Myocardial Protection Of Diazoxide-delayed Preconditioning Mediated By UQCRC1: A Study Of The Role And Mechanisms

Objective:In recent years, with the aging population increase and medical technology developmentin our country, the number of patients with elder age or heart disease for non-cardiac surgeryand patients for heart surgery is increasing. The risk of myocardial ischemia/reperfusion (I/R)injury in perioperative period rises accordingly. Myocardial I/R injury is an important causeof the complications and mortality for patients during perioperative period[1]. Therefore, it isstill difficult for clinicians to provide patients with effective myocardial protection inperioperative. Ischemia preconditioning (IPC) is an effective way to enhance the ischemic orhypoxic tolerance of cardiac myocytes through triggering body’s endogenous anti-injurymechanism. The protection of IPC is a biphasic process, the first phase of which is the fastphase of preconditioning, appearing at several minutes of preconditioning and lasting up to1-3h, and the second phase of which calls second window of protection (SWOP), appearing in24-96h after the first ischemia. Studies shows the delayed phase is the time point of strongestmyocardial protection in IPC[2][3][4]. Anesthetic drugs such as morphine, isoflurane andsevoflurane can provide preconditioning cardioprotection by SWOP[5][6][7]. However, themechanism of SWOP has not been completely elucidated, so the widely application of SWOPin clinic is limited. Proteomics research had shown that SWOP can enhance the expression of manymitochondrial respiratory chain proteins in myocardium[1], which suggests that mitochondrialrespiratory chain maybe play an important role in SWOP. Ubiquinol-cytochrome c reductasecore protein1(UQCRC1) is a subunit of mitochondrial respiratory chain complex Ⅲ withimportant physiological function. Our preliminary studies showed the expression of UQCRC1protein has positive correlation with SWOP in rat myocardium. Consequently, we speculatethat UQCRC1may play an important role in SWOP, it could be another new target proteinwith cardioprotection.Mitochondrial KATPchannel is the end-effectors of pharmacological preconditioningsuch as morphine, sufentanil, sevoflurane and isoflurane[2][3][4][5]. Mitochondrial KATPchannel opening is the common signaling pathway in either pharmacologicalpreconditioning or ischemia preconditioning to produce cardioprotection. Diazoxide (DZ) isa specific opener of mitochondrial KATPchannels. Therefore, it is a good choice to usediazoxide preconditioning to investigate the mechanisms of SWOP mediated by UQCRC1.In our research, the protection of UQCRC1was investigated on anti-ischemia/reperfusion injury in H9C2cardiac myocytes during diazoxide delayed preconditioning,UQCRC1gene expression was up-regulated by adenovirus vector transfection anddown-regulated by RNA interference technology in rat H9C2cardiac myocytes. Oxygenglucose deprivation (OGD) model was used to simulate ischemia/reperfusion injury. Thoseresults of this study would provide theoretical basis to develop myocardial protective drugstargeting at UQCRC1and expound the mechanism of SWOP cardioprotection.Method:1. Three kinds of siUQCRC1sequences were synthesized. The expression of UQCRC1gene and protein was detected by RT-PCR and Western Blot respectively in H9C2cardiacmyocytes after transfection with different siUQCRC1sequence or different concentration of siUQCRC1. The most effective siUQCRC1sequence and the best transfectionconcentration were selected.2. The adenovirus vector of UQCRC1was constructed. The expression of UQCRC1protein in H9C2cardiac myocytes was detected by Western Blot after UQCRC1overexpression or at the first phase and second phase of diazoxide preconditioning.5-hydroxydecanote (5-HD), a mitochondrial KATPchannel specific blocker was used at thesame time of diazoxide preconditioning to observe the relationship of UQCRC1expressionwith mitochondrial KATPchannels opening.3. To observe the influence of UQCRC1expression at SWOP on H9C2cardiac cellsagainst OGD injury, CCK-8method, Hoechst33342fluorescent dye andtetramethyrhodamine ester(TMRE) fluorescent dye were used respectively to detectsurvival rate, apoptotic rate and mitochondrial membrane potential in H9C2cardiacmyocytes.4. To observe the influences of UQCRC1at SWOP on the expression of protectiveproteins including Bcl-2and cyclooxygenase-2(Cox-2), pro-apoptotic protein activecaspase-3and the activation of Akt/GSK-3β signal after OGD injury, Western Blot was usedto detect the expression of those proteins including Bcl-2, Bax, Cox-2, active caspase-3,pho-Akt(Ser473), Akt, pho-GSK3β(S9) and GSK-3β protein after OGD injury.Results:1. Expression of UQCRC1mRNA in H9C2cardiac myocytes was most significantlyinhibited by transfection with siUQCRC1sequence with targeting sequence5′-CCGUUGCUGUAGCUAACAAdTdT-3′, and the concentration with the best effectivity is200nmol/L.2. Diazoxide preconditioning can significantly increase UQCRC1protein expression atSWOP induced by opening mitochondrial KATPchannels; when mitochondrial KATPchannelswere closed by5-HD during the process of diazoxide preconditioning, both the up-expressionof UQCRC1and cardioprotection induced by diazoxide delay preconditioning can becompletely eliminated.3. Up-expression of UQCRC1at SWOP can significantly increase the survival rate andmitochondrial membrane potential, but reduce apoptotic rate after OGD injury. However,down-regulated expression of UQCRC1at SWOP can completely eliminate those protective effects.4. SWOP can increase the expression of protective proteins including Bcl-2and Cox-2,raise ratio of Bcl-2/Bax, and achieve cardioprotection by up-regulating UQCRC1expressionin H9C2cardiac myocytes, but the up-expression of Bcl-2and Cox-2protein at SWOP incardiac myocytes can be completely eliminated by down-regulated UQCRC1expression.5. The up-expression of UQCRC1at SWOP in cardiac myocytes can inhibit theexpression of active caspase-3, and increase the expression of pho-GSK3β (S9) andpho-Akt(Ser473) protein significantly after OGD injury; however, down-regulated expressionof UQCRC1can completely eliminate the up-expression of phosphorylated GSK-3β,phosphorylated Akt and the down-expression of active caspase-3, and also lose thecardioprotection after OGD injury.Conclusion:1. siUQCRC1sequences targeting for CCGUUGCUGUAGCUAACAAdTdT caneffectively inhibit UQCRC1expression in H9C2cardiac myocytes at the transfectionconcentration of200nmol/L.2. Diazoxide delayed preconditioning can protecte cardiac myocytes fromischemia/reperfusion injury through up-regulating the expression of UQCRC1proteininduced by opening mitochondrial KATPchannels.3. UQCRC1can produce cardioprotection at SWOP by up-regulating expression ofBcl-2and Cox-2, inhibiting the activation of caspase-3apoptosis signaling pathway andactivating Akt/GSK-3βcell protective signaling pathway after ischemia/reperfusion injury.4. UQCRC1is another protective protein in SWOP and it may be a new target incardioprotection.