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Analysis of global mRNA expression in human skeletal muscle during recovery from endurance exercise

D. J. Mahoney^*, G. Parise, S. Melov, A. Safdar and M. A. Tarnopolsky^,1

Departments of
^* Medical Sciences
Kinesiology
Pediatrics and Medicine, McMaster University, Hamilton, Ontario, Canada; and
Buck Institute for Aging Research, Novato, California, USA

¹Correspondence: Department of Pediatrics, Rm. 4U4, McMaster University Medical Center, 1200 Main St. W., Hamilton L8N 3Z5, Ontario, Canada. E-mail: tarnopol{at}mcmaster.ca

SPECIFIC AIM

The purpose of our study was to use cDNA microarrays to search for novel genes and transcriptional pathways that are activated in human skeletal muscle after endurance exercise.

PRINCIPAL FINDINGS

1. Endurance exercise alters mRNA expression in skeletal muscle
To examine the transcriptional response to endurance exercise, we measured global mRNA expression in skeletal muscle from healthy, young, sedentary males before, 3 h, and 48 h after an exhaustive bout of high-intensity cycling. All microarray comparisons were made within individual subjects (nonpooled; n=4), and we used repeated measures significance analysis of microarray (SAM) to determine statistically significant differential mRNA expression. The expression of 223 genes was significantly altered post-exercise. To confirm the population-wide validity of these mRNA expression changes, we used real-time RT-PCR and examined selected genes in a different cohort of subjects (n=10). We were able to confirm 8 of 9 genes whose expression was significantly altered by > 2-fold, while we were unable to confirm 3 genes whose expression was significantly altered by < 2-fold. We set our parameters for defining "differential mRNA expression" as both statistically significant and altered by > 2-fold. Given these parameters, expression of 118 genes differentially increased 3 h post-cycling and 8 decreased. At 48 h, the expression of 29 genes differentially increased and 5 decreased. 1) Endurance exercise rapidly induced the expression of a large number of genes, but repressed the expression of very few; and 2) elevated mRNA expression post-endurance exercise was generally transient, with the expression of relatively few genes being elevated at 48 h (to view the dataset, visit http://www.ncbi.nlm.nih.gov/geo/).

2. Endurance exercise induced mRNA expression of genes involved in metabolism and mitochondrial biogenesis
Endurance exercise induced the expression of 13 transcriptional regulators and nontranscriptional regulators involved in metabolism and mitochondrial biogenesis (Table 1 ). The transcriptional regulators consisted of transcription factors and coactivators such as the forkhead transcription factor O1 (FOXO1), peroxisome proliferator activated receptor (PPAR), delta (PPAR), and gamma (PPAR), nuclear receptor binding factor-2 (NRBF-2), and PPAR coactivator 1 (PGC1). The expression of these genes was each elevated at 3 h (1.7- to 5.2-fold), and only NRBF-2 remained elevated at 48 h. The nontranscriptional regulators consisted of proteins involved in IL-6 signaling (IL-6 receptor; gp130), glucose metabolism (pyruvate dehydrogenase 4, PDK4; ras-related associated with diabetes), heme biosynthesis (aminolevulinate synthase 2), and mitochondrial translation (mitochondrial ribosomal protein L2). The expression of each of these genes was moderately elevated 3 h post-endurance exercise (2.3- to 3.5-fold), and none of them were significantly altered at 48 h. Taken together, endurance exercise led to the rapid, transient, and coordinate induction of transcriptional programs for regulating various aspects of metabolism and stimulating mitochondrial biogenesis.

3. Endurance exercise induced mRNA expression of genes involved in oxidant stress and signaling
Endurance exercise induced the expression of all seven metallothionein (MT) genes measured on our array at 3 h, each of which returned toward baseline by 48 h (Table 1) . The increases were very consistent between these genes (4.2- to 7.5-fold), suggesting a coordinate induction, and were amongst the largest observed in the study. Endurance exercise also increased the expression (3 h) of a superoxide-responsive transcription factor (interferon regulatory factor 1), an enzyme involved in DNA repair from free radical damage (tyrosyl-DNA phosphodiesterase 1), and an immediate early gene that forms part of the AP-1 complex (junB). These data describe the rapid, transient, and coordinate activation of an expression program involved in oxidant stress management and signaling.

4. Endurance exercise induced mRNA expression of genes involved in electrolyte transport
Endurance exercise induced the expression of genes involved in transporting Ca²⁺ into the sarco/endoplasmic reticulum (sarco/endoplasmic reticulum Ca²⁺ ATPase 3, SERCA 3), Na⁺ and K⁺ across the plasma membrane (Na⁺/K⁺ ATPase ß3), and Cl^– across the plasma membrane (chloride channel 4), as well as other miscellaneous membrane carriers (Table 1) . These increases were moderate (2.2- to 4.5-fold), except for the NMDA receptor, whose expression increased 25.9-fold at 48 h. These results describe a wide-ranging expression program for electrolyte transport across cell membranes.

