Study On Effects Of RYR Channel With Its Regulators And Caveolin-1 In Skeletal Muscle Fibers Of Daurian Ground Squirrels (Spermophilus Dauricus) During Different Hibernation Periods

Background:According to the previous research of our group,the concentration of free calcium in the extensor digitorum longus and soleus muscle fibers of the ground squirrel(Spermophilus dauricus)increased significantly(56%and 119%,respectively)during interbout arousal compared with pre-hibernation.However,its detailed molecular regulation mechanism is still unclear.Researchers argued that the increase of cytosolic calcium concentration was mainly due to the release of Ca2+ from the cell endoplasmic reticulum.Ryanodine receptor 1(RYR1),is a receptor-channel protein of skeletal-muscle sarcoplasmic reticulum,which is not only a receptor for intracellularsecond messenger but also a major calcium release channel on sarcoplasmic reticulum.RYR1 is regulated by varieties of signals,the most important ones of which are the dihydropyridine receptor(DHPR)coupled with the triple tube and the cyclic adenosine phosphate ribose(cADPR).DHPR is a voltage detector located on the transverse tube membrane of skeletal muscles.DHPR would detect the voltage changes and subsequently change the conformation when cells are stimulated,and then open L-type calcium channel on plasma membrane to make extracellular calcium influx causing cytosolic calcium concentration increase.Meanwhile the changes of DHPR also make coupled RYR1 allosteric.Opening calcium release channel assists the sarcoplasmic reticulum Ca2+ influx to the cytoplasm,which would also increase the intracellular calcium concentration of skeletal muscle fibers.The position-coupling relationship between the DHPR and the RYR1 is the key to realize the sarcoplasmic reticulum calcium release,but some studies have found that the coupling function between DHPR and RYR is selective to muscle fiber type.The cADPR is generated with hydrolysis and cyclization of NAD+ by ADP ribose cyclase CD38(cluster of differentiation 38)in mammalian cells and then transported into the cell.CD38 is the transmembrane glycoprotein on the cell membrane,which could promote ADP cyclization and produce cADPR.And the cADPR could make calcium ions released from sarcoplasmic reticulum to the cytoplasm by opening RYR1 as the second messenger.Caveolin-1 is the cell membrane skeleton protein,which is the marker of cell membrane invagination.It is important to cell remodeling.Our-previous research of the hibernant ground squirrels illustrated that expression of Caveolin-1 in extensor digitorum longus during post-hibernation period was significantly lower than that of pre-hibernation.And the research also shown that coupling relationship between DHPR and RYR would be weakened by the silence of Caveolin-1,but the specific regulatory mechanism is not yet clear.The purpose of this study was to investigate the effect of Caveolin-1 for maintaining calcium homeostasis of skeletal muscle fibers during hibernation.To explore the role of RYR1 and its regulators in resisting calcium homeostasis in skeletal muscle of hibernation ground squirrels by studying the changes of expression levels of RYR1,DHPR,CD38 and Caveolin-1,and the colocalization coefficients of DHPR and RYR1.Objective:1.To explore the influence of individual differences to hibernation physiology research by observing and comparing the effects of different body weight and gender for Daurian ground squrriels on hibernation bout frequency.2.To explore the role of RYR1 in resisting calcium homeostasis in hibernation process by studying the changes of mRNA and protein expression level and protein location of RYR1 and its main regulators in soleus and extensor digitorum longus in Daurian ground squirrels for different periods of hibernation,further to studied the possible mechanisms of anti-muscle atrophy of hibernators.3.To explore the possible active mechanism of Caveolin-1 in resisting calcium homeostasis in hibernators' skeletal muscle by comparing the Caveolin-1 mRNA and protein expression level in Daurain ground squirrels' skeletal muscle during different periods of hibernation.Methods:1.The relative expression level of RYR1,DHPR,CD38 and Caveolin-1 mRNA of soleus muscle and extensor digitorum longus for different periods of hibernation were tested by real-time fluorescent quantitative PCR technique.2.The colocalization coefficients of RYR1 to Myosin and RYR1 to DHPR of soleus muscle and extensor digitorum longus for different periods of hibernation were tested by immunofluorescence histochemistry technique.3.The relative protein expression level of CD38 and Caveolin-1 of soleus muscle and extensor digitorum longus for different periods of hibernation were tested by Western Blot technique.Results:1.Effects of ground squirrels' body weight and gender on hibernation bout frequency There were little differences in hibernation bout frequency among ground squirrels with different body weight after months of hibernation,but there was no statistical significance(P>0.05).There was no linearity between body weight and times of interbout arousal.The times of interbout arousal in males are more than that in females,but there was no statistical significance(P>0.05),too.2.Changes of RYR1 and DHPR mRNA expression level and protein colocalization coefficients in Daurain