Eccentric exercise activates novel transcriptional regulation of hypertrophic signaling pathways not affected by hormone changes

Abstract

Unaccustomed eccentric exercise damages skeletal muscle tissue, activating mechanisms of recovery and remodeling that may be influenced by the female sex hormone 17beta-estradiol (E2). Using high density oligonucleotide based microarrays, we screened for differences in mRNA expression caused by E2 and eccentric exercise. After random assignment to 8 days of either placebo (CON) or E2 (EXP), eighteen men performed 150 single-leg eccentric contractions. Muscle biopsies were collected at baseline (BL), following supplementation (PS), +3 hours (3H) and +48 hours (48H) after exercise. Serum E2 concentrations increased significantly with supplementation (P<0.001) but did not affect microarray results. Exercise led to early transcriptional changes in striated muscle activator of Rho signaling (STARS), Rho family GTPase 3 (RND3), mitogen activated protein kinase (MAPK) regulation and the downstream transcription factor FOS. Targeted RT-PCR analysis identified concurrent induction of negative regulators of calcineurin signaling RCAN (P<0.001) and HMOX1 (P = 0.009). Protein contents were elevated for RND3 at 3H (P = 0.02) and FOS at 48H (P<0.05). These findings indicate that early RhoA and NFAT signaling and regulation are altered following exercise for muscle remodeling and repair, but are not affected by E2.

Figure 1. Expression fold changes in mRNA expression of genes in muscle from baseline after exercise protocol.

Graph A – RCAN1 (N = 18). Graph B – HMOX1 (N = 18). 3H = 3 hours post exercise, 48H = 48 hours post exercise. Values are mean ± SEM. **Significant difference vs. baseline when collapsed across supplementation (P<0.01). ***Significant difference vs. baseline when collapsed across supplementation (P<0.001).

Figure 2. Expression fold changes in mRNA expression of CAPZA1 in muscle from baseline after exercise protocol.

3H = 3 hours post exercise, 48H = 48 hours post exercise. N = 18. Values are mean ± SEM. *Significant difference vs. baseline when collapsed across supplementation (P<0.05).

Figure 3. Fold change of phosphorylated/total ratio of signaling pathways from baseline after eccentric exercise.

BL  =  baseline, 3H  =  3 hours post exercise, 48H = 48 hours post exercise. Graph A – p38MAPK (Thr ¹⁸⁰/Tyr ¹⁸²) (N = 18). Graph B – GSK-3β (Ser⁹) (N = 18). Values are mean ± SEM. **Significant difference vs. baseline when collapsed across supplementation (P<0.01).

Figure 4. Western blot analysis of RND3 and FOS in skeletal muscle after eccentric exercise.

BL  =  baseline, 3H = 3 hours post exercise, 48H = 48 hours post exercise. Graph A – RND3 (N = 18). Graph B – FOS (N = 14). Values are mean ± SEM. *Significant difference vs. baseline when collapsed across supplementation (P<0.05).

Figure 5. Schematic representation of the transcriptionally active pathways following exercise induced muscle damage.

Eccentric exercise promoted greater expression of targets within the STARS/RhoA/AP1 and NFAT/AP1 signaling pathways for hypertrophy and actin biogenesis and organization.

Figure 6. Regulatory and downstream targets of STARS transcriptionally active following a single bout of eccentric exercise.

RCAN – regulator of calcineurin; HMOX1 – hemeoxygenase 1; ARHGEF7 and ARHGEF12 – Rho guanine nucleotide exchange factor 7 and 12; ARHGAP24 – Rho GTPase activating protein 24; RND3 – Rho family GTPase 3; DIAPH1 – diaphanous homologue 1; CORO1C – Coronin, actin binding protein 1; FLNB – Filamin B, beta; CAPZA1 – capping protein (actin filament) muscle Z-line alpha 1; ACTA2 – actin, alpha 2, smooth muscle, aorta; ACTN1 – actinin, alpha 1; AP1 – activator protein 1; FOS – FBJ murine osteosarcoma viral oncogene homologue; FOSB – FBJ murine osteosarcoma viral oncogene homologue B; JUND – Jun D proto-oncogene.

Figure 7. Regulatory and downstream targets of NFAT transcriptionally active following a single bout of eccentric exercise.

p38MAPK – p38 mitogen activated protein kinase; GSK-3β – glycogen synthase kinase 3 beta; MAF – v-maf musculoaponeurotic fibrosarcoma oncogene homologue (avian).