Exercise alters mRNA expression of telomere-repeat binding factor 1 in skeletal muscle via p38 MAPK

Abstract

Telomeres protect chromosome ends and shorten with age in most tissues. Integral to the maintenance of telomeres is the protein complex shelterin. The gene expression regulation of shelterin proteins to physiological stressors is not understood in vivo. We have recently reported increased telomere-repeat binding factor 1 (TRF1) protein expression and longer telomere length in skeletal muscle of sedentary compared with chronically active mice. These provocative observations led us to examine the effects of acute physiological stress on shelterin expression in vivo in mice and to further define potential mechanisms associated with gene regulation of shelterin. Three groups of female C57Bl/6 mice were studied: one control group and two groups that underwent a 30-min treadmill running bout and were killed either immediately following or 1-h after the exercise. Following the exercise bout, mRNA expression of Trf1 was significantly reduced in the plantaris muscle, and this reduction was paralleled by significant increases in p38 MAPK phosphorylation. To determine if p38 mediated the decreases in Trf1 mRNA expression, C2C12 myotubes were treated with the calcium ionophore, A23187. In response to the A23187, Trf1 gene expression was significantly reduced, coupled with significant increases in p38 phosphorylation, similar to in vivo data. C2C12 myotubes pretreated with a p38 inhibitor (SB-202190) prevented the A23187-induced decrease in Trf1 mRNA expression, indicating a link between Trf1 gene expression and p38 MAPK activation. While it is too early to definitively report the effect of exercise on telomere biology in rodents or humans, these data provide important mechanistic insights into the paradoxical telomere shortening that occurs in skeletal muscle in response to chronic exercise in mice.

Fig. 1.

Acute exercise reduced shelterin component gene expression in the plantaris. A: Trf1 gene expression was significantly reduced in time point 1 (TP1) animals compared with baseline (BL) animals (P = 0.02), tended to remain reduced at time point 2 (TP2) (P = 0.07), and was not different between TP1 and TP2 (P = 0.58). *TP1 significantly different than BL (P < 0.05). B: Trf2 mRNA expression tended to decrease in TP1 animals compared with BL (P = 0.1), but was not different between BL and TP2 (P = 0.3) or between TP1 and TP2 (P = 0.5). C and D: Pot1a and Po1tb mRNA expression, respectively, was not different between any groups (P = 0.3 and P = 0.4, respectively). E: Pgc1a was not different between BL and TP1 or TP2 (P = 0.4 for both), but tended to be higher at TP2 compared with TP1 (P = 0.08). Values are means ± SE. mRNA abundance was assessed with RT-PCR, corrected for Gapdh, and expressed relative to the BL group. BL, n = 6; TP1, animals killed immediately following acute exercise bout, n = 8; TP2, animals killed 1 h after the acute exercise bout, n = 8. See text for definition of genes.

Fig. 2.

Gene expression of shelterin components in tibialis anterior (TA) muscle following acute exercise. A: Trf1 gene expression was not different in TP1 or TP2 animals compared with BL animals (P = 0.3 and P = 0.7, respectively) and was not different between TP1 and TP2 (P = 0.1). B: Trf2 gene expression was higher in TP1 compared with TP2 animals and tended to be higher than BL (P = 0.03 and P = 0.08, respectively), but was similar between BL and TP2 (P = 0.8). C and D: Pot1a and Po1tb mRNA expression, respectively, was not different between any groups (P = 0.2 and P = 0.2, respectively). E: Pgc1a gene expression was not different in BL compared with TP1 and TP2 (P = 0.8), but TP2 had significantly higher expression compared with TP1 (P = 0.02). Values are means ± SE. mRNA abundance was assessed with RT-PCR, corrected for Gapdh, and expressed relative to the BL group. BL, n = 6; TP1, n = 8; TP2, n = 8. #TP1 significantly different than TP2 (P < 0.05).

