| Background: Mitochondrial calcium overload will increase the production of reactive oxygen species(ROS),and accompanied by the risk of increasing nonspecific intimal permeability,depolarizing mitochondrial membrane potential,leading to apoptosis or necrosis of cells and muscle atrophy.The previous research of our research group found that the mitochondrial calcium concentration of typical slow muscle soleus muscle of hibernating animals(Spermophilus dauricus)did not change significantly during different hibernating periods,and the typical extensor digitorum longus muscle showed significant calcium overload phenomenon during hibernating,and did not recover to the pre-hibernating level when post-hibernation,and the change law and regulation mechanism of mitochondrial calcium concentration of other skeletal muscles in different hibernating periods were unknown.One-way transporters complex is the most important complex in mitochondrial calcium uptake channels.Mitochondrial calcium transporters(MCU)are the key channel proteins in the complex,which can uptake calcium ions along chemical gradients.Mitochondrial calcium uptake protein 1(MICU 1)and mitochondrial calcium uptake protein 2(MICU2)are the regulatory proteins of MCU,and the regulatory mechanism is affected by calcium concentration.Leucine-dependent zipp-ef-hand transmembrane protein 1(let m1)is a mitochondrial membrane protein that mediates that release of calcium ion and plays a key role in the dynamic balance of mitochondrial calcium.In this study,the changes of mitochondrial calcium concentration and the expression of calmodulin MCU,calmodulin MICU1 and MICU2,as well as the gene and protein level of calcium excretion protein Letm1 in skeletal muscle of daurian ground squirrel were determined to explore the changes of mitochondrial calcium concentration and its regulation mechanism.Objective: 1.By detecting the changes of mitochondrial calcium concentration of daurian ground squirrel in different hibernating periods,the changes of mitochondrial calcium concentration of daurian ground squirrel in different periods were explored.2.To explore the regulatory mechanism of mitochondrial calcium concentration change by measuring the expression of calcium intake MCU,calcium intake regulatory proteins MICU1 and MICU2,and calcium excretion protein Letm1 gene and protein level in daurian ground squirrel during hibernation.Research methods: 1.The animals were separated into six groups,summer active group(SA),pre-hibernation group(PRE),early torpor group(ET),late torpor group(LT),inter-bout arousal group(IBA)and post-hibernation group(POST).The plantaris longus(PL),gastrocnemius(GAS)and semitendinosus(ST)were used for all experiments.The changes of mitochondrial calcium concentration in different skeletal muscles of gerbils were measured by single fiber separation and laser confocal microscopy.2.The changes of mitochondrial calcium channel protein MCU,MICU,MICU2 and Letm1 in gastrocnemius,plantar and semitendinosus muscle were detected by real-time fluorescence quantitative PCR.3.The protein levels of mitochondrial calcium channel protein MCU,MICU1 and Letm1 in gastrocnemius,plantar and semitendinosus were determined by western blot.Results: 1.Changes of mitochondrial calcium concentration in skeletal muscle of daurian ground squirrel in different periods Compared with IBA group,the concentration of mitochondrial calcium in the gastrocnemius muscle of the rats in EL group decreased by 4.34 %(P < 0.05),and there was no significant difference among the other groups.The mitochondrial calcium concentration in the plantar muscles of daurian ground squirrel increased slightly during IBA,but there was no statistical significance.There was no significant difference in hibernating period.Compared with pre group and IBA group,the mitochondrial calcium concentration of semitendinosus in EL group increased by 5.99 %(P < 0.01)and 3.29 %(P < 0.01),respectively.2.Gene and protein expression of mitochondrial calmodulin in skeletal muscle of daurian ground squirrel at different stages.