| Objective:Postconditioning is verified to have cardioprotection, and is associated with state of mitochondrial permeability transition pore. But classic postconditioning is unfeasible. Many studies suggested that transient acidosis during reperfusion also had cardioprotection and the mechanism of protection may be also related to the mitochondrial permeability transition pore (mPTP). The aim of this study was to analyze whether cyclosporine A postconditioning (a potent inhibitor of mitochondrial permeability transition), acidic buffer postconditioning or plus cyclosporine A can show the same cardioprotective effect as classic postconditioning, and their cardioprotection with the state of mPTP in isolated rat hearts after cardioplegic arrest.Methods:Langendorff-perfused Sprague-Dawley rat hearts were perfused for 20 min with Krebs-Henseleit buffer followed by 30 min of normothermic crystalloid cardioplegia and 60 min of reperfusion. Control hearts (n=12) were reperfused with Krebs-Henseleit buffer. Postconditioning hearts (Ipo; n=12) were with 6cycles of 10s re-occlusion separated by 10s reperfusion before reperfusion. Cyclosporine A postconditioning hearts (CsA; n=12) were reperfused with Krebs-Henseleit buffer containing 0.8 u mol/L cyclosporine A at first 5 min of reperfusion. Acidic buffer postconditioning hearts (Low pH; n=12) were reperfused with acidic Krebs-Henseleit buffer (pH 6.8) for the first 3 min of reperfusion. Acidic buffer plus cyclosporine A hearts (Low pH+CsA; n=12) were perfused with Krebs-Henseleit acidic buffer (pH 6.8) containing cyclosporine A (0.2±μmol/L) for the first 3 min of reperfusion. Cardiac function was digitalized and analyzed by powerlab softwares. Mitochondrial membrane permeability was evaluated by measurement of cytochrome C release into the cytosol by western blot. The opening of the mPTP was determined at 25℃under de-energized conditions by following the decrease in light scattering monitored. Assessment of myocardial apoptosis was using TUNEL assay.Results:Compared with Con group, myocardial performance (left ventricular develop pressure, maximum positive and negative first derivatives of left ventricular pressure and heart rate) at baseline,5,15,30, and 60 min after reperfusion was better preserved in Ipo, CsA, Low pH and Low pH+CsA groups. Coronary artery flow was decreased at reperfusion in all groups compared with the baseline. But there were no significant differences among groups. Ipo, CsA, Low pH and Low pH+CsA groups had less cytochrome C into the cytosol, decreased sensitivity of mPTP-opening to Ca2+ and reduced rate of apoptotic myocytes compared with Con group after reperfusion.Conclusion:These findings suggested that cyclosporine A, acidic buffer or plus cyclosporine A postconditioning could show the same cardioprotective effect as classic postconditioning. The mechanism is related to inhibit apoptosis-related mitochondrial permeabilization after cardioplegic arrest. This protection was correlation with improved myocardial performance. But addition of cyclosporine A postconditioning did not show much better performance than acidic buffer postconditioning alone. Objective:'Conditioning'the heart to render it more resistant to an episode of acute myocardial ischaemia—reperfusion (I/R) injury is an endogenous cardioprotective strategy which can be readily applied to the clinical setting of cardiac surgery, to reduce myocardial injury and preserve left ventricular systolic function. MicroRNAs (miRNAs) are endogenous 20-23 nucleotide (nt) small noncoding RNAs that negatively regulate gene expression in various eukaryotic organisms. The miRNAs activity is involved in the control of a wide range of biological functions and processes, such as development, differentiation, metabolism, growth, proliferation and apoptosis. Some studies revealed that cardiac miRNA-1, miRNA-21 and miRNA-24 were significantly increased by late IPC. The IPC-miRNAs treatment caused an increase in eNOS and HSP70 protein versus control recently. In the present study, we observed the expression change of miRNA-1 and miRNA-21 and their target protein HSP70 and programmed cell death (PDCD4) after three'condition'protocols in an isolated rat heart I/R model.Methods:The hearts were randomly assigned into one of the four groups. Control hearts (CON, n=12) were perfused for a 20 min of stabilization followed by 30 min of global ischemia at 37℃and 60 min of reperfusion. The IPC hearts (IPC, n=12) were achieved via two cycles of 2 min occlusion/2 min reperfusion cycles of the aortic root during stabilization period. RIPC hearts (RIPC, n=12) were achieved via there cycles of 5 min occlusion and 5 min of reperfusion of the bilateral femoral artery during induction of anesthesia period. The lower-limb ischemia has been verified by means of pulse oxymetry modified for application in the rats. IPO hearts (IPO, n=12) were via 12 cycles of 5s occlusion and 5s reperfusion of the aortic root at the beginning of reperfusion. Cardiac function was digitalized and analyzed by powerlab softwares. The expression of miRNA-1 and miRNA-21 was detected by real-time RT-PCR. The expression of HSP70, PDCD4, Bcl-2 and Bax was detected by western blot. Assessment of cardiac infarct size and myocardial apoptosis was using TTC assay and TUNEL assay respectively.Results:LVDP,±dP/dtmax, heart rate and coronary artery flow were recorded. At baseline, there were no differences among groups. During reperfusion, LVDP and±dp/dtmax in IPO group was significantly better preserved compared with CON group at 10min of reperfusion.+dp/dtmax in RIPC group was significantly better preserved compared with CON group at 10min of reperfusion. Heart rate in IPC group was better preserved than CON group at 10min of reperfusion. Coronary artery flow was decreased at reperfusion in all groups compared with the baseline. But there were no significant differences in IPC, RIPC and IPO groups compared with CON group. The IPC protocol caused a significant increase of miRNA-1(233±45%) and miRNA-21(356±33%) as compared with CON group. But the expression of miRNA-1 was significantly decreased in RIPC (61±16%) and IPO (61±13%) groups compared with CON group. The expression of miRNA-21 was decreased in RIPC (68±23%) and IPO (68±11%) groups, there was no statistical significance compared with CON group. Western blotting analysis conformed a significant upregulation in HSP70 (121±12%) and PDCD4 (130±11%) in CON group compared with the other "conditioning" groups [HSP70:IPC (74±5%), RIPC (81±6%), IPO (67±11%). PDCD4:IPC (74±11%), RIPC (81±16%), IPO (83±12%)] after 60min of reperfusion. Bcl-2 protein was decreased (54±12%) and Bax protein was increased significantly (63±8%) in CON group compared with the other "conditioning" groups. [Bcl-2:IPC (73±8%), RIPC (79±16%), IPO (87±18%). Bax:IPC (27±6%), RIPC (21±3%), IPO (27±4%)]. Myocardial infarct size was determined by TTC assay. The results showed that myocardial infarct size was significantly decreased in IPC (31.7±4.1%); RIPC (29.6±6.19%) and IPO (32.8±4.71%) compared with CON (51.77±4.3%). Positive staining was observed in Con, IPC, RIPC and IPC groups (32.8±3.96% vs 15.2±4.21% vs 17.2±1.92% vs 15.6±4.04%), respectively. The apoptotic rate was significantly higher in CON group than the other "conditioning" groups.Conclusion:In the current study, we used the Langendorff-perfusion apparatus to establish an I/R model in vitro and observed the miRNA-1 and miRNA-21 expressions in different "conditioning" protocols.The expression of the miRNA-1 and miRNA-21 are different in IPC, RIPC and IPO protocols, which revealed that the different miRNAs are regulated in different "conditioning" protocols, and the cardioprotective proteins may be regulated by multiple miRNAs. |
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