Study On The Function Of Sarcoplasmic Reticulum Of Skeletal Muscle Fiber In Maintaining Intracellular Calcium Homeostasis In Hibernating Daurian Ground Squirrels

Background:Calcium(Ca²⁺)homeostasis is an important aspect to maintain intracellular environment in mammals.Under normal condition,intracellular Ca²⁺concentration is accurately regulated and maintain in a steady level.However,skeletal muscle disuse?e.g.spaceflight,hind limb unloading and bed rest?leads to the disturbance of intracellular Ca²⁺homeostasis,and mainly exhibits cytoplasmic Ca²⁺overload.Intracellular Ca²⁺overload plays an important role in the mechanisms of disuse-induced muscle atrophy.Calcium-activated proteases?calpains?,which can be activated by elevated intracellular Ca²⁺concentration,contributed to the degradation of filaments and initiation of most proteolytic pathways?e.g.the ubiquitin–proteasome pathway?in disuse.Increased protein degradation triggered by calpains is considered as one of the most important pathways in disuse-induced muscle atrophy.Hibernation is an important strategy for survival under low environmental temperatures and food scarcity during the winter season.Facing prolonged periods of inactivity and mechanical unloading,numerous hibernators like Daurian ground squirrels?Spermophilus dauricus?evade significant loss of muscle mass and force during long periods of hibernation inactivity,thus providing a natural model to study mechanisms of how disuse-induced skeletal muscle atrophy could be prevented.And after months of hibernating inactivity,there was no cytoplasmic calcium overload in different types of skeletal muscle fibers.Obviously,compared with non-hibernating animals,hibernating animals have remarkable ability in maintaining cytosolic calcium homeostasis and it also contributed to the ability of anti-disuse muscular atrophy in hibernators.However,the mechanism of skeletal muscle fibers maintain cytosolic calcium homeostasis remains unclear.In skeletal muscle,endoplasmic reticulum,as one of the most important calcium pools,is specialized into sarcoplasmic reticulum?SR?.On the one hand,SR can achieve a fluctuating balance with cytoplasmic Ca²⁺concentration,On the other hand it can reduce the calcium concentration of SR through its internal Ca²⁺buffering.One of the main reasons for skeletal muscle cytoplasmic calcium overload in disuse state is the Ca²⁺excessive release in sarcoplasmic reticulum caused by up-regulation of ryanodine receptor?RyR?,a release channel located in sarcoplasmic reticulum.The rate of Ca²⁺uptake in SR depends on Ca²⁺pump?SR calcium transport ATPase,SERCA?which transporting Ca²⁺from cytoplasm to SR.Except the regulation of calcium pool,the homeostasis of intracellular calcium also depends on the dynamic balance between free calcium and bound calcium.Under normal conditions,more than 90%of Ca²⁺were stored in different buffering in the form of bound calcium.Based on these contents,our study mainly explored the changes of calcium concentration in cytoplasm and sarcoplasmic reticulum during hibernation,and focused on the changes of SERCA and RyR channels in sarcoplasmic reticulum.In addition,we also focused on the changes of regulatory proteins of calcium pump and RyR channel during hibernation and their effects on channel proteins.We hypothesized that RyR protein expression or regulation was up-regulated in later torpor,which promoted calcium overload in the cytoplasm;while the protein expression or regulation of sarcoplasmic reticulum calcium pump is up-regulated through interbout arousal,which can alleviate cytoplasmic calcium overload.To test this hypothesis,we studied the cytosolic and SR Ca²⁺concentration,protein and mRNA expressions of SERCA and RyR signal pathway?including SERCA1,PLB,SLN,?-AR2,CaMK2,RyR1,DHPR and FKBP12?,protein co-localization level of SERCA and RyR with its distinct proteins and expressions of Ca²⁺binding protein?CSQ,CaM?,further explored the molecular mechanism that SR involved in the regulation of calcium homeostasis in mammalian hibernators.Method:?1?The concentration of resting Ca²⁺in cytoplasm and sarcoplasmic reticulum of skeletal muscle was measured by single muscle fiber separation technique and laser confocal microscopy.?2?Western blot was used to detect the protein level of target molecules involved.?3?Real-time PCR?Quantitative Real-time PCR?was used to detect the transcriptional level of important target molecules involved.?4?Frozen sections and immunofluorescence histochemistry?IFC?were used to quantify the degree of protein overlap between target molecules.Research contents:In this study,48 Daurian ground squirrels were selected for different hibernation periods?summer,pre-hibernation,late torpor,interbout arousal,early torpor and post-hibernation?,and different types of skeletal muscles?Soleus represents slow muscle,extensor digitorum longus represents fast muscle,gastrocnemius represents mixed muscle?were selected for quantitative analysis.?1?The concentration of resting Ca²⁺in the cytoplasm and sarcoplasmic reticulum of different types of skeletal muscle during different hibernation periods was measured,which served as the basic data for this study.?2?The protein and transcriptional levels of RyR 1,an important Ca²⁺release channel in sarcoplasmic reticulum,and its key inhibitor protein FKBP12 and DHPR were quantitatively detected.The degree of protein overlap the two molecules was quantitatively measured to measure the ability of sarcoplasmic reticulum to release Ca²⁺during different periods of hibernation.?3?The protein and transcription levels of Phospholamban?PLB?and Sarcolipin?SLN?,the most important Ca²⁺uptake pathway in skeletal