Acute Exercise and Ca2+ Stimulation Regulate Enzymes Involved in DNA Methylation in Human Skeletal Muscle

Abstract

Adaptations to exercise occur at the organ, cellular, protein, and molecular levels. At the molecular level, epigenetics are defined as non-sequence changes to the DNA that regulate gene expression. Two types of epigenetics modifications are chromatin remodeling and DNA methylation and while recent studies clearly demonstrate that exercise results in covalent modifications of histone in the chromatin, less is known about whether exercise changes DNA methylation at specific promoters. Changes in DNA methylation occur through DNA methyltransferase enzymes and putative demethylase enzymes. PURPOSE: To determine whether exercise is capable of altering mRNA levels of enzymes that modify DNA methylation levels. METHODS: Healthy young men (n=8) performed 3 hours of cycle ergometer exercise at 60% of VO2max and muscle biopsies were taken at 0 (pre), 3 (immediately post), 4.5, 6, 12, and 24 hours. Primary muscle cultures were established in vitro from healthy young males, subjected to a variety of stimuli and inhibitors. Both studies utilized SYBR green qPCR to examine the mRNA levels of Dnmt3a, Dnmt3b, GADD45b, and GADD45a. RESULTS: In vivo the mRNA level of Dnmt3b was decreased by 50% (p<0.05) immediately following exercise, while GADD45b (17 fold) and GADD45a (6 fold) both peaked at 6 hrs (p<0.05). In culture, stimulation of proliferating myoblasts with either AICAR, epinephrine, of ionomycin (a Ca2+ ionophore) failed to alter mRNA levels of the Dnmt3a/b, while ionomycin increased GADD45b (∼4-5 fold) and GADD45a (∼7-9 fold). Similarly, in differentiated myotubes GADD45b and GADD45a were upregulated (3 and 2 fold respectively, p<0.05)) by Ca ionomycin, however Dnmt3a and Dnmt3b were both downregulated by about 40% following stimulation by ionomycin. Stimulation by ionomycin while inhibiting p38/MAPK during further increased GADD45b and increased Dnmt3a/b to control levels. CONCLUSIONS: In vivo exercise and in vitro stimulation of primary human muscle cultures can increase the mRNA of GADD45b/a, a putative DNA demethylase, while simultaneously decreasing Dnmt3a/b, two de novo DNA methyltransferases. Thus, future experiments are addressing whether simultaneous changes in DNA methylation of promoters of genes regulated during acute exercise also occurs.