Effects Of Calcium Handling Of Sarcoplasmic Reticulum In Skeletal Muscle Fatigue And Recovery

Skeletal muscle is an important part of the movement system. Protection and keeping the normal function of skeletal muscle is crucial significance for the movement ability of body. The maximal force of contraction progressively decreases with the shortened interval of intermittent stimulations in skeletal muscles. The gradual decline in tetanic force is known as fatigue. Except the gradual decline in tetanic force, the relaxation duration of tetanic contraction shows a prolongation during repeated short tetani. The mechanisms contributes to skeletal muscle fatigue are still unknown, which involved metabolic and nonmetabolic effects. Although it is widely recognized that the changes of calcium relaease and uptake by sarcoplasmic reticulum play a key role in skeletal muscle fatigue, but the precise course needs to be clarified. The higher muscle fatigue places one limit on the physical capability of muscles during normal activities such as running, dowstairs or upstairs. Higher fatigability in aging muscle increases the risk of fell down, which would cause to fracture more probability. Long-term spaceflight also results in muscle fatigue which is very dangerous. To elucidate the specific mechanisms of skeletal muscle fatigue could laid the function to establish the methords of alleviating fatigue and speeding the recovery of contractile function, which has important effect and significance for protecting movement ability and promoting manned space activities.For this purpose, we aimed to the changes in calcium handling of sarcoplasmic reticulum in skeletal muscle fatigue and recovery. We established soleus muscle strip preparation and used measuring technique of SERCA activity, western blotting. The important contraction parameters in soleus obtained from normal rats, tail-suspened rats and hyperthyroid rats were observed and compared during fatigue and recovery, the mechnisms maybe involved were also discussed fringly. The main results are fllowing.Feasible electric stimulate conditions for isolated soleus The isolated soleus strips displayed a significant dependency on voltage, duration and frequency of electric stimulation, especially in the range of 8²0 V, 200μs⁵ ms and 20⁸0 Hz. But the frequency at maximal tension was not affected by changes of voltage.Prolongation ratio of TR₇₅ and calcium reslease function of sarcoplasmic reticulum Prolongation ratio of T75 in CON group is 2.6 in titanic contraction. After 5 mmol/L caffine perfusion, which could enchance the calcium release from sarcoplasmic reticulum, the value increased to 3.0. In contrast, when treated with 5 mmol/L Mg₂SO₄, the value decreased to 2.1 due to inhibiting the calcium realease function of sarcoplasmic reticulum. Moreover, when we set the duty cycly as 30 %, the stimulate voltage as 20 V and the frequency as 100 Hz, the prolongation ratio of TR₇₅ was 2.5. Henceforce, stimulate frequency increased to 120 Hz or 140Hz, the value were 2.7 and 2.3, respectively, which have no significant difference compare with 100 Hz. It can be explained by no more increase of calcium reslease, because calcium realease ability of sarcoplasmic reticulum has already reached a maximal value at 100Hz. Prolongation ratios of TR₇₅ were still decrease to 1.4 and 1.9 after 5 and 10 min of recovery, due to the inhibiti of calcium reslease. Along with the recovery, prolongation ratio of TR₇₅ at the 60^th min was 2.4, which has no significant difference compare with the initial value. It is confirmed that prolongation ratio of TR₇₅ was closely related to calcium reslease function of sarcoplasmic reticulum. This result suggests that TR₇₅ maybe used to indirectly reflect calcium reslease function of sarcoplasmic reticulum.TR₇₅ and SERCA activity The curve of frequency-force shifted to right and the force declined rate in hyperthyroid rats was more susceptible to fatigue compared with CON group. In additionally, TPT and TR₇₅ of twitch and TR₇₅ of titanic contraction were obviously shorten. The TR₇₅ of T4 group and CON group prolonged significantly after perfusion with CPA, the specific inhibitor of SERCA, and the percentages of prolongation have no significant difference between two groups. The FI of T4 group also increaseed to the level near to CON group, and the effect was most notable after 5.0μmol/L CPA perfusion. It is interesting that the prolongation ratio of TR₇₅ have no significant difference between T4 and CON groups, which implicated that T4 treatment did not affect calcium reslease function of sarcoplasmic reticulum. Therefore, the changes of TR₇₅ mainly resulted from SERCA.Bi-directional regulation effect of SERCA activity on fatigue SERCA activity of T4 group increased 36.5 % compare with CON group. CPA, the