Exercise And Skeletal Muscle Gene Expression

SUMMARY

1.Skeletal muscle is a complex and heterogenous tissue capable of remarkable adaptation in response to exercise train-ing. The role of gene transcription, as an initial target to control protein synthesis, is poorly understood.

2.Mature myo?bres contain several hundred nuclei, all of which maintain transcriptional competency, although the local-ized responsiveness of nuclei is not well known. Myo?bres are capable of hypertrophy. These processes require the activation and myogenic differentiation of mononuclear satellite cells that fuse with the enlarging or repairing myo?bre.

3.A single bout of exercise in human subjects is capable of activating the expression of many diverse groups of genes.

4.The impact of repeated exercise bouts, typical of exercise training, on gene expression has yet to receive systematic investigation.

5.The molecular programme elicited by resistance exercise and endurance exercise differs markedly. Muscular hypertrophy following resistance exercise is dependent on the activation of satellite cells and their subsequent myogenic maturation. Endurance exercise requires the simultaneous activation of mitochondrial and nuclear genes to enable mitochondrial biogenesis.

6.Future analysis of the regulation of genes by exercise may combine high-throughput technologies, such as gene-chips, enabling the rapid detection and analysis of changes in the expression of many thousands of genes.

Key words: mRNA, myo?bre, satellite cells, training, tran-scription.

INTRODUCTION

Mature skeletal muscle is a remarkably adaptive tissue, able to demonstrate signi?cant regeneration, hypertrophy and metabolic adaptation.1One of the most powerful stimuli for inducing skeletal muscle cellular re-organization is exercise training. In adults, depending on the type and duration of training, there can be appre-ciable changes in the size of the muscle mass and ?bre-type com-position, in addition to increased contractile activity and metabolic characteristics of the myo?bre population.2Considerable research has focused on the phenotypic nature of the adaptations, measured as alterations in protein abundance and activity, metabolic pathway ?ux and anatomical alterations. Yet, surprisingly few studies have systematically addressed the role and importance of gene expres-sion, despite the sometimes marked increase in protein levels. In the present review, the role of gene expression in skeletal muscle adaptability will be examined. Attention will be given to the dif-ferential gene programmes elicited by varied exercise interventions, the hypothetical adaptations in gene expression following repeated exercise (training) and the emerging technologies that hold promise in analysing the responsiveness of many thousands of genes to exer-cise interventions.

REGULATION OF GENE TRANSCRIPTION The draft sequence of the human genome, published February 2001, had as a major ?nding that the human genome contains approxi-mately 30000 genes,3one-quarter of the number predicted only 12 months earlier.4The smaller than anticipated gene number encodes approximately 85000 different mRNAs, due to alternative mRNA splicing and variable polyadenylation.5Messenger RNA synthesis or gene transcription is catalysed by multisubunit RNA polymerase II, which is under the interacting and complex regula-tion of the chromatin, histone acetylation and many nuclear proteins, including ‘transcription factors’.6Transcription factors comprise a diverse population of DNA-binding proteins, which, in many instances, are the terminal protein linking a complex signalling sequence with the activation of gene transcription. The activation of gene transcription results in the transient synthesis of mRNA, which may undergo modi?cation to generate the mature transcript. The mature transcript exits the nucleus via the nuclear pores and, when coupled to the translational machinery of the ribsome, is trans-lated into the encoded peptide.

UNIQUE REGULATION OF GENE TRANSCRIPTION IN SKELETAL MUSCLE Skeletal muscle is a complex tissue containing predominantly multi-nucleated mature myo?bres. Each myo?bre arises from the fusion of many hundred mononucleated progenitor cells, known as satel-lite cells, which are capable of undergoing myogenic programming

Proceedings of the Australian Physiological and Pharmacological Society Symposium: Integrative Physiology of Exercise

EXERCISE AND SKELETAL MUSCLE GENE EXPRESSION

David Cameron-Smith

School of Health Sciences, Deakin University, Melbourne, Victoria, Australia Correspondence: Dr David Cameron-Smith, School of Health Sciences,

Deakin University, 221 Burwood Highway, Burwood, Victoria 3125,

Australia. Email: davidcs@https://www.360docs.net/doc/f412889542.html,.au

Presented at the Australian Physiological and Pharmacological Society

Symposium Integrative Physiology of Exercise, November 2000. The papers

in these proceedings have been peer reviewed.

Received 21 July 2001; revision 10 September 2001; accepted

11 September 2001.

Clinical and Experimental Pharmacology and Physiology (2002) 29, 209–213

210 D Cameron-Smith

and differentiation into mature myocytes.7Satellite cells remain present within skeletal muscle, even into old age, retaining their proliferative potential.8Satellite cell proliferation and subsequent differentiation into myocytes enables repair and hypertrophy of exist-ing myo?bres or the generation of new myo?bres.

Within mature myo?bres, the many nuclei remain transcription-ally competent and theoretically capable of upregulated gene syn-thesis.9However, signi?cant compartmentalization of gene expression is evident, with genes for the ?-subunit of the acetyl-choline receptor and ?-dystrobrevin (involved in synapse matura-tion) predominantly expressed by nuclei in close proximity to the neuromuscular junction.10This is further supported by the limited diffusion of mRNA within structurally complex cells.11Yet, the expression of both endogenous muscle-speci?c genes and house-keeping genes is not uniform along the length of a myo?bre, despite shared cytoplasm and equal protein requirement along the myo?-bre.9The differences in individual nuclei action may be explained in terms of instability of the transcriptional complexes, such that small ?uctuations in low-abundance enhancers or transcription factors increase stability and regulate gene expression stochastically, in an off/on mode, rather than regulating the rate of transcription.12 To date, it has yet to be determined whether exercise elicits the widespread activation of nuclei or, alternatively, localized nuclei have sustained periods of transcriptional activity. However, the large number of nuclei and the involvement of the entire length of the myo?bre in contractile events suggest marked synthesis of new mRNA species is possible in myo?bres.

EXERCISE REGULATES GENE

TRANSCRIPTION

Numerous studies have now demonstrated that, following a single exercise bout, signi?cant elevations in the concentration of mRNA species, including metabolic,13–15coordinatory16,17and immuno-modulatory18genes, are observed in muscle samples from healthy human subjects. In response to exercise, gene transcription may be activated within seconds of contraction initiation through to hours after the cessation of exercise or following the restoration of nutri-ent stores.19For example, the mRNA abundance of the fos proto-oncogene family (c-fos and fosB) has been reported to be more than 20-fold higher 4min after the commencement of treadmill running in adult subjects.16Yet, the majority of studies have demonstrated that gene expression is most signi?cantly enhanced in the recovery period, following the completion of the exercise bout.19Using a novel run-on protocol to measure nuclear mRNA abundance, Pilegaard et al.20have demonstrated widespread activation of genes in the hours following varying modes of exercise. Thus, the pre-dominant transcriptional response to exercise may be present in the recovery phase of exercise. However, during exercise, the expres-sion of many genes may be suppressed. In preliminary data from our laboratory, the expression of 184 genes was analysed simulta-neously using gene-array technology (Research Genetics, Human Gene?lter, Huntsville, AL, USA) in human subjects from whom biopsies were collected prior and immediately at the end of 40min cycling exercise (70% V O2max). Of the genes analysed, more than 85% demonstrated a greater than 1.5-fold reduction in abundance (D Cameron-Smith, unpubl. data, 2001), demonstrating the pre-dominant action may be the inhibition of transcription or degrada-tion of mRNA during exercise prior to selective transcriptional activity once exercise has ceased.

EXERCISE TRAINING AND GENE

TRANSCRIPTION

The impact on gene expression of exercise performed without prior familiarization or training is likely to differ markedly from the response to repeated exercise bouts or the trained response. Few studies have systematically examined the changes in mRNA abun-dance following repeated bouts of the same exercise protocol, either maintained at the same absolute workload or matched relative to the improvements in exercise performance observed with training. Furthermore, the contribution of transcriptional (mRNA synthesis) versus translational (mRNA stability or translation ef?ciency) adaptations to a training-induced increase in protein levels are poorly understood. It is dif?cult to correlate mRNA with protein levels due to both the transient nature of mRNA following exercise and the protracted synthesis and longer half-lives of proteins. Therefore, caution must be applied to the analysis of adaptive changes in both mRNA responses to exercise and the impact of transcriptional com-pared with translational events.

