Hydrogen Sulfide Postconditioning Activates Mitochondrial K(ATP) And PI3K/Akt To Protect Ischemic Rat Hearts

ObjectiveHydrogen sulfide(H₂S)is an endogenous gaseous mediator,produced by cystanthionine-γ-lysase(CSE)in the cardiovascular system during cysteine metabolism. Hydrogen sulfide given before ischemia can decrease myocardial ischemia and reperfusion injury.If hydrogen sulfide postconditioning can protect heart against ischemia reperfusion injury and the endogenous mechanism are not clear.The present study established isolated ischemia and reperfusion rat heart Langendorff model and hypoxia/reoxygenation cardiomyocytes model to study the effects of hydrogen sulfide postconditioning on ischemia and reperfusion myocardium.The aims are to investigated:(1)If hydrogen sulfide given at early reperfusion could decrease myocardial ischemia and reperfusion injury;(2)If the protective effects of hydrogen sulfide were related to mitochondrial ATP-sensitive K⁺(K(ATP))channels opening;(3) If the protective effects of hydrogen sulfide were related to PI3K-Atk-GSK3βpathway; (4)If mitochondrial K(ATP)opening and PI3K-Atk-GSK3βmodulated mitochondrial permeability transition pore(MPTP)in hydrogen sulfide postconditioning.Methods1.Hydrogen sulfide given at early reperfusion protects rat heart against myocardial ischemia and reperfusion injury.All isolated Spragre-Dawley(SD)rat hearts underwent an ischemia-reperfusion protocol of 30 min global ischemia followed by 90 min reperfusion.Postconditioning consisted of 4 cycles for 15s global ischemia or NaHS treatmeants followed by 15 s oxygenic K-H buffer at the onset of reperfusion.Rats were randomized into following six groups,each group included 8 rats:Ischemic/reperfusion(CON),Ischemic postconditioning(Pcon),0.1μmol/LNaHS postconditioning(NP0.1),1μmol/LNaHS postconditioning(NP1),10μmol/LNaHS postconditioning(NP10),100μmol/LNaHS postconditioning(NP100).The hemodynamic measurements,CK and LDH assays and infarct size determinations were performed.2.Role of mitochondrial K(ATP)channels in hydrogen sulfide postconditioning.The perfusion protocol was same as experiment one.Rats were randomized into following seven groups,each group included 8 rats:0.2%dimethyl sulfoxide(DMSO) (Vehicle),ischemic/reperfusion(CON),1μmol/LNaHS postconditioning(NP1), 100μmol/L 5-HD and NP1(5-HD+NP1),10μmol/L Glibenclamide and NP1(Gli+NP1), 100μmol/L 5-HD(5-HD),10μmol/L Glibenclamide(Gli).5-HD and Glibenclamide were perfused simultaneously for 10 min from the beginning of reperfusion.The hemodynamic measurements,CK and LDH assays and infarct size determinations were performed.3.The role of PI3K-Akt-GSK3βpathway in hydrogen sulfide postconditioning.The perfusion protocol was same as experiment one.Rats were randomized into following five groups,each group included 8 rats:0.2%dirnethyl sulfoxide(DMSO) (Vehicle),ischemic/reperfusion(CON),1μmol/LNaHS postconditioning(NP1), 15μmol/L LY294002 and NaHS postconditioning(LY+NP1),15μmol/L LY294002 (LY).The hemodynamic measurements,CK and LDH assays and infarct size determinations were performed.Immunohistochemistry and Western blot detected p-Akt and p-GSK3βexpression. 4.The role of mitochondrial permeability transition pore(MPTP)in hydrogen sulfide postconditioning.A 2h hypoxia and 2h reoxygenation cardiomyocytes model was used,newborn SD rat cardiomyocytes cultured for 72 h randomized into following six groups: hypoxia/reoxygenation(CON),1μmol/LNaHS postconditioning(NP1),100μmol/L 5-HD and NP1(5-HD+NP1),15μmol/L LY294002 and 1μmol/L NariS postconditioning(LY+NP1),100μmol/L 5-HD(5-HD),15μmol/L LY294002(LY). 5-HD and LY294002 treatments for 30 rain were performed from the onset of reoxygenation.Mitochondrial free Ca²⁺concentration,mitochondrial reactive oxygen species,mitochondrial permeable transition(MPT),mitochondrial inner membrane potential(MMP),mitochondrtial ultrastructure and cell viability was detected at the end of reoxygenation.5.Statistical analysis.All results were expressed as means±S.D..Statistical analysis was performed by software SPSS13.0 for windows.Differences between groups were analyzed by one-way ANOVA followed by Student-Newman-Keuls test.Statistical significance was set at P＜0.05.Results1.Hydrogen sulfide postconditioning protects isolated rat hearts against ischemia and reperfusion injuryLeft ventricular developed pressure was significantly improved at 60 min and 90 rain of reperfusion in Pcon(69±5,66±7mmHg),NP0.1(65±6,63±5mmHg),NP1 (67±6,65±7mmHg),NP10(61±5,60±6mmHg)groups compared with those in CON(48±8,43±6 mmHg)respectively(P＜0.05).Pcon reduced the infarct size (21.7±6.2%)vs CON(47.6±5.3%,P＜0.05).Postconditioning with NaHS produced a concentration-dependent limitation of infarct size(NP0.1,34.2±5.1%,NP1,23.4± 4.6%,NP10,25.1±7.2%,P＜0.05 vs CON respectively),100μmol/L NaHS(NP100) did not decrease the infarct size(49.4±8.1%).Ischemic postconditioning(Pcon) limited the CK and LDH release from coronary effluent versus CON(P＜0.05).