Protection And Mechanism Of Diazoxide Against Heart In Hypothermic Preservation Induced Injury Mediated By Hsp90

BackgroundHeart transplantation is a surgical transplant procedure performed on patients with end-stage heart failure or severe coronary artery disease. Successful organ preservation is the premise for clinical organ transplantation. However, hypothermic preservation of the heart has a limit of about 4-6 h, which is much lower than that for the liver, kidney, and pancreas.Prolonged donor heart preservation becomes more important in cardiovascular surgery. We have reported that diazoxide as a supplementation in Celsior preservation solution, can improve myocardial function and decrease apoptosis during long- term hypothermic preservation. However, the exact anti-apoptotic mechanism of diazoxide is still unknown. The heat shock proteins (Hsps) are highly conserved families of proteins, the cytoprotective function of Hsp90 is largely explained by their anti-apoptotic function, since Hsp90 has been shown to interact with different key apoptotic proteins.ObjectivesTo investigate whether heat shock protein 90 (Hsp90) plays an important role in the anti-apoptotic effect of diazoxide in hypothermic preservation rat hearts.Methods(1) In vitro hypothermic heart preservation model and cardiac function assay: Male SD rat hearts were quickly removed and washed in cold Krebs-Henseleit (KH) solution. Then hearts were mounted on the Langendorff perfusion apparatus, and perfused reversely at 76mmHg with KH solution at 37℃and gased with 95%O2-5%CO2. After balancing for 30 min, the left ventricular developed pressure was recorded as the basal value. After preservation for certain time periods, the hearts were reloaded onto the Langendorff perfusion apparatus and reperfused for another 60 min. LVDP was recorded during reperfusion.(2) Cell apoptosis assay:At the end of reperfusion, the cardiac apex of the left ventricle was used. Cell apoptosis was detected by TUNEL assay.(3) The expression of Bid:At the end of reperfusion, the cardiac apex of the left ventricle was used. The protein expression of Bid and tBid were assessed by Western Blot.(4) The expression of Hsp90:At the end of reperfusion, the cardiac apex of the left ventricle was used. The mRNA expression of Hsp90 was assessed by RT-PCR and the protein expression was detected by Westren Blot.Results(1) Compared with control group, LVDP recovery rate significantly decreased and cardiomyocyte apoptosis index increased after 3-9 h of hypothermic preservation in a time dependent manner. When compared with the 9 h preservation group, supplement Celsior solution with diazoxide significantly (30μM) enhanced the LVDP recovery rate and decreased the apoptosis index.(2) The cleavage of Bid increased after 9 h of hypothemic preservation, which was inhibited by supplying Celsior solution with diazoxide.(3) Compared with control group, hypothermic preservation of rat hearts for 3 h increased the expression of Hsp90 mRNA and protein. But, the expression of Hsp90 mRNA and protein was decreased when the hypothermic preservation time was prolonged to 6-9 h. After supplement with diazoxide, the expression of Hsp90 mRNA and protein was significantly increased in 9 h preserved rat hearts.(4) Hsp90 inhibitor 17-AAG inhibited the diazoxide-induced decrease in tBid. Meanwhile,17-AAG also partly abolished the diazoxide-induced improvement of cardiac function and decrease of apoptosis.Conclusion Hsp90 might mediate diazoxide-induced cardioprotection against apoptosis in hypothermic preservation heart by preventing the cleavage of Bid.