The Role Of FAF1 In HSP70 Inhibiting Fas Pathway In Stress-induced Cardiomyocyte Apoptosis And Its Molecular Mechanism

Stress is the body’s psychological and physiological response and reaction to the imbalances between the actual or perceived needs and the ability to cope with unfavorable factors in the living environment. Long-term high stress load can lead to cardiac dysfunction, hypertension, atherosclerosis and other serious cardiovascular diseases. Myocardial apoptosis is an important cellular basis of cardiovascular disease, the Fas signaling pathway is an important pathway of cardiomyocyte apoptosis. FAF1(Fas-associated factor-1) is an important component of death-inducing signaling complex(DISC), and it is also an important regulatory element in FAS apoptotic pathway. Studies have shown that there is an overlap between the binding domain of FAS and HSP70 binding to FAF1, which suggests the possibility of competitive inhibition in combination with FAF1. Therefore, it is necessary to study the role of FAF1 in HSP70’s inhibitory of stress-induced myocardial apoptosis. We need to exam whether the interaction between HSP70 and FAF1 could affect that of FAS and FAF1 or it could also affect the formation of DISC. To study the molecular basis of the HSP70 inhibition of Fas signal pathway would be helpful to clarify the biological basis of stress-induced myocardial apoptosis. And the study of HSP70 inhibitory mechanism of myocardial apoptosis induced by stress would also be helpful to explore and discover new prevention medical approaches on stress-induced cardiovascular injury. 1. The expression of FAF1 in stress-induced myocardial apoptosis model. According to the previous studies in our laboratory, we chose Wistar rats weighed 180-200 g to construct the stress model. We used purpose-made metal cabin to restrain Wistar rats for 6h per day to build the restraint stress model. The restraint lasted 1w, 2w and 3w for the whole experiments. After the modeling is complete, the rat plasma glucocorticoid and norepinephrine levels in 2w and 3w stress-group was significantly higher than the control group. TUNEL staining of rat myocardium indicated that the apoptosis ratio in the stress group increased significantly compared with the control group, which proved that the restraint stress-induced myocardial injury model was successfully established. According to the NE level in restraint-stressed rat plasma, we 10 treated H9c2 cell with different concentrations of NE to induce cardiomyocyte apoptosis and to establish the cell model of stress. In NE treated H9c2 cells we used flow cytometry to detect the apoptosis ratio of cells. The results showed that, NE could induce apoptosis in H9c2 cells at a dose effect. Stress-induced cardiomyocyte apoptosis model was constructed successfully. And we chose 50 μM as the optimal concentration of NE to induced apoptosis in H9c2 cell line. In NE-induced cardiomyocyte model and restraint-stress model, we detected the expression of FAF1 using western blot assay. FAF1 expression gradually increased with the severity of stress, and the apoptosis ratio was significantly increased in stress group compared with control, which indicated the correlation between myocardial apoptosis and FAF1 level. 2. Biological function of FAF1 in stress-induced cardiomyocyte apoptosis. In the NE-induced myocardial apoptosis model we detected the expression of FAS using the western blot assay and detected the caspase-8 activity using the caspase-8 activity detection kit. The cardiomyocyte apoptosis ratio was measured by flow cytometry. The results showed that stress can significantly increase FAS expression in myocardial cells, and significantly increased caspase-8 activity in cardiomyocytes under stress conditions, which prompted the Fas pathway was activated. High expression vector of FAF1 was constructed and transfected into H9c2 cells and the expression of FAF1 was significantly increased. FAF1 interfering RNA sequence was designed and synthesized to specificity knockdown FAF1. After the high-expression vector or si RNA sequence was transfected into H9c2 cells, we measured the FAF1 level and apoptosis ratio. FAS expression and caspase-8 activity were also detected to study the activity of Fas signal pathway. The results showed that high expression of FAF1 would significantly increase caspase-8 activity and apoptosis ratio than the control group in myocardial cells; After suppression of FAF1 expression using si RNA-FAF1, caspase-8 activity and apoptosis ratio was significantly decreased. This indicates FAF1 level positively correlated with caspase-8 activity and apoptosis ratio of myocardial cells. FAF1 plays a pro-apoptotic role in apoptosis of myocardial cells. 3. The role of HSP70 in stress-induced myocardial apoptosis process. In the NE-induced myocardial apoptosis model, expression of HSP70 was up-regulated or down-regulated by transfection of HSP70 high-expression vector or heat shock protein inhibitor KNK437. Detection of FAS level was carried out with western blot assay. And we also measured the caspase-8 activity using the caspase-8 activity detection kit. Data showed that high expression of HSP70 could lower FAS level and reduce the activity of caspase-8 in NE-induced myocardial apoptosis model. HSP70 inhibitor can reduce the expression of HSP70 effectively. The FAS expression and caspase-8 activity increased after KNK437 treated. HSP70 could significantly inhibit the activation of Fas signal pathway, thereby reduce the apoptosis by stress. HSP70 has an important protective effect in stressed cardiomyocyte. 4. The role of FAF1 in HSP70 inhibiting Fas pathway in stress-induced cardiomyocyte apoptosis and its molecular mechanism HSP70 and FAF1 plays an important role in myocardial apoptosis process. Coimmunoprecipitation results indicated that there was interaction between HSP70 and FAF1. FAF1 interacted with FAS, too. We constructed the deletion mutants p EGFPN1/r Hsp70△N-(121–642) and full-length plasmid of HSP70 and transfected them into H9c2. The co-immunoprecipitation results showed that FAF1 did not interact with the deletion mutants. This indicated that HSP70 interacted with FAF1 by its N-ter minal region of 1-120 a mino acid. High expression of FAF1 raised the apoptosis ratio of cardiomyocyte, while high expression of HSP70 can alleviate FAF1 induced apoptosis ratio increase. Research results showed that there was an overlap in the binding sites of HSP70, FAS and FAF1. There is a competition mechanism between the two molecules in combination with FAF1. HSP70 occupied the binding sites of FAS and FAF1, thereby reducing the level of FAS interacted with FAF1, resulting in the reduction of deathinducing signaling complex(DISC) formation. This reduction affected the activation of Fas apoptotic pathway and reduced the apoptosis ratio of cardiomyocytes. FAF1 and HSP70 level has an important impact on FAS-FAF1 interaction and DISC formation in cardiomyocyte apoptosis process. In this study we validated the protective effect of HSP70 on stressed myocardial cells by constructing stress-induced apoptosis model and found that HSP70 and FAF1 played important roles in myocardial apoptosis induced by stress. We explored the molecular basis of myocardial Fas apoptosis pathway and biological effects. The study revealed that the interactions between HSP70 and FAF1 played an important regulatory role in activation of apoptosis Fas pathway. HSP70 weakened the interaction between FAS and FAF1 by competitively binding to FAF1, which inhibited the formation of DISC and regulated the activation of Fas pathway. FAF1 played an important regulatory role in the cardiomyocyte apoptosis. This revealed the mechanism of HSP70’s myocardial protection effects. The results of this study has important practical value to elucidate the biological basis of myocardial stress, to develop new medical approaches for prevention and treatment of stress-induced cardiovascular damage.