Molecular adaptations of neuromuscular disease-associated proteins in response to eccentric exercise in human skeletal muscle

Abstract

The molecular events by which eccentric muscle contractions induce muscle damage and remodelling remain largely unknown. We assessed whether eccentric exercise modulates the expression of proteinases (calpains 1, 2 and 3, proteasome, cathepsin B+L), muscle structural proteins (alpha-sarcoglycan and desmin), and the expression of the heat shock proteins Hsp27 and alphaB-crystallin. Vastus lateralis muscle biopsies from twelve healthy male volunteers were obtained before, immediately after, and 1 and 14 days after a 30 min downhill treadmill running exercise. Eccentric exercise induced muscle damage as evidenced by the analysis of muscle pain and weakness, creatine kinase serum activity, myoglobinaemia and ultrastructural analysis of muscle biopsies. The calpain 3 mRNA level was decreased immediately after exercise whereas calpain 2 mRNA level was increased at day 1. Both mRNA levels returned to control values by day 14. By contrast, cathepsin B+L and proteasome enzyme activities were increased at day 14. The alpha-sarcoglycan protein level was decreased immediately after exercise and at day 1, whereas the desmin level peaked at day 14. alphaB-crystallin and Hsp27 protein levels were increased at days 1 and 14. Our results suggest that the differential expression of calpain 2 and 3 mRNA levels may be important in the process of exercise-induced muscle damage, whereas expression of alpha-sarcoglycan, desmin, alphaB-crystallin and Hsp27 may be essentially involved in the subsequent remodelling of myofibrillar structure. This remodelling response may limit the extent of muscle damage upon a subsequent mechanical stress.

Figure 1. Eccentric exercise induces delayed onset muscle soreness

A, maximal power of lower limbs. At the indicated time points, subjects performed a force velocity test (see ‘Testing of the muscle function’ in Methods for details). B, serum creatine kinase activity and myoglobinaemia. Values are means ± s.e.m. (n = 12). Asterisks denote statistical difference from control values: * P < 0.05.

Figure 2. Electron micrographs of longitudinal sections illustrating muscle damage following eccentric exercise in human skeletal muscle

All muscle sections were obtained from the same subject. Normal ultrastructural profiles were observed in control (a) and in day 14 muscle biopsies (b). Eccentric muscular contractions gave rise to extensive disorganization of the myofibrillar ultrastructure immediately after exercise (c) and at day 1 post exercise (d, e and f). Overall, eight subjects experienced muscle damage (n = 2, post exercise; n = 8, day 1). Streaming, disruption and dissolution of the Z-disk were frequently observed. Severe Z-disk damage was associated with A-band disruption and misalignment of myofibrils (c, d, e and f). Disturbances of the cross-striated band pattern were focalized along the longitudinal sections (c). Scale bars: 1 μm.

Figure 3. Eccentric exercise differentially regulates the mRNA levels of calpains 1, 2 and 3

RT-PCR was used to quantify mRNA levels of calpains 1, 2 and 3 (see ‘Total RNA extraction and real-time quantitative RT-PCR’ in Methods for details). Values are means ± s.e.m. (n = 9) and are expressed relative to control mRNA level. * P < 0.05; *** P < 0.001 vs. control.

Figure 4. Effects of eccentric exercise on α-actin, α-sarcoglycan and desmin protein levels

A, representative immunoblots of α-actin, α-sarcoglycan and desmin protein levels in control, post exercise, day 1 and day 14 muscle biopsies. B, immunoblotting quantification. Values are means ± s.e.m. (n = 8-12) and are expressed relative to control protein level. * P < 0.05 and *** P < 0.001 vs. control, # P < 0.001 vs. post exercise and day 1.

Figure 5. Effects of eccentric exercise on αB-crystallin and Hsp 27 protein levels

A, representative immunoblots of the small heat shock proteins, Hsp27 and αB-crystallin protein levels in control, post exercise, day 1 and day 14 muscle biopsies. B, immunoblotting quantification. Values are means ± s.e.m. (n = 9-10) and are expressed relative to control protein level. * P < 0.05.