(1)Gastrocnemius muscle protein level: compared with sa group,the expression of MCU,MICU1 and Letm1 protein in IBA group decreased by 44.69 %(P < 0.01),34.15 %(P < 0.001),52.8 %(P < 0.01)respectively,but there was no significant difference in post group.Compared with PRE group,MICU1(IBA(23 %,P < 0.05),ET(40.23 %,P < 0.001),LT(39.1 %,P < 0.001),POST(34.48 %,P < 0.001)),Letm1(IBA did not change,ET(30.66 %,P < 0.01),LT(25.78 %,P < 0.05),POST(23.72 %,P < 0.05))Letm1 protein expression decreased in different degrees,compared with IBA group,(2)Gastrocnemius muscle m RNA level: compared with SA group,MCU m RNA increased in LT(70.06 %,P < 0.01)and POST(91.53 %,P < 0.001).The m RNA expression of MICU1(IBA(31.2 %,P < 0.05),ET(49.35 %,P < 0.01),MICU2(IBA(55.97 %,P < 0.01),ET(66.05 %,P < 0.001)),Letm1(IBA(47.64 %,P < 0.05),ET unchanged)decreased in IBA and ET periods.The m RNA expression of MICU1,MICU2 and Letm1 increased in different degrees during LT and POST compared with SA and PRE.(3)Plantar protein level: compared with SA,there was no change in IBA and MCU(IBA,et(39.58 %,P < 0.01)),MUIC1(IBA(64.98 %,P < 0.05),ET(81.09 %,P < 0.05)),Letm1(IBA(65.70 %,P < 0.001),ET(70.13 %,P < 0.001))protein expression increased in different degrees.(4)m RNA level of plantar muscle: compared with SA and pre groups,the expression of MCU,MICU1 and MICU2 m RNA decreased in IBA and ET periods,and the expression of Letm1 m RNA did not change.Compared with SA group,m RNA expression of MCU,MICU2 and Letm1 LT group increased by 48.93 %(P < 0.05),107.8 %(P < 0.001)and 91.67 %(P < 0.05)respectively.In POST group,the m RNA expression of MCU(49.4 %,P < 0.05)and MICU2(73.35 %,P < 0.01)was significantly higher than that of MICU1 and Letm1.(5)Semitendinosus protein level: Compared with SA group,MCU protein expression did not change significantly in each period.MICU1 protein expression increased by 14.27 % in POST group(P < 0.05),letm1 protein expression increased in different degrees in ET(32.47 %,P < 0.05),LT(47.69 %,P < 0.01),POST(44.06 %,P < 0.01).Compared with IBA,MCU protein expression increased by 22.23 %(p < 0.05)in ET group and 23.52 %(P < 0.05)in LT group.(6)Semitendinosus m RNA level: compared with SA group,three groups of MCU(IBA(67.49 %,P < 0.001),ET(79.25 %,P < 0.001),LT(73.18 %,P < 0.001),MICU1(IBA(56.63 %,P < 0.01),ET(66.80 %,P < 0.001),LT(33.20 %,P < 0.01)),Letm1(IBA(76.92 %,P < 0.001),ET(81.62 %,P < 0.001).The m RNA expression of MCU,MICU1 and MICU2 in the hibernation group returned to the summer level,and Letm1 decreased by 41.03 % compared with the summer group(P < 0.001).Conclusions: 1.There was no mitochondrial calcium overload in plantar muscle during hibernation,and mitochondrial calcium overload in gastrocnemius muscle and semitendinosus muscle during hibernation,but returned to the level of summer group when sleeping,indicating that the three muscles had good mitochondrial calcium homeostasis.2.The gastrocnemius muscle of daurian ground squirrel may increase the concentration of mitochondrial calcium by decreasing the protein expression of Letm1.Mitochondria may play the role of calcium storage and relieve the calcium overload in cytoplasm during LT period.During hibernation,the metatarsal muscle may increase the expression of calcium uptake protein and calcium excretion protein at the same time,and maintain mitochondrial calcium homeostasis.Semitendinosus may increase mitochondrial calcium concentration by increasing the protein expression of calmodulin MCU during hibernation,suggesting that in daurian ground squirrel may alleviate cytoplasmic calcium overload by increasing mitochondrial calcium uptake during hibernation. |
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