Effect Of Skeletal Muscle Contraction Modes On Phosphorylation Of P38/Akt And Recovery Rate After Fatigue

Insulin resistance of skeletal muscle is a key defect in the development of Type 2 diabetes. It is well established that exercise training can have beneficial effects on glucose uptake in insulin-resistant states. The molecular mechanism for enhanced glucose uptake after exercise training may be related to increased expression and activity of key proteins known to regulate glucose metabolism in skeletal muscle, like Akt in the PI3K-Akt pathway. Exercise also leads to an insulin-independent increase in glucose transport, mediated in part by the mitogen-activated protein kinase signaling cascades, and two of the important proteins in this cascade is p38 and ERK. Understanding the activation of Akt, p38 and ERK after skeletal muscle contraction may provide novel entry points for new strategies to enhance glucose uptake and for improved health in the general population. Besides, little is currently known about the effects of different modes of exercise on the phosphorylation of Akt, p38 and ERK. Moreover, the recovery rate after tetanic fatigue differs owning to different contraction modes. So the optimal contraction modes that both effectively phosphorylate the key proteins like p38 and pose relatively small impact on recovery rate are to be found out.As to the recovery rate after fatigue, there are still few investigations on the mechanism modulating it. Additionally, unloading induces an atrophy in the skeletal muscle with reduced recovery rate after tetanic contraction. The mechanism for the slower recovery rate in atrophic soleus is still unclear. So it's important to clarify the factors modulating recovery rate after fatigue and to seek the reasons causing slower recovery rate in atrophic muscles.Isolated soleus muscle strips were perfused ex vivo and different modes of contraction were induced by electrical stimulation. The phosphorylation on p38,ERK and Akt were detected by Western Blot analysis. The main results are following.1) The level of phosphorylation of p38 was higher at the optimal contraction modes, but these modes could not increase the level of phosphorylation of ERK and Akt. Muscle contraction led to an increase in p38 phosphorylation, with the greatest effect observed after 5 minutes of 10 % DC(duty cycle) contraction and 5 minutes of 1 % DC contraction. However, phosphorylation of ERK and Akt were not altered by the two contraction modes.2) As contraction intensity raised, the fatigability of skeletal muscles increased too. The sarcoplasmic reticulum Ca²⁺-ATPase was inhibited. As the contraction duty cycle became higher, skeletal muscles became easier to fatigue and slower to recovery. After 5 minutes of fatigue, the contraction tension was decreased to 16 % of the maximal tetanic tension (PO) in the 30 % DC group. In the 10 %DC group and 1 %DC group, 5 minutes of fatigue reduced the contraction tension to 32 %PO and 84 %PO, respectively. TR75 (time from peak tension to 75 % relaxation) is an indication of sarcoplasmic reticulum Ca²⁺-ATPase. TR75 was prolonged during the fatigue course. The level and mode of prolongation was related to duty cycle. In 1 % DC contraction mode, TR75 was prolonged from 165 ms to 220 ms in 5 minutes of fatigue. In 10 %DC contraction mode, TR75 was prolonged from 165 ms to 222 ms in the first 4 minutes of fatigue, and then it was shortened to 218 ms in the fifth minute. In 30 %DC contraction mode, TR75 was prolonged from 119 ms to 253 ms in the first 2 minutes of fatigue, and then shortened to 140 ms in the fifth minute.3) As the contraction intensity enhanced, the recovery rate after fatigue became slower.a. Slight fatigue mainly induced an inhibition to myofibril, and it had little effect on the recovery rate. After slight fatigue (contraction tension decreased by 10 % during fatigue course), the tetanic tension recovered to nearly 100 % PO at the twentieth minute, and the recovery rate was not affected by fatigue time. Perfusion with ruthenium red (an inhibitor of Ca²⁺ release channels in sarcoplasmic reticulum) slowed down recovery rate. This suggested that slight fatigue induced an inhibition to myofibril.b. Moderate fatigue mainly inhibited sarcoplasmic reticulum Ca²⁺ release channels, and it slowed down the recovery rate. After moderate fatigue (contraction tension decreased by 50 % during fatigue course), the recovery rate at the twentieth minute was 90-95 % Po. The recovery rate was related to the tetanic contraction duration. Perfusion with caffeine (an opener of Ca²⁺ release channels in sarcoplasmic reticulum) made near 100 % recovery at the fifth minute. This suggested that moderate fatigue induced an inhibition to sarcoplasmic reticulum Ca²⁺ release channels.c. Hypoxia, exogenous oxygen free radical and degradation of TnI (troponin I) all reduced recovery rate with different features. Perfusion with CoCl2 and Na2S2O4 (chemical hypoxia agent) during recovery course did not influence recovery rate at the first 10 minutes. But after that, the contraction tension decreased instead of increasing. Perfusion with H2O2 increased the recovery rate at the first few minutes, then the contraction tension declined. It might be related to TnI degradation after H2O2 perfusion.d. Endogenous oxygen free radical had no effect on recovery rate. Perfusion of Tiron (oxygen free radical remover) did not affect recovery rate. It indicated that endogenous oxygen free radical did not influence recovery rate.e. Change of sarcoplasmic reticulum Ca²⁺-ATPase did not affect recovery rate. TR75 indicates the activity of sarcoplasmic reticulum Ca²⁺-ATPase. Shorter TR75 indicates better activity of sarcoplasmic reticulum Ca²⁺-ATPase. In all modes of contraction, TR75 was prolonged during fatigue course and shortened during recovery course. So the activity of sarcoplasmic reticulum Ca²⁺-ATPase increased during recovery course. But the increase of sarcoplasmic reticulum Ca²⁺-ATPase did not correlate with recovery rate. Thus change of sarcoplasmic reticulum Ca²⁺-ATPase did not affect recovery rate.4) The mechanism of slower recovery rate in atrophic soleus mainly involved the easier inhibition of calcium releasing channels. In one-week tail-suspended rats, soleus muscles showed a 40 % of atrophy. After different modes of fatigue, the recovery rate in unloaded soleus was significantly slower than their synchronous groups. Sarcoplasmic reticulum Ca²⁺-ATPase showed increased activity in unloaded soleus, so it did not contribute to slower recovery rate in atrophic soleus. Inventions of different drugs showed that hypoxia and oxygen free radical were not the reason for the slower recovery rate in atrophic soleus either.On the whole, contraction modes with low duty cycle and short fatigue time could phosphorylate p38 effectively, and these modes had little influence on recovery rate. The inhibition of myofibril and sarcoplasmic reticulum Ca²⁺ release channels, hypoxia, exogenous oxygen radicals and degradation of TnI could affect the recovery rate after fatigue. The slower recovery rate in atrophic soleus was mainly due to the easier inhibition of sarcoplasmic reticulum calcium releasing channels.