase sion during the immediate-response period (< 8 minutes) its metabolic rate from basal levels by up to 60-fold under has not, to our knowledge, previously been reported in racing conditions [3]. A critical component for athletic skeletal muscle following exercise in any species. Also, performance is muscle and it is notable that the Thor- global gene expression changes in equine skeletal muscle oughbred has a high skeletal muscle mass comprising following exercise have not been reported. Therefore to over 55% of total body mass [4]. identify novel genes and key regulatory pathways respon- sible for exercise adaptation we have used equine-specific The biological importance of skeletal muscle is reflected cDNA microarrays to examine global mRNA expression in in its remarkable structural and functional plasticity that skeletal muscle from a cohort of Thoroughbred horses (n enables rapid alterations to phenotype following repeated = 8) at two time points (immediately, and four hours bouts of exercise [5]. A single bout of acute exercise post-exercise) following a standardised incremental-step induces multiple stresses in skeletal muscle, including exercise test on a high-speed equine treadmill. increased demand for ATP and mechanical stress [6,7]. The responses to these stressors can be divided into two Results and Discussion broad categories: the return to homeostasis, and the adap- Experiment overview tive response. The principle processes associated with Eight four-year old unconditioned Thoroughbred horses homeostatic recovery are glucose sparing, elevated fat oxi- (castrated males) were exercised to maximum heart-rate