Fig. 3.

p38 MAPK is activated following acute treadmill exercise in the plantaris. We measured the phosphorylation status of the MAPKs ERK1/2 (A) and p38 (B) following acute treadmill exercise. ERK1/2 was not different among any groups (ERK1 p44, P = 0.6 and ERK2 p42, P = 0.8). p38 MAPK had significantly increased phosphorylation at TP1 compared with BL (P = 0.03), TP1 and TP2 were similar (P = 0.2), as were BL and TP2 (P = 0.3). Densitometric analysis and representative immunoblot images (right) are shown. Values are means ± SE. Phosphorylated (p)-to-total (t) protein content ratios were derived and then expressed relative to the BL group for comparisons. GAPDH was a loading reference. BL, n = 6; TP1, n = 8; TP2, n = 8. *TP1 significantly different than BL (P < 0.05).

Fig. 4.

p38 MAPK is not activated following acute treadmill exercise in the TA. A: ERK1 (p44) phosphorylation was not significantly altered in the TA due to treadmill exercise (P = 0.4). ERK2 (p42) phosphorylation was greater at TP1 compared with TP2 (P = 0.04) and tended to be greater than BL (P = 0.08), but BL and TP2 were similar (P = 0.8). #Significantly different than TP2 (P < 0.05). B: p38 MAPK phosphorylation was not significantly altered by treadmill exercise (P = 0.6). Densitomeric analysis and representative immunoblot images (right) are shown. Values are means ± SE. Phosphorylated (p)-to-total (t) protein content ratios were derived and then expressed relative to the BL group for comparisons. GAPDH was a loading reference. BL, n = 6; TP1, n = 8; TP2, n = 8.

Fig. 5.

Calcium ionophore stimulation in C2C12 myotubes results in downregulation of Trf1 gene expression. Myotubes after 72 h of differentiation were treated with calcium ionophore (A23187, 1 μM) for 24 h to induce a similar reduction in Trf1 gene expression, as observed in vivo following treadmill running. All experiments were performed in duplicate on at least 3 separate occasions, in serial passage. Values are means ± SE. mRNA abundance of Trf1 and Gapdh were assessed with RT-PCR, corrected for Gapdh, and expressed relative to the BL group. *A23187 treated significantly different than BL (ethanol, P < 0.05).

Fig. 6.

Calcium ionophore stimulation results in p38 MAPK phosphorylation in C2C12 myotubes. To determine whether calcium ionophore treatment resulted in activation/phosphorylation of p38 MAPK, myotubes were treated for 3 h with 1 μM A23187 or control, and protein was isolated 30 min and 24 h following treatment. Calcium ionophore treatment resulted in significant phosphorylation of p38 MAPK compared with BL. Densitometric analysis (top) and representative immunoblot images (bottom) are shown. Values are means ± SE. Phosphorylated-to-total protein content ratios were derived and then expressed relative to the BL group for comparisons. GAPDH was a loading reference. BL, vehicle treated; A23187, treated for 3 h, and protein isolated 30 min posttreatment; 24H post, myotubes treated for 3 h, protein isolated 24 h posttreatment. All experiments were performed in duplicate on at least 3 separate occasions, in serial passage. *A23187 significantly different than BL (P < 0.05). #A23187 significantly different than 24H post (P < 0.05).

Fig. 7.

Inhibition of p38 MAPK prevents reduction of Trf1 gene expression. To determine whether p38 MAPK activation was directly related to Trf1 gene expression, we treated myotubes with calcium ionophore and in combination with p38 MAPK inhibitor SB-202190 (note: this inhibitor does not affect phosphorylation at Thr180-X-Tyr182, but inhibits p38 activity by competitively binding to the ATP binding site). Myotubes were treated with control (vehicle), calcium ionophore (A23871, 1 μM), or pretreated with SB-202190 p38 MAPK inhibitor (10 μM) for 30 min before 24 h of treatment with A23187. Treatment with p38 MAPK inhibitor prevented calcium ionophore-induced decrease in Trf1 gene expression. All experiments were performed in duplicate on at least 3 separate occasions. Values are means ± SE. mRNA abundance of Trf1 and Gapdh was assessed with RT-PCR, corrected for Gapdh, and expressed relative to the BL group. BL, vehicle treated (DMSO and ETOH); A23187, ionophore treated; SB-202190, myotubes treated in combination with A23187 and p38 MAPK inhibitor. *A23187 significantly different than BL. #A23187 significantly different than SB-202190.