Although few studies have examined the transient changes in mRNA levels following an initial exercise bout and again after an adaptation or training period, signi?cant adaptation is likely to occur. Of the available data, it has been shown that 8 weeks of leg–knee extensor training markedly attenuated (three-fold) the expression of vascular endothelial growth factor (VEGF) mRNA at a workload adjusted to increase as exercise capacity improved with training.21 An alternative adaptive response to repeated exercise may be the selective activation of mRNA species that require repeated bouts of exercise prior to any measurable increase in mRNA abundance. In our laboratory, the expression of several genes involved in skeletal muscle fat transport (fatty acid translocase) and oxidation (carnitine palmitoyltransferase 1) were not increased following a single exercise bout, but demonstrated increased basal and post-exercise expression following 9 days of 1h cycle training (RJ Tunstall, unpubl. data, 2001). Similarly, adaptations, such as inhi-bitions in gene expression, requiring repeated bouts of exercise have also been shown. After a single exercise bout, no measurable impact has been observed on the expression of the myosin heavy chain (MHC) IIa isoform, yet 7 days of training led to reduced mRNA levels after the last exercise bout, demonstrating that suppression was evident only after repeated exercise bouts.22Further studies are clearly required to elucidate the differential impact exercise train-ing has on the expression of genes and the contribution this exerts in adaptive alterations in the cellular protein concentrations.

POST-TRANSLATIONAL EVENTS AND

EXERCISE TRAINING

In experimental models of increased muscle loading, an increased protein synthesis rate proceeds changes in mRNA abundance, imply-ing that the ef?ciency of protein synthesis is enhanced.23Several studies have now examined the activation of the rate-limiting step of protein synthesis, the initiation step in which the mRNA tran-script is coupled to the ribosomal machinery. Several kinases involved in the initiation step of protein synthesis, including; the

Exercise and gene expression211

70kDa AS6 protein kinase (p70S6K), protein kinase B (PKB/Akt) and terminal members of the mitogen-activated protein kinase kinase pathway (extracellular signal-regulated kinase and p38), have all recently been identi?ed to be upregulated by exercise.24

The extensive remodelling of muscle with exercise training also requires the activation of protein degradation pathways for the removal of proteins no longer required. The ubiquitin–proteasome pathway is the major mechanism of selective protein degradation.25 Chronic motor nerve stimulation for 28 days of the rabbit tibialis anterior muscle markedly increased total proteasome activity of muscle extracts, with upregulated protein abundance of the 20S proteasome subunit and two regulatory proteins, namely PA700 and PA28.26

TRANSCRIPTIONAL REGULATION IN

EXERCISE

Resistance exercise

A pronounced adaptive response to high-intensity or weight-bearing exercise interventions is muscle hypertrophy. The increased mass of active muscle groups is achieved by an increase in the volume of individual myo?bres.27The enlarged myo?bre can only expand with the insertion of new nuclei, because a constant ratio of nuclei to cytoplasmic volume is maintained throughout all hyper-trophic responses.28Thus, hypertrophy is dependent on the prolif-erative activation of satellite cells and their myogenic differentiation7 prior to fusion with the existing myo?bre.29

Progression of satellite cells into myoblasts involves the regula-tion of muscle-speci?c proteins belonging to the basic-helix-loop-helix family of transcription factors. Members include MyoD, myogenin, myf-5, myogenic regulatory factor (MRF)-4 and myocyte enhancer factor (MEF)-2, which collectively function as dominant activators of skeletal muscle differentiation.30In the quiescent state, these myogenic factors are expressed at very low levels in satellite cells but, once activated, their abundance increases markedly.31 Considerable progress has been made in elucidating the signalling pathways responsible for the activation of the myogenic differenti-ation pathway, highlighting the pivotal role of endogenously derived factors regulated by shear or stress forces, including integrin-linked kinases,32and increased intracellular calcium ?ux, activating the calcineurin intracellular signalling pathway.33 Exogenous hormone activation of insulin-like growth factor-1, which may participate in the activation of calcineurin,34in addition to angiotensin II35and ?broblast growth factors,36may also coordinate myogenic differentiation of satellite cells.

