The Molecular Regulatory Mechanism Of TR3 Translocation To Mitochondria In Stress Induced Cardiomyocytes Apoptosis

Objective:To clarify the influence of MAPK phosphorylation and Hsp70 on TR3 translocation to mitochondria and illuminate the molecular mechanism modulating TR3 translocation during stress-induced cardiomyocytes apoptosis.Methods:Intervening cardiomyocytes by using 10-5mol/L glucocorticoid. Detecting Caspase 3 enzyme activity and the rate of cardiomyocytes apoptosis by using flow cytometry(FCM) and Hochest 33342 staining,Studying cardiomyocytes survival by using MTT method. Exploring the levels of MAPK and TR3 protein expression and phosphorylation by using Western blotting and immunoprecipitation metheds.Investigating the influence of SP600125,PD98059 and SB203580 on TR3 protein phosphorylation and translocation to mitochondria in stressed cardiomyocytes by Western blotting and immunoprecipitation metheds.Modulating Hsp70 protein expression by using KNK437,chemical inhibitor of Hsp70 protein expression,studying the change of Hsp70 protein expression.Results:After 24 hours of cardiomyocytes intervered by 10-5mol/L GC,it was found that both Caspase-3 enzyme activity and cardiomyocytes apoptosis rate were greatly increased.DNA fragments in the fracture were also observed inside cardiomyocytes nuclei.And the survival rate of cardiomyocytes decreased significantly.MAPK signol pathway actived greatly after cardiomyocytes intervered by GC, including the enhanced phosphorylation of JNK protein at 10 to 90 minutes and ERK1/2 at 10 to 60 minutes.While distint change of p38 protein phosphorylation level was not observed.The level of TR3 protein phosphorylation was greatly higher at 40 to 60 minutes.After inhibiting JNK protein phosphorylation,we found that both the level of TR3 protein phosphorylation and the quantity of TR3 protein in cardiomyocytes mitochondria were significantly reduced.It was found that Caspase-3 enzyme activity and the apoptosis rate of stressed cardiomyocytes were decreased clearly. The level of TR3 protein phosphorylation was not reduced obviously after inhibiting ERK1/2 protein phosphorylation by using PD98059.So we can conclude that there existed an importent role of activated JNK to TR3 exporting from nuclei and translocation to mitochondria.The expression of Hsp70 protein was up-regulated in stressed cardiomyocytes,enhancing the interaction between Hsp70 and TR3.After inhibiting the expression of Hsp70 by using KNK437,we found that the level of TR3 protein in stressed cardiomyocytes mitochondria reduced distinctly,while the enchanced Caspase-3 enzyme activity and boosted apoptosis rate and decleased survival rate of stressed cardiomyocytes were not found.It can be concluded that Hsp70 relived the injury of cardiomyocytes induced by stress.Conclusion:During the process of stress-induced cardiomyocytes apoptosis,JNK was activated apparently,promoting TR3 protein phosphorylated and transferring to mitochondria,spurring the occurrence of cardiomyocytes apoptosis.Increased Hsp70 proteins assisted TR3 transferring to mitochondria by interacting with TR3 in stressed cardiomyocytes.