Mechanism Of CRT-CaM-PMCA1 Signaling Pathway In Stress-induced Myoblast Apoptosis

Objective:Apoptosis of myoblast cells plays an important role in the adaptive reconstruction of muscle tissue under orthopedic force.Intracellular calcium concentration has been proved to be closely related to cell state.Calcium reticulin CRT and membrane calcium ion pump PMCA1 play a core regulatory role in intracellular calcium ion concentration.However,the mechanism of how CRT and PMCA1 synergistically regulate intracellular calcium ion concentration to induce apoptosis is still not fully studied.Therefore,this study aims to explore the signaling pathway of myoblast apoptosis under stress conditions,and explore the deep molecular mechanism of myoblast apoptosis under stress conditions.The purpose of this study is to provide a new basis for revealing the cellular structure and molecular mechanism of muscle tissue adaptive remodeling in the process of functional orthopedics.Methods:In this study,we mainly discussed the relationship between stress stimulation,CRT,PMCA1,calcium homeostasis and myoblast apoptosis.The stress stimulation model of myoblast culture in vitro was constructed,and the rat L6 myoblast cell line was stimulated by periodic tensile stress of 10%,15%and 20%tensile deformation rate.The myoblasts were cultured for 24 hours,and the 0 h group was used as the control.The intracellular free Ca²⁺concentration of myoblasts was detected by calcium fluorescence probe Fluo-4,AM,and the apoptosis of myoblasts was detected by TUNEL staining and Annexin V-FITC/PI.q RT-PCR,Western blot and other methods were used to detect the changes of calcium-related proteins CRT,PMCA1,apoptosis,ERS and other related factors.The activity of membrane protein PMCA1 was detected by ATPase detection kit.After selecting the optimal stretching rate,si RNA and recombinant plasmid targeting CRT were designed and synthesized for transfection,and the correlation among CRT,PMCA1 and intracellular calcium homeostasis was analyzed,and then the relationship between CRT,PMCA 1 and apoptosis was proved.Results:1.L6 myoblast cells were subjected to cyclic tensile stress stimulation with a deformation amplitude of 10-20%and a frequency of 10 cycles/min for 24h,the intracellular Ca²⁺ concentration increased significantly,accompanied with obvious apoptosis.The intracellular Ca²⁺concentration and apoptosis rate were positively correlated with tensile deformation amplitude,and reached the highest value at 20%.2.The expression levels of ERS-related factors GRP78,CHOP,apoptosis-related factors Caspase 3,PARP and calreticulin（CRT）were significantly increased with the increase of deformation amplitude,and reached the highest value at 20%.The expression level and protein activity of PMCA1 decreased with the increase of deformation amplitude.3.In CRT silenced L6 myoblasts under stress stimulation,ERS was down-regulated, apoptosis rate was reduced,the expression and activity of PMCA1 were increased,and intracellular calcium content was significantly reduced.4.In CRT overexpressed L6 myoblasts under stress stimulation,ERS was significantly activated,the apoptosis rate was increased,the expression and activity of PMCA1 were decreased,and the content of intracellular calcium was increased.5.CaM inhibitors can negatively regulate the expression and activity of PMCA1 and activate ERS,and simultaneously increase the apoptosis rate and intracellular calcium content,while ERS inhibitors can positively regulate the expression and activity of PMCA1 and decrease the apoptosis rate and calcium content.Conclusions:1.The amplitude of tensile deformation is 10-20%,the frequency is 10 cycles per minute, and the stress stimulation lasting 24 hours promotes myoblast apoptosis.2.Endoplasmic reticulum stress（ERS）occurs in cells under stress loading,and ERS participates in stress-mediated myoblast apoptosis.3.Periodic tensile stress affects calcium homeostasis of myoblasts by regulating PMCA1.4.Periodic tensile stress induces apoptosis of myoblasts mediated by endoplasmic reticulum stress through CRT-CaM-PMCA1 signaling pathway.