Sevoflurane Postconditioning Induces Cardioprotection In Rat Hearts In Vitro: The Involvement Of Signaling Pathway Of Mitochondrial Permeability Transition Pore And Mitochondrial Adenosine Triphosphate-sensitive Potassium Channels

Purpose To investigate cardioprotection induced by sevoflurane ischemic postconditioning(IPO) in rats with Langendorff apparatus in vitro,to survey if postconditioning prevents opening of the mitochondrial permeability transition pore (mPTP) through protein kinase B(PKB/Akt) - glycogen synthase kinase 3β(GSK3β) signaling pathway,to explore the role of the mitochondrial ATP-sensitive potassium channel(mK_ATP channel) in salvaging myocardium from infarction and inspect the relationship of mPTP and mK_ATP in sevoflurae induced IPO.Method 120 of Male Wistar rats,275-325 g body weight(BW),were randomly assigned into one of the 8 groups(n=15/group):Control(time matched perfusion group),ISCH(ischemia and reperfusion alone served as ischemic control group), ISCH + ATR(ischemia + atractyloside(ATR) group),ISCH + 5-HD(ischemia + 5-hydroxydecanoate(5-HD) group),Vehicle(dimethyl sulfoxide(DMSO) group), IPO(sevoflurane postconditioning group),IPO + ATR(sevoflurane postconditioning + ATR group),and IPO + 5-HD(sevoflurane postconditioning + 5-HD group).All rats were heparinized(500 IU/kg BW) via the femoral vein and anesthetized with 50 mg/kg BW of sodium pentobarbital administered by intraperitoneal injection.Each rat heart was rapidly excised through a median sternotomy and immersed into ice-cold heparinized saline solution during preparation,and then mounted on the stainless steel cannula of the modified Langendorff perfusion apparatus via the aorta.Aortic perfusion was initiated at a constant perfusion pressure of 80 mmHg with gassed (95%O₂,5%CO₂) Krebbs-Henseleit bicarbonate buffer(K-H).A water-filled latex balloon was inserted into the left ventricle cavity through a small incision in the left atrium and connected to a pressure transducer by rigid polyethylene tubing.The balloon volume was adjusted to produce 10 mmHg of diastolic pressure. Hemodynamic data from the latex balloon were analyzed using a real-time data acquisition and analysis system(PowerLab data acquisition system,Chart 5 Recorder Software) and displayed on a monitor during the experiment.The left ventricular developed pressure(LVDP),the maximum rate of increase or decrease of left ventricular pressure(±dp/dtmax),the heart rate(HR) and the coronary flow(CF) were recorded.In the blocker experiments,20μmol/L of the specific mPTP opener atractyloside(Sigma-Aldrich,St.Louis,MO) or 10μmol/L of the specific mKATP channels inhibiter 5-Hydroxydecanoate(5-HD)(Sigma-Aldrich,St.Louis,MO) dissolved in dimethyl sulfoxide(DMSO)(Sigma-Aldrich,St.Louis,MO) at a final concentration of less than 0.1%was used to inhibit ischemic postconditioning during the first 15 rain of reperfusion.Hearts subjected to ischemia and reperfusion alone served as ischemic control.Myocardial TