| Introduction/Background:The mammals skeletal muscle will occur serious disuse atrophy under the long term limb fixation,and the muscle atrophy will cause cytoplasmic calcium overload.A large amount of calcium ions enter the mitochondria and cause mitochondrial calcium overload with the cytoplasmic calcium concentration continuously increase of skeletal muscle,and then lead to mitochondric dysfunction,damage,disintegration,and apoptosis.The previous study of our group showed that a unique protective mechanism of hibernating animals,which can maintain the calcium homeostasis of skeletal muscle fibers during the long period of inactivity and inhibit the related damage caused by wasting muscle atrophy.However,the role of the mitochondria to maintain the cytoplasmic calcium and the mitochondrial calcium regulation mechanism of hibernating animal muscle is unclear.Objective:To explore the molecular mechanism of mitochondrial calcium regulation and measure the mitochondrial calcium level and the related transport proteins relative expression in soleus muscle(SOL)and extensor digitorum longus(EDL)of hibernating ground squirrels(Spermophilus dauricus).Methods:1.To measure the concentration of mitochondrial calcium ion in soleus muscle and extensor digitorum longus by laser scanning confocal microscope.2.The relative expression level of absorb calcium channels MCU,MICU1,MICU2 and discharge calcium channels Letml mRNA of SOL and EDL for different periods of hibernation were tested by real-time fluorescent quantitative PCR technique.3.The relative protein expression level of absorb calcium channels MCU,MICU1 and discharge calcium channels Letml of SOL and EDL for different periods of hibernation were tested by Western Blot technique.Results:1.Changes of the muscle wet weight/body weight(MWW/BW)in ground squirrels for different peiord of hibernation:Compared with the pre-hibernation group(PRE),the MWW/BW ratio in hibernation group(HIB)showed no significant differences both in SOL and EDL.And compared with PRE,HIB and interbout arousal group(IBA),the MWW/BW ratio in the SOL of Post-hibernation group(POST)increased by 55.65%(P<0.01),38.22%(P<0.01)and 31.34%(P<0.01).The MWW/BW ratio in the EDL of POST increased by 23.02%(P<0.01),32.07%(P<0.01)and 18.97%(P<0.01).2.Hibernation bout of daurian ground squirrel:The average hibernation time of squirrels was 138.60 ± 23.81 days,and the torpor time was about 89.75%,and the awakening time was about 10.25%.The average time of hibernation bout was 11.84 ±6.94 days.The time of interbout was 1.36 ± 0.86 days.The numaber of interbout was 9.00 ± 1.77.3.The changes of the mitochondrial Ca2+ level:There was no significant change in mitochondrial Ca2+ level in SOL of different periods of hibernation(P>0.05).Compared with PRE,the mitochondrial Ca2+ level in EDL of HIB and POST increased by 3.11%(P<0.01)and 3.61%(P<0.01).Compared with HIB,the mitochondrial Ca2+ level decreased by 3.54%(P<0.01).Compared with IBA,the mitochondrial Ca2+ level increased by 4.12%(P<0.01).4.The relative mRNA expression of MCU,MICU1,MICU2 and Letm1 in different periods of hibernation:SOL:Compared with PRE,the MCU mRNA expression in the SOL of HIB increased 14.70%(P<0.01),decreased 34.71%(P<0.01)and 82.73%(P<0.01)of IBA and POST.Compared with HIB,the expression of IBA and POST decreased 43.08%(P<0.01)and 84.94%(P<0.01).Compared with IBA,the expression of POST decreased 73.55%(P<0.01).Compared with PRE,the MICU1 mRNA expression in the SOL of HIB,IBA and POST increased 137.00%(P<0.01),207.00%(P<0.01)and 165.00%(P<0.01).Compared with HIB,the expression of IBA increased 29.18%(P<0,01),Compared with IBA,the expression of POST decreased 13.59%(P<0.01).Compared with PRE,the MICU2 mRNA expression in the SOL of HIB,IBA and POST decreased 54.56%(P<0.01),45.51%(P<0.01)and 74.09%(P<0.01).Compared with HIB and IBA,the expression of POST decreased 42.98%(P<0.01)and 52.45%(P<0.01).Compared with PRE,the Letml mRNA expression in the SOL of HIB,IBA and POST decreased 52.79%(P<0.01),41.99%(P<0.01)and 81.89%(P<0.01).Compared