(0.1,1, 10μmol/L)NariS postconditioning produced a concentration-dependent limitation of CK and LDH release except NP 100 versus CON respectively(P＜0.05).2.Hydrogen sulfide postconditioning opens mitochondrial K(ATP) channels and confers cardioprotectionBased on the dose-dependent results,we select 1μmol/L NariS as representative dose to further test if K(ATP)channel blockers can abrogate the cardiac protection by NariS.Both Glibenclamide and 5-HD completely abrogated the infarct-limiting effect of 1μmol/L NariS postconditioning respectively(Gli + NP1,46.5±8.6%;5-HD + NP1, 42.0±10.3%,P＜0.05 vs NP1 respectively,23.4±4.6%).10μmol/L Glibenclamide or 100μmol/L 5-HD given during early reperfusion blocked the beneficial effects on left ventricular developed pressure by exogenous 1μmol/L NaHS postconditioning during reperfusion respectively(P＜0.05).They also completely reversed of the CK and LDH release limiting effects of NP1.3.The cardioprotective effects of hydrogen sulfide postconditioning involves PI3K-Atk-GSK3βpathway activationCompared with CON(infarct size 47.6±5.3%),1μmol/L NariS postconditioning reduced infarct size(23.4±7.2%,P＜0.05)and limited the CK,LDH release respectively (P＜0.05).NP1 markedly improved the expression of p-Akt(Ser473)and p-GSK-3β(Ser9)compared with CON respectively(P＜0.05)detected with western blot. 15μmol/L LY294002 abrogated the infarct-limiting effect of 1μmol/L NaHS postconditioning,LY+NP1(44.5±6.7%)vs NP1(P＜0.05).LY294002 given during early reperfusion blocked the beneficial effect on left ventricular developed pressure by exogenous 1μmol/L NariS postconditioning during reperfusion(P＜0.05).LY294002 also completely blocked of the CK(72.3±13.3U/ml)and LDH(52.3±11.6 U/ml) release limiting effects of NP1(CK 28.9±9.1,LDH 25.5±3.8 U/ml)(P＜0.05). Meanwhile LY+NP1 also markedly decreased the expression of p-Akt(Ser473)and p-GSK-3β(Ser9)compared with NP1 respectively(P＜0.05).4.Mitochondrial K(ATP)and PI3K-Atk-GSK3βmodulated MPTP in hydrogen sulfide postconditioningNP1 improved the cell viability at the end of reoxygenation compared with CON O9.1±3.8%vs 31.9±4.1%,P＜0.05).Compared with CON,NP1 decreased mitochondrial free Ca²⁺concentration(424.3±14.7 vs 712.8±18.4 nmol/mg.pro, P＜0.05),decreased mitochondrial reactive oxygen species(8.1±0.6 vs 13.2±0.7 fluorescence units/sec.mg.pro,P＜0.05),increased Calcein fluorescence intensity (indicate MPT inhibition)(61.7±7.5%vs 39.4±5.2%,P＜0.05),improved MMP (73.6±4.5%vs 51.6±6.1%,P＜0.05),increased the Cytochrome C content in mitochondria(P＜0.05)and decreased the Cytochrome C content in cytoplasm(P＜0.05) at the end of reoxygenation,protected the mitochondrial integrity.100μmol/L 5-HD abolished all the beneficial effects of 1μmol/L NariS postconditioning(P＜0.05).While 15μmol/L LY294002(LY+NP1)abolished the beneficial effects of 1μmol/L Nails postconditioning on MPT,MMP,mitochondrial Cytochrome C content and cell viability(P＜0.05),but did not abolished the beneficial effects on mitochondrial free Ca²⁺and ROS.Conclusions(1)Hydrogen sulfide postconditioning can protect ischemia and reperfusion rat heat against reperfusion injury.Hydrogen sulfide postconditioning decreased mitochondrial free Ca²⁺concentration,decreased mitochondrial reactive oxygen species,inhibited MPT,improved MMP and increased the Cytochrome C content in mitochondria at the end of reoxygenation.Subsequently protected the integrity of mitochondria,improved the cell viability,reduced the infarct size and improved myocardial systolic function. (2)The cardioprotective mechanism of hydrogen sulfide postconditioning involves mitochondrial K(ATP)channels opening.(3)Hydrogen sulfide postconditioning opening mitochondrial K(ATP)channels inhibits mitochondrial free Ca²⁺and ROS,subsequently inhibites MPTP opening.(4)The cardioprotective mechanism of hydrogen sulfide postconditioning involves PI3K-Atk-GSK3βpathway activation,GSK3β(ser9)phosphorylation subsequently modulate MPTP opening.