ground squirrels' skeletal muscle during different periods of hibernationReletive mRNA expression level in RYR1 and DHPR:Extensor digitorum longus:Compared with Pre-hibernation group and Hibernation group,the expression of RYR1 mRNA in Interbout arousal group increased by 357.77%(P<0.001)and 741.04%(P<0.001),respectively,while compared with Interbout arousal group,the level decreased by 83.01%(P<0.001)in Post-hibernation group.Compared with Pre-hibernation group and Hibernation group,the expression of DHPR mRNA in Interbout arousal group increased by 1312.3%(P<0.001)and 1234.94%(P<0.001),respectively,while compared with Interbout arousal group,the level decreased by 99.09%(P<0.001)in Post-hibernation group.Soleus:Compared with Pre-hibernation group and Hibernation group,the expression of RYR1 mRNA in Interbout arousal group increased by 202.7%(P<0.001)and 1513.4%(P<0.001),respectively,while compared with Interbout arousal group,the level decreased by 85.24%(P<0.001)in Post-hibernation group.Compared with Pre-hibernation group and Hibernation group,the expression of DHPR mRNA in Interbout arousal group increased by 81.94%(P<0.05)and 168.04%(P<0.01),respectively,while compared with Interbout arousal group,the level decreased by 75.56%(P<0.01)in Post-hibernation group.Colocalization coefficients:Extensor digitorum longus:Compared with Pre-hibernation group and Hibernation group,the colocalization coefficients of RYR1 to fast-myosin in Interbout arousal group decreased by 27.4%(P<0.05)and 62.17%(P<0.001),respectively,while compared with Interbout arousal group,the level increased by 155.71%(P<0.001)in Post-hibernation group.Compared with Hibernation group,the colocalization coefficients of RYR1 to DHPR in Interbout arousal group increased by 16.59%(P<0.05),while compared with Interbout arousal group,the level decreased by 38.97%(P<0.001)in Post-hibernation group.Soleus:Compared with Pre-hibernation group,the colocalization coefficients of RYR1 to slow-myosin in Hibernation group increased by 63.79%(P<0.01),and there was no significant differences among other groups.There is no significant difference in colocalization coefficients of RYR1 to DHPR among all groups(P>0.05).3.Changes of CD38 mRNA and protein expression level in Daurain ground squirrels'skeletal muscle during different periods of hibernationExtensor digitorum longus:Compared with Pre-hibernation group and Hibernation group,the expression of CD38 mRNA in Interbout arousal group increased by 743.74%(P<0.001)and 482.19%(P<0.001),respectively,while compared with Interbout arousal group,the level decreased by 97.27%(P<0.001)in Post-hibernation group.Compared with Hibernation group,the expression of CD38 protein in Interbout arousal group was decreased by 50.63%(P<0.01).Soleus:Compared with Pre-hibernation group and Hibernation group,the expression of CD38 mRNA in Interbout arousal group increased by 367.74%(P<0.001)and 1184.22%(P<0.001),respectively,while compared with Interbout arousal group,the level decreased by 93.75%(P<0.001)in Post-hibernation group.Compared with Hibernation group,the expression of CD38 protein in Interbout arousal group decreased by 50.43%(P<0.01),while compared with Interbout arousal group,the level increased by 249.31%(P<0.001)in Post-hibernation group.4.Changes of Caveolin-1 mRNA and protein expression level in Daurain ground squirrels' skeletal muscle during different periods of hibernationExtensor digitorum longus:Compared with Pre-hibernation group and Hibernation group,the expression of Caveolin-1 mRNA in Interbout arousal group increased by 758.88%(P<0.001)and 968.44%(P<0.001),respectively,while compared with Interbout arousal group,the level decreased by 81.46%(P<0.001)in Post-hibernationgroup.Compared with Pre-hibernation group and Hibernation group,the expression of f Caveolin-1 protein in Interbout arousal group increased by 192.47%(P<0.001)and 72.51%(P<0.001),respectively,while compared with Interbout arousal group,the level decreased by 80.53%(P<0.001)in Post-hibernation group.Soleus:Compared with Pre-hibernation group,the expression of Caveolin-1 mRNA in Hibernation group,Interbout arousal group and Post-hibernation group increased by 174.81%(P<0.001),162.49%(P<0.001)and 83.17%(P<0.001),respectively.Compared with Pre-hibernation group and Hibernation group,the expression of Caveolin-1 protein in Interbout arousal group increased by 73.22%(P<0.05)and 54.88%(P<0.05),respectively.Conclusion:1.There is no significant effect of Daurain ground squirrels' body weight and gender on hibernation bout frequency and the physiological and biochemical indexes in skeletal muscle of this study.2.The upregulations of relative mRNA level of RYR1 and DHPR and their colocalization coefficient are likely to be the main reasons why intracellular calcium concentration increased during interbout arousal.3.The relative mRNA expression level of CD38 was upregulated in reserve during interbout arousal.However,the regulation of the CD38 singal pathway has not playing a major role in the regulation during interbout arousal.4.The upregulation of expression level of Caveolin-1 is likely to be the reason why the colocalization coefficient of DHPR and RYR1 increased during interbout arousal,so that Caveolin-1 may participate in the regulation of cell calcium homeostasis in ground squirrels' extensor digitorum longus during hibernation.