with HIB,the expression of IBA increased 22.89%(P<0.01)and decreased 61.64%(P<0.01)of POST.Compared IBA,the expression of POST decreased 68.78%(P<0.01).EDL:Compared with PRE,the MCU mRNA expression in the EDL of HIB and POST decreased 20.20%(P<0.01)and 79.08%(P<0.01).Compared with HIB,the expression of IBA incerased 17.18%(P<0.01)and decreased 73.78%(P<0.01)of POST.Compared with IBA,the expression of POST decreased 77.62%(P<0.01).Compared PRE,the MICU1 mRNA expression in the EDL of IBA and POST decreased 14.47%(P<0.05)and 67.20%(P<0.01).Compared with HIB,the expression of IBA and POST decreased 12.41%(P<0.01)and 66.41%(P<0.01).Compared with IBA,the expression of POST decreased 61.65%(P<0.01).Compared with PRE,the MICU2 mRNA expression in the EDL of IBA and POST decreased 29.43%(P<0.01)and 69.53%(P<0.01).Compared with HIB,the expression of IBA and POST decreased 23.08%(P<0.01)and 66.78%(P<0.01).Compared with IBA,the expression of POST decreased 56.82%(P<0.01).Compared with PRE,the Letml mRNA expression in the EDL of HIB and POST decreased 20.74%(P<0.01)and 74.04%(P<0.01).Comapred with HIB,the expression of IBA increased 20.50%(P<0.01)and dereased 67.25%(P<0.01).Compared with IBA,the expression of POST decreased 72.82%(P<0.01).5.Changes of MCU,MICU1 and Letml relative protein expression in different periods of hibernation:SOL:Compared with PRE and HIB,the MCU expression in the SOL of IBA decreased 58.08%(P<0.01)and 63.64%(P<0.01).Compared with IBA,the expression of POST increased 233.54%(P<0.01).Compared with PRE,the MICU1 expression in the SOL of HIB,IBA and POST increased 125.22%(P<0.01),83.52%(P<0.05)and 384.31%(P<0.01).Comapred with HIB and IBA,the expression of POST increased 115.04%(P<0.01)and 163.91%(P<0.01).Compared with PRE,the Letml expression in the SOL of IBA decreased 44.42%(P<0.01)and increased 217.80%(P<0.01)of POST.Compared with HIB and IBA,the expression of POST increased 349.27%(P<0.01)and 471.8%(P<0.01).EDL:Compared with PRE,the MCU expression in the EDL of HIB increased 60.76%(P<0.01).Compared with HIB,the expression of IBA and POST decreased 43.50%(P<0.01)and 56.11%(P<0.01).Compared with PRE,the MICU1 expression in the EDL of HIB increased 19.51%(P<0.05).Compared with HIB,the expression of POST decreased 26.21%(P<0.01).Compared with PRE,the Letml expression in the EDL of HIB increased 78.60%(P<0.01),but the expression of IBA and POST decreased 41.34%(P<0.01)and 39.23%(P<0.01).Compared with HIB,the expression of IBA and POST decreased 67.15%(P<0.01)and 65.98%(P<0.01).Conclusion:1.The ratio of MWW/BW showed no significant difference(P>0.05)of HIB compared with PRE,but there was a significant difference of POST.This was a direct evidence that daurian ground squirrel skeletal mucle did not atrophy during hibernation.2.The mitochondrial cacium level was no significant change between the groups of SOL in the different periods of hibernation.However,the significant difference in the EDL,the mitochondrial cacium level was higher during HIB and POST.When cytoplasmic calcium is overloaded,mitochondria can store cytoplasmic calcium.Give priority to the use of fast muscle mitochondria to store calcium,to ensure slow muscle function and calcium steady state maintenance.3.The mRNA expression of mitochondrial calcium transporter MCU in the IB A was downregulated(SOL)or maintained(EDL),downregulated mRNA expression of MICU1 and MICU2,and upregulated the expression of Letml,compared with HIB,which may be one of the ways to maintain the calcium homeostasis of skeletal muscle mitochondria.4.The results showed that the protein expression levels of MCU and MICU1 were significantly increased during HIB,suggesting that mitochondria were involved in the regulation of cytoplasmic calcium homeostasis.And downregulated absorb calcium channels MCU and MICU1 was an important mechanism to avoiding the Ca2+ overload of mitochondrian.Maintain(SOL)or uptake(EDL)the protein expression level of Letml may be another way to avoid the Ca2+ overload of mitochondrian. |
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