Tetanic Contraction Impairs Atrophic Soleus Via Calpain Pathway

The dysfunction and atrophy of skeletal muscle has been regarded as one of the major adverse effects of microgravity exposure, and there are still no effective countermeasures. In addition, the improper exercise will further impair the anti-gravity muscles when the astronauts return to the ground. It has been reported that calpain pathway may participate in the regulation of muscle fiber in the dystrophic or disused atrophy. However, the cellular mechanisms remain unknown.In present study, we want to investigate the precise mechanisms of calpain pathway in regulating sloeus remodeling with simulated microgravity rats. We found that calpain pathway, such as the protein expression and activity of calpain, intracellular Ca2+, and Desmin are associated with the structural and functional changes of soleus in simulated microgravity rats. However, tetanic contraction could further impair function and structure of soleus in simulated microgravity rat through calpain pathway.1. Ultrastructure changes in soleus of simulated mocrogravity. The tail-suspended, hindlimb-unweighting rat model was used to simulate the atrophic effects of microgravity. The diameter of soleus became thinner in 2-week tail-suspended rats (SUS) as compared with simultaneous control (CON). However, there are no significant changes in the myofibrils arrangement and Z-line flow between two 2-week SUS and CON. In further study, we found that the soleus atrophy became obvious and the myofilaments began to dissolve in 4-week SUS as compared with simultaneous CON. When the rats were forced to exercise after continuous 4-week hind limb unloading, severe injuries were observed: the myofibrils were malalignment and the Z-lines were in breakages. Interestingly, when the rat was injected with the calpain inhibitor, PD150606, before the forced exercise, the ultrastructure was less impaired. These results indicated that simulated microgravity induced the soleus atrophy and succedent exercise further impaired the atrophic soleus.2. The concentration of intracellular calcium increased in atrophied SOL The concentration of intracellular calcium increased in SUS rats as compared with CON, which was detected by laser confocal microscope. Moreover, caffeine induced a higher concentration of intracellular calcium in SUS as compared with CON, which suggested that the atrophic soleus in SUS showed a stronger sensitivity to caffeine.3. Calpain 1 activity increased after repeated tetanic contraction. The protein expressions and activity of Calpain 1 were increased in SUS as compared with CON. However, no significant changes of calpain1 or 2 expressions were observed after treatment with repeated tetanic contraction in both groups. Particularly, the activity of Calpain 1 increased significantly after treatment with tetranic contraction in SUS, which can be significantly inhibited by calpain inhibitor PD150606. 4. Desmin was degraded after repeated tetanic contraction. The degradation of Desmin in SUS was observed by Western blot. After treatment with repeated tetanic contraction, both SUS and CON groups showed an increased tendency in Desmin degradation. However, there are also significant changes in Desmin degradation between SUS and CON group. The intervention of inhibitor PD150606 can reduce the amount of Desmin degradation in both groups.5. Effect of Ca2+, the upstream signal molecular of calpain pathway, on isolated muscle strip. Caffeine and H2O2 increased the intracellular Ca2+ concentration and then degraded the skeleton proteins, TnI. Meanwhile, caffeine and H2O2 increased the resting tension, decrease the developed tension and fatigue resistance in soleus strips. It has been proven that the ratio of maximal TR75 to initial TR75 (R-TR75) can be an index for SR Ca2+ release function in muscle fibers. Increased stimulating voltage, repeated tetanic contraction, shorten interval of recovery and unloading SOL can lead to Ca2+ concentration increase within atrophy muscle. Consequently, muscle contraction function decreased.In summary, suspended rat SOL suffered significant atrophy in ultrastructure and PD150606 intervention can significantly reduce the degree of ultrastructural damage. Ca2+ concentration and calpain expression and activity increased in suspended rat SOL muscle fibers. In repeated tetanic contraction, calpain 1 activity increased significantly, and PD150606 intervention can significantly reduce the calpain 1 activity. It's suggested that calpain is a key factor inducing skeletal muscle atrophy. Increased intracellular Ca2+ level is a major factor to increase calpain activity in muscle fibers. In present study, we want to investigate the precise mechanisms of calpain pathway in regulating sloeus remodeling with simulated microgravity rats. We found that calpain pathway, such as the protein expression and activity of calpain, intracellular Ca2+, and Desmin are associated with the structural and functional changes of soleus in simulated microgravity rats. However, treatment with tetanic contraction could further impair function and structure of soleus in simulated microgravity rat through calpain pathway.