muscle sarcoplasmic reticulum,and the degree of protein overlap calcium pump and SLN were quantitatively detected to measure the ability of calcium pump to absorb Ca²⁺in cytoplasm during different periods of hibernation.?4?The protein expression of beta adrenergic receptor 2 and Camodulin kinase 2,which can up-regulate the activity of calcium pump upstream,and the phosphorylation level of Camodulin kinase 2were quantitatively detected in order to better explore the regulation mechanism of calcium pump in hibernation.?5?The transcription and protein levels of Ca²⁺binding protein Camodulin in cytoplasm and Ca²⁺binding protein in sarcoplasmic reticulum were quantitatively detected to measure the ability of skeletal muscle to reduce intracellular free Ca²⁺during hibernation.Results:?1?In late torpor?hibernation>60d,after interbout arousal and into torpor again>5d?,the resting free calcium concentration of the distinct skeletal muscles was significantly overloaded by 106-213%?P<0.001?,while the SR calcium concentration decreased by68-80%?P<0.001?;in the early torpor?after inter-bout arousal and into torpor again<24h?,the concentration of calcium in cytoplasm and SR of skeletal muscle fibers returned to levels in the summer active group.These results indicate that both cytoplasmic and sarcoplasmic reticulum calcium homeostasis are in dynamic equilibrium during hibernation,and the interarray awakening may be an important process for the recovery of calcium homeostasis.?2?During the whole hibernation period?including late torpor,interbout arousal and early torpor?,the protein expression level of ryanodine receptor 1(RyR1,major Ca²⁺release pathways in SR)was significantly increased by 37-107%?P<0.05?.In hibernation,the level of its mRNA in the three muscles was not higher than that in summer control group,and decreased by 25-50%?P<0.05?.The protein expression of DHPR in hibernation group was significantly lower?23-40%,P<0.05?than that in summer group.The co-localization level of RyR1 and DHPR was significantly lower in the early torpor group and the late torpor group than in the summer group.FKBP12,a key factor inhibiting the activity of RyR1,increased both in the SOL and EDL muscles during hibernation,but decreased in the GAS muscle.The level of its mRNA in three kinds of muscles was lower than that in summer control group?37-69%,P<0.05?.The results showed that the co-localization level of RyR1 and FKBP12increased only in late torpor of GAS among different hibernating periods of three muscles.The protein co-localization level of CSQ and RyR1 in hibernation group was significantly lower than that in summer group.This suggests that the increase of RyR1 protein expression and the decrease of DHPR protein expression and their co-localization may lead to the increase of calcium release from sarcoplasmic reticulum mediated by RyR1,which may be one of the reasons for the occurrence of cytoplasmic calcium overload in late torpor.?3?During the whole hibernation period?including late torpor,interbout arousal and early torpor?,the protein expression level of SERCA1(SERCA1,major Ca²⁺uptake pathways in SR)was significantly increased by 28-103%?P<0.05?.Phospholamban and sarcolipin,as two key regulators of inhibiting calcium pump activity,increased significantly or unchanged in all three types of muscle during hibernation.Phosphorylation of phosphoprotein increased significantly by 14-134%in the inter-bout arousal of SOL and in the early torpor of GAS?P<0.05?.The co-localization level between sarcolipin and calcium pump increased in early torpor and interbout arousal of EDL and GAS?P<0.05?.This suggested that calcium pump-mediated sarcoplasmic reticulum uptake of calcium may be enhanced during hibernation,which may be one of the reasons for the recovery of calcium homeostasis of sarcoplasmic reticulum and cytoplasmic reticulum during interbout arousal or early torpor.?4?The protein expression of Beta adrenergic receptor 2 increased by 42-50%during hibernation of GAS,which could up-regulate calcium pump activity through intracellular signal cascade reaction?P<0.05?.The phosphorylation level of Camodulin kinase 2increased by 30%in the inter-bout arousal of SOL,which could relieve the inhibition of phospholamban upon calcium pump?P<0.05?.This indicates that the way of up-regulating sarcoplasmic reticulum calcium pump has significant specificity in different types of muscles.?5?Calsequestrin?CSQ?and camodulin1?CaM?,two kinds of Ca²⁺-binding protein located in the SR and cytoplasm,respectively,increased by 85-323%and 48-136%during the whole hibernation period?P<0.05?.These results indicate that the three skeletal muscles in hibernation can significantly reduce the free calcium concentration in the whole cell.Conclusion:These findings first confirmed that the protein expression levels of SERCA1,RyR1,CaM and CSQ are significantly increased at different states of hibernation?torpor or euthermy?and the expression of factors that inhibit?phospholamban and sarcolipin?and up-regulate?beta adrenergic receptor 2 and Camodulin kinase 2?calcium pump activity were also maintained or even higher than that in the summer active group,which suggested that the function of SR in skeletal muscle cells may be more active during hibernation?hypothermy state?than that during the non-hibernation?homoiothermy state?.Up-regulating the calcium pump activity of sarcoplasmic reticulum through different signaling pathways,plus dramatically increasing the expression of CaM and CSQ which combined with the free Ca²⁺,from multiple dimensions,constituted the major mechanism of hibernators to maintain intracellular calcium homeostasis during hibernation.