specifici inhibitor of SERCA, could recover the FI of T4 group to the level of CON group when the concentration was 1.0μmol/L and higher than CON group when the concentration was 5.0μmol/L. But when the concentration of CPA was 10.0μmol/L, the effect of enchance FI was dramaticly diminished. These results suggest that it is beneficial for strenghten resistance of fatigue by moderate inhibiting of SERCA, but excessively inhibit would leds to opposite result.The effect of unloaded and CPA on fatigue The decrease rate of tetanic force in unloaded soleus strips was more faster than CON group. Perfusion with 1-, 2-, 5-μmol/L CPA, the decrease rate of tetanic force increased both in SUS and CON groups. On the contrary, 10μmol/L CPA could postpone the decrease of titanic force. No matther what kind of concentraction of CPA perfusion, the decrease rate of tetanic force in unloaded soleus strips was slower than that of CON group which perfused with same concentraction of CPA, and cameback to the level of CON group without CPA perfusion. The prolongation of twitch duration induced by CPA had no significant difference between SUS and CON groups. Compared with the values before CPA treatment, TPTs of twitch contraction were prolonged by 4.2%, 13.1%, and 26.7% in the control soleus, and by 4.0%, 11.3%, and 24.2% in the unloaded soleus following 1-, 2-, and 5-μmol/L CPA treatment, respectively. TR₇₅s of twitch contraction were prolonged by 9.7%, 53.4%, and 121.6% in the control soleus, and by 9.3%, 55.6%, and 117.5% in the unloaded soleus after 1-, 2-, and 5-μmol/L CPA treatment, respectively. TR₇₅ of tetanic contraction was reduced by 38% in the unloaded soleus compared with control. Compared with the values without CPA treatment, TR₇₅s of tetanic contraction were prolonged by 19.3%, 52.7%, and 70.5% in the control soleus after 1-, 2-, and 5-μmol/L CPA perfusion, respectively. Interesting, the percentages of prolongation in TR₇₅ of tetanic contraction was 7.6%, 37.4%, and 73.1% in the unloaded soleus.Effects of temperature on force decline rate and contraction duration in control soleus Twitch duration (TPT and TR₇₅) of the soleus muscle strip during 27°C perfusion was setup as 100%. There were 50% or 70% increase in TPT or TR₇₅ at 22°C, but 37.5% or 52.3% decrease at 35°C. The force at 22°C was higher than those at 27°C during 5-minute repeated short tetani. However, the force at 35°C was much lower than those at 27°C. The average TR₇₅ of tetanic contraction was 345 ms at 22°C perfusion, and significantly shortened at 27°C and 35°C in the control soleus. Furthermore, Tiron (5 mmol/L), a reactive oxygen species scavenger, did not elicit any overt effect on the force decline rate of repeated short tetani at the fifth minute in the control and unloaded soleus strips at 35°C perfusion temperature.Modulations of caffeine, elevated extracellular Ca²⁺, and A23187 to force decline rate of repeated short tetani Caffeine (1 or 5 mmol/L) had no effect on the force decline rates in either control or unloaded soleus muscles. The tetanic force tended to be elevated (although not significant) during 3.5 mmol/L of extracellular Ca²⁺ perfusion in muscle strips of the control and unloaded soleus compared with 2.25 mmol/L Ca²⁺ perfusion. The tetanic force was significantly increased following 5-min exposure to A23187 (1μmol/L) in both control and unloaded soleus muscles.Unloaded soleus displayed dependency on extracellular calcium The titanic force obsviously decreased in unloaded soleus after 5 min of perfusion without calcium. At the 20^th min, it decreased to 82.8±5.7 % of initial force, meanwhile, the titanic force in CON soleus was 95.6±2.6 % of initial force. After reperfusion with 2.25 mmol/L calcium, the titanic force in CON soleus increased slightly at the 20^th min, differently, the titanic force in unloaded suleus increased to 147.4±9.66 %.Changes of expression of DHPRs and RyR in unloaded soleus 4 week of tail-suspension induced DHPRα1c overexpression compared with CON group. Accordingly, expression of DHPRα1s was reduced obviously compared with the control group. Percentage of expression of DHPRα1s in total amount of DHPRs also decreased significantly in unloaded soleus compared with control group. Moreover, expression of RyR decrease to the level near to EDL and heart, also dramatically lower than control group.In summary, the function of calcium relaese of sarcoplasmic reticulum and activity of SERCA have important effect on skeletal muscle contraction, especially on the parameters such as force, duration, which are closely relative to fatigue. They also regulate the resistence of fatigue by bi-directional regulation effect. In the unloaded soleus, uncoupling of ECC induced by changes of DHPRs and RyR may contribute to an increase in susceptibity to fatigue.