Endurance exercise

In contrast, endurance exercise typically results in a shift in myo-?brillar components towards an increased abundance of slow isoform proteins, together with upregulated mitochondrial and oxida-tive metabolism enzyme levels.37Interestingly, induction of slow myo?brillar isoform genes may also be dependent on calcium and the calcineurin pathway.38Thus, an apparent anomalous situation arises in which calcium activation of calcineurin is pivotal in two vastly differing transcriptional programmes, either hypertrophy or the activation of the slow myo?bre cellular pattern and mito-chondrial biogenesis. A partial explanation is evident in the pattern of intracellular calcium released by tonic low-frequency motor nerve activity typical of endurance exercise, as compared with the high-amplitude and short-duration response of intense hypertophic phys-ical activity.39The resultant differing patterns of activation may result in the recruitment of alternative calcium-responsive kinases40or depend on kinases being activated simultaneously to stimulate an integrative downstream regulator.41Such a downstream integration of calcium signalling may be controlled by the myogenic transcription factor MEF-2, the activity of which is regulated by multiple signal cascades, including calcineurin and calmodulin-dependent protein kinase pathways.41However, much is to be dis-covered about how these pathways may intersect with those necessary for mitochondrial proliferation, which requires the coordination of both nuclear genes and the genes contained within the mitochondria’s own DNA.42

ANALYSIS STRATEGIES

Current individual mRNA species are analysed using either hybridization techniques (northern blotting or RNAse protection) or polymerase chain reaction (PCR) ampli?cation of individual genes. Normalization of gene abundance is based on comparisons with genes that have a constant gene expression, namely the house-keeping genes. These techniques enable the semiquantitative analysis of single or small numbers of genes and are labour inten-sive. Given that any phenotypic adaptation may require the activa-tion and inhibition of many thousands of genes simultaneously, these techniques will provide limited new data on the scope or complex-ity of the adaptation.

New technologies, particular high-throughput gene-scanning tools (including gene-chips), will provide dramatic insights into the complex patterns of gene expression necessary for the phenotype adaptation in muscle. The use of gene-chip technology to probe for patterns of gene expression have been applied to the analysis of ?bre-type differences in gene expression,43skeletal muscle ageing and species differences44and the molecular pathophysiology of muscular dystrophies.45Gene-chip technology, not withstanding the potential pitfalls,46will enable the mass screening of genetic response to a wide variety of stimuli, including different exercise inter-ventions. This technique holds particular promise with respect to the sensitivity and reproducibility of gene analysis, enabling quanti-tative detection of many regulatory genes that are often expressed at relatively low abundance.47Furthermore, the inclusion on gene-chips of expressed sequence tags, which represent partial gene sequences of genes of no described function, may aid in the identi-?cation of new and novel genes involved in the adaptive response of exercise to physical activity.

FUTURE ISSUES

There is much to be discovered in the pathways of gene regulation in response to exercise. Currently, little is known of the complex integration of competing signalling cascades prior to the activation of exercise-sensitive genes. Further confounding these investigations are the large differences in individual responsiveness to exercise. The population differences in exercise capacity are unlikely to be due to polymorphisms of single genes, although the recent exami-nation of the angiotensin-converting enzyme gene has yielding inter-esting associations with performance in several,48,49but not all,50

212 D Cameron-Smith

studies. The recently published genomic scan of the HERITAGE family training study identi?ed a number of potential genetic markers that correlate with changes in body composition.51Such gene-scanning studies will increasingly de?ne areas of the human genome that are linked to individual differences in exercise respon-siveness. Beyond these issues will be the dif?cult task of convert-ing genetic ?ndings into outcomes that can bene?t the health of diseased individuals, including the obese and diabetic.

ACKNOWLEDGEMENTS

The author acknowledges the collaborations and support of Professor Mark Hargreaves, Professor Greg Collier and Dr Rodney Snow (School of Health Sciences, Deakin University).

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