5. Endurance exercise induced mRNA expression of genes involved in other miscellaneous functions
Endurance exercise induced the expression of genes involved in cell stress management, proteolysis, apoptosis, cell growth and differentiation, as well as genes involved in transcription and other miscellaneous functions (not shown). These expression changes describe programs of gene expression that represent: 1) increased capacity for protein folding and intracellular transport, pain management, and metal handling; 2) inflammatory cell recruitment; 3) regulation of proteolysis, apoptosis, and cell growth and differentiation. One particularly striking finding was that endurance exercise increased the expression of all three members of the nuclear receptor subfamily 4, group A (NRS4A1-3; also known as Nur77, Nurr1 and Nor1, respectively).

CONCLUSIONS AND SIGNIFICANCE

This is the first study to use DNA microarrays to examine global mRNA expression in skeletal muscle during recovery from endurance exercise. Given the major stressors faced by working skeletal muscle during exercise and the major adaptations that occur after a period of endurance training, we speculate that many of these expression changes describe transcriptional programs geared toward reestablishing muscle homeostasis, and inducing the adaptations that occur in skeletal muscle in response to endurance training (Fig. 1 ).

View larger version (30K): [in this window] [in a new window]   Figure 1. The transcriptional response to endurance exercise. This schematic illustrates the potential cellular significance of the expression changes observed in our study. A) A bout of endurance exercise induced the expression of genes involved in various aspects of metabolism, mitochondrial biogenesis, oxidant stress and signaling, electrolyte shuttling, stress management, proteolysis, death, growth, and transcription. B) At a cellular level, these expression changes can be predicted to participate in reestablishing muscle cell homeostasis post-endurance exercise, as well as contributing to the adaptations that occur in muscle after a period of endurance training (ETC: electron transport chain; NO: nitric oxide).

One such program involves a group of genes that regulate metabolism and mitochondrial biogenesis. Among these were seven genes that encode for regulatory proteins, including the "master regulators" of skeletal muscle glucose sparing, fatty acid metabolism, and mitochondrial biogenesis (PDK4, PPAR, PGC1, respectively).

Elevated expression of PPAR, PPAR, FOXO1, and NRBF2 has not previously been reported post-endurance exercise, and we speculate that these regulatory proteins are integral to the recovery and adaptive programs outlined above. Particularly exciting among them is PPAR, whose forced expression in skeletal muscle has very recently been shown to induce a strikingly similar phenotype to that of endurance training, such as increased skeletal muscle oxidative capacity, mitochondrial biogenesis, elevated type I muscle fibers, and enhanced performance on a treadmill running test. This finding led to the suggestion that PPAR is an important regulator of the major adaptive changes that occur in skeletal muscle in response to endurance training. Recently, PPAR protein content was shown to be elevated after endurance training in mice, and together with our observations in humans, support the suggestion that PPAR may be an important determinant of adaptation to endurance training. FOXO1, PPAR, and NRBF-2 elevation were also exciting findings, as each of these transcriptional activators are involved in regulating various aspects of lipid and glucose metabolism, and may be involved in stimulating mitochondrial biogenesis. Importantly, all 4 of these proteins are linked in various ways to PDK4, PGC1, and PPAR. Taken together, the rapid and relatively coordinate induction of each of these regulatory proteins, as well as their known function, lead us to speculate that they respond together and link endurance exercise stress to metabolic recovery and adaptation.

A second transcriptional program activated after our endurance protocol is a potential antioxidant program defined by the rapid and coordinate induction of all seven metallothionein (MT) genes measured on our arrays. Such a pronounced response suggests that MT induction is biologically important during, or during the recovery from, endurance exercise. Although their definitive cellular function is not known, there is growing interest in the antioxidant role of MTs. MTs protect various cell types from reactive oxygen species (ROS)-mediated damage, and ROS are strong activators of MTs. Given that endurance exercise induces elevated ROS generation, MTs may participate in skeletal muscle free radical management during or after endurance exercise. Glucocorticoids and IL-6 are also known to activate MT gene transcription, and may have mediated MT induction in the present study.

In conclusion, we have identified a large number of genes whose mRNA expression is differentially expressed in skeletal muscle during recovery from endurance exercise. Given the known function of these genes, we speculate that many of them are involved in various aspects of metabolism, mitochondrial biogenesis, oxidant stress and signaling, electrolyte handling, cell stress management, proteolysis, and cell growth/death. These observations expand our current understanding of the transcriptional response to endurance exercise, and generate exciting new hypotheses regarding the cellular programs involved in reestablishing homeostasis after endurance exercise and inducing adaptation after a period of endurance training.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3149fje;

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