The Role Of TR3 In Mitochondrial Mechanism Of Stress Induced Cardiomyocyte Apoptosis And Its Molecular Basis

Many researches showed that stress induced the cardiovascular disease, which happened basis on the cardiomyocytes apoptosis. Mitochondria play a key role in cardiomyocytes apoptosis induced by stress. However, up to now, the regulatory molecular mechanism by which stress induces mitochondrial apoptosis pathway opening in cardiomyocyte remains unknown. Recent investigation indicates that treated with apoptosis inducers, TR3 translocate from nucleus to cytoplasm. At the same time, apoptotic cascade is initiated and Cyt c released. In our previous study, we found that stress induced the translocation of TR3 from nucleus to mitochondria resulting in an increase in TR3 content of mitochondria in cardiomyocytes. Thus, TR3 may have a crucial influence on mitochondrial mechanism of cardiomyocyte apoptosis under stress. The role of TR3 and its molecular basis under stress, however, is not clear. To explore the key molecular mechanism in regulating the stress-induced mitochondria in cardiomyocyts, the present study is therefore designed to examine the change of the expression and subcellular localization of TR3 in cardiomyocytes under stress and its role in inducing cell apoptosis.1. The role of TR3 targets mitochondria in cardiomyocyte apoptosis induced by stress.The animal model and cell model of stress-induced cardiomyocyte injury were established. The Western blotting method and confocal microscopy method were used to investigate the subcellular location of TR3 in cardiomyocytes under stress. The results show that Stress induced the increase of TR3 content in the mitochondria of cardiomyocytes and the translocation of TR3 from the nucleus to the mitochondria.Treatment of stressed cardiomyocytes with leptomycin B and PKA Inhibitor (To block nuclear export of TR3), resulted in nuclear retention of TR3 and abrogated its ability to induce cardiomyocyte apoptosis. Indicate that TR3 targets mitochondria is an important molecular mechanism in cardiomyocyte apoptosis.2. Identification of mitochondrial function proteins in stressed cardiomyocytes associated with TR3.Clues to the regulatory mechanism by which NGFI-B targets mitochondria and induces cardiomyocyte apoptosis was revealed by Blue Native page electrophoresis. Three proteins bands complex were found to associated with TR3. Eight proteins were successfully identified in three bands by MALDI-TOF MS analysis. They include Vimentin-1/2, lactate dehydrogenase B, acetyl-CoA synthetase, citrate synthase, Butyryl-CoA dehydrogenase, glucose-regulated protein, malate dehydrogenase and heat shock protein 65. 6 proteins associated with NGFI-B participate directly in mitochondrial energy metabolism. Studies of mitochondrial respiratory efficiency and ATP synthase activity strongly support the findings.Co-immunonoprecipitation of complexes captured using antibody against NGFI-B shown that GRP75 is bound to TR3 in mitochondria of stressed cardiomyocytes. But they were not finding to bind together in mitochondria of unstressed cardiomyocytes. In vitro, the two-way interaction of GRP75 and TR3 was not finding yet. Those indicate that the interaction between GRP75 and TR3 is an important molecular effect in mitochondria of stressed cardiomyocytes.3. The role of GRP75 in cardiomyocytes apoptosis induced by TR3 under stress.Eukaryotic expression and siRNA vector of TR3 and GRP75 were established. The gene expression regulation of them was identified in H9c2 cells. High expression of TR3 in H9c2 induced decline of mitochondrial membrane potential, activation of caspase cascade reaction and increase of apoptosis rate. High expression of GRP75 could inhibit the activition of apoptosis pathway induced by TR3. Suggested that promotion apoptosis of TR3 was implemented through GRP75.To investigate the effect of TR3 on physiological function of GRP75, both of TR3 and GRP75 expression were regulated. The results showed that over expression of TR3 in H9c2 cells induced the disorder of MPTP and energy metabolism. GRP75 protected H9c2 cells against the disorder of MPTP and energy metabolism induced by over expression of TR3 under stress. The results indicated that TR3 induced apoptosis by mediates the disorder of MPTP and energy metabolism through GRP75.In conclusion, TR3 targets mitochondria is an important molecular mechanism in cardiomyocyte apoptosis under stress. The interaction between GRP75 and TR3 is an important molecular effect in mitochondria of stressed cardiomyocytes. The interaction of GRP75 and TR3 significantly regulate the cardiomyocytes apoptosis under stress. TR3 mediates apoptosis under stress by regulating the disorder of MPTP and energy metabolism through GRP75. Thus, GRP75 in mitochondria is the key target point, by which TR3 mediates cardiomyocytes apoptosis.