| Part Ⅰ Differential expression of calcium regulatory proteins in cardioprotection by sevoflurane postconditioning and delayed remote ischemic preconditioningObjective:Calcium regulatory proteins-L-type Ca2+ channel (LTCCs), ryanodine receptor2 (RyR2), and Na+/Ca2+ exchanger isoform 1 (NCX1) have been recognized as important protective mechanisms during myocardial ischemia-reperfusion injury (I/RI). Both sevoflurane postconditioning (SevoPoC) and delayed remote ischemic preconditioning (DRIPC) have been shown to protect the heart against I/RI. In this study, we explored the effect of SevoPoC and DRIPC on the expression of three calcium regulatory proteins in an isolated rat heart model.Methods:After 30-minute balanced perfusion (the hemodynamics were stable), isolated hearts were subjected to 30-minute ischemia followed by 60-minute reperfusion,40 isolated hearts were randomly assigned to 4 groups (n=10):(1) Time control (TC) group: continuous perfusion for 120 minutes; (2) I/RI group:after 30-minute balanced perfusion, ischemia for 30 minutes followed by 60-minute reperfusion; (3) SevoPoC group:after 30-minute balanced perfusion and ischemia for 30 minutes, perfused with 3%(v/v) sevoflurane-bubbled KHBs oxygenated with 95% oxygen for 10 minutes at the onset of reperfusion and then with normal KHBs for the remaining 50 minutes; (4) DRIPC group: 4 cycles of 5-minute occlusion and 5-minute reflow at unilateral hind limb once at the day before heart isolation. Ischemia was confirmed using modified pulse oxymetry for the rats. The effect of SevoPoC (3% v/v) and DRIPC were observed. Heart rate (HR), left ventricular developed pressure (LVDP), and maximum LVDP increase (+dp/dt) and decrease (-dp/dt) rate were continuously collected. Myocardial infarct size, cardiac troponin I (cTnI) level and heart function were measured. The protein and messenger RNA (mRNA) levels of LTCCs, RyR2, and NCX1 were determined.Results:Both SevoPoC and DRIPC significantly improved the recovery of LVEDP, RPP, and ±dp/dt. Compared with the SevoPoC group, the significant decreases in RPP were observed in the DRIPC group (P< 0.05). Meantime, they reduced cTnI release after I/RI. The decrease in infarct size was more significant in the SevoPoC group than that in the DRIPC group(16.50% ± 4.54% vs 22.34% ± 4.02%, P<0.001). SevoPoC, but not DRIPC significantly inhibited the activity of NCX1 (0.59 ± 0.09 in the I/RI group vs 0.32 ± 0.16 in the SevoPoC group, P<0.01; vs 0.57 ± 0.14 in the DRIPC group, P>0.05). No statistical differences were observed in the expression of LTCCs and RyR2 between SevoPoC and DRIPC.Conclusions:SevoPoC and DRIPC could confer similar cardioprotection, manifested as the improved recovery of myocardial function, reduced cTnI release,and the decrease in infarct size. However, SevoPoC may be more effective in the deactivation of NCX1 than DRIPC.Part II Differential expression of calcium regulatory proteins in cardioprotection and effects on reperfusion arrhythmias by sevoflurane preconditioning and sevoflurane postconditioningObjective:1. To investigate the effect of SevoPC and SevoPoC on the expression of LTCCs, RyR2 and NCX1 in an isolated rat heart model.2.To investigate the effect of SevoPC and SevoPoC on the reperfusion arrhythmias in an isolated rat heart mode and explore the related mechanism.Methods:After 30-minute balanced perfusion, isolated hearts were subjected to 20-minute ischemia followed by 60-minute reperfusion,50 isolated hearts were randomly assigned to 5 groups (n=10):(1) Time control (TC) group:continuous perfusion for 110 minutes; (2) I/RI group:After 30-minute balanced perfusion, ischemia for 20 minutes followed by 60-minute reperfusion; (3) SevoPC group:After 15-minute balanced perfusion, perfused with 3% (v/v) sevoflurane-bubbled KHBs oxygenated with 95% oxygen for 15 minutes before 20 minutes of global ischemia and 60 minutes of reperfusion; (4) SevoPoC group: After 30-minute balanced perfusion and 20 minutes of global ischemia, perfused with 3% (v/v) sevoflurane-bubbled KHBs oxygenated with 95% oxygen for 15 minutes at the onset of reperfusion and then with normal KHBs for the remaining 45 minutes; (5) PCPo group:After 15-minute balanced perfusion, perfused with 3% (v/v) sevoflurane-bubbled KHBs oxygenated with 95% oxygen for 15 minutes before 20 minutes of global ischemia and then perfused with 3%(v/v) sevoflurane-bubbled KHBs oxygenated with 95% oxygen for 15 minutes at the onset of reperfusion and then with normal KHBs for the remaining 45 minutes. The effect of SevoPC and SevoPoC were observed. Heart rate (HR), left ventricular developed pressure (LVDP), and maximum LVDP increase (+dp/dt) and decrease (-dp/dt) rate were continuously collected. Myocardial infarct size, cTnl level heart function and reperfusion arrhythmias were measured. The protein and messenger RNA (mRNA) levels of LTCCs, RyR2, and NCX1 were determined.Part II Differential expression of calcium regulatory proteins in cardioprotection and effects on reperfusion arrhythmias by sevoflurane preconditioning and sevoflurane postconditioningObjective:1. To investigate the effect of SevoPC and SevoPoC on the expression of LTCCs, RyR2 and NCX1 in an isolated rat heart model.2.To investigate the effect of SevoPC and SevoPoC on the reperfusion arrhythmias in an isolated rat heart mode and explore the related mechanism.Methods:After 30-minute balanced perfusion, isolated hearts were subjected to 20-minute ischemia followed by 60-minute reperfusion,50 isolated hearts were randomly assigned to 5 groups (n=10):(1) Time control (TC) group:continuous perfusion for 110 minutes; (2) I/RI group:After 30-minute balanced perfusion, ischemia for 20 minutes followed by 60-minute reperfusion; (3) SevoPC group:After 15-minute balanced perfusion, perfused with 3% (v/v) sevoflurane-bubbled KHBs oxygenated with 95% oxygen for 15 minutes before 20 minutes of global ischemia and 60 minutes of reperfusion; (4) SevoPoC group: After 30-minute balanced perfusion and 20 minutes of global ischemia, perfused with 3% (v/v) sevoflurane-bubbled KHBs oxygenated with 95% oxygen for 15 minutes at the onset of reperfusion and then with normal KHBs for the remaining 45 minutes; (5) PCPo group:After 15-minute balanced perfusion, perfused with 3% (v/v) sevoflurane-bubbled KHBs oxygenated with 95% oxygen for 15 minutes before 20 minutes of global ischemia and then perfused with 3%(v/v) sevoflurane-bubbled KHBs oxygenated with 95% oxygen for 15 minutes at the onset of reperfusion and then with normal KHBs for the remaining 45 minutes. The effect of SevoPC and SevoPoC were observed. Heart rate (HR), left ventricular developed pressure (LVDP), and maximum LVDP increase (+dp/dt) and decrease (-dp/dt) rate were continuously collected. Myocardial infarct size, cTnl level heart function and reperfusion arrhythmias were measured. The protein and messenger RNA (mRNA) levels of LTCCs, RyR2, and NCX1 were determined.Results:1.Compared with I/RI goup, SevoPC group, SevoPoC group and PCPo group could improve the isolated rat heart function after reperfusion 30 min and 60 min, manifested by improved LVDP、+dp/dt、-dp/dt and HR(P<0.05),reduced LVEDP(P<0.05). SevoPC group and SevoPoC group decrease IS caused by ischemia reperfusion injury(17.92% ±2.12%, SevoPC; 17.22% ±2.30%, SevoPoC; vs 23.83% ±2.07%, I/RI, P<0.05) and a combination of SevoPC and SevoPoC confered more pronounced effect on the decrease of IS than SevoPC or SevoPoC alone (P<0.001). Compared with TC group, cTnI release at the end of reperfusion was elevated significantlly (P<0.001), SevoPC group and SevoPoC group could reduced cTnI release after I/RI (P<0.001 vs I/RI), there were no differences between SevoPC group and SevoPoC group on the cTnI release at the end of reperfusion (P>0.05).2. SevoPC and SevoPoC could improve the occurring of reperfusion arrhythmias manifested by the decrease of the total number of VPB incidences of VF and reperfusion arrhythmias score, and the reduction of lasting of VT and VF.3. In SevoPC group, the expression of RyR2 at the levels of transcription was siginificantly decreased; In SevoPoC group, the expression of LTCCs, RyR2 and NCX1 at the levels of transcription and the expression of RyR2 and NCX1 at the levels of translation were siginificantly decreased.Conclusion:1.SevoPC and SevoPoC could improve the isolated rat heart function after ischemia reperfusion, there were no differences on the effect of regulation of heart function between SevoPC and SevoPoC,both of which could decrease cTnl and IS caused by ischemia reperfusion injury, a combinationof SevoPC and SevoPoC confered more pronounced effect on the decrease of IS than SevoPC or SevoPoC alone.2. SevoPC and SevoPoC could improve the occurring of reperfusion arrhythmias.3.The effects of SevoPC and SevoPoC on the reperfusion arrhythmias may be due to the inhibition of LTCCs, RyR2 and NCX1.4. SevoPC could inhibit the expression of RyR2 at the levels of transcription. SevoPoC could inhibit the expression of LTCCs, RyR2 and NCXl at the levels of transcription and the expression of RyR2 and NCX1 at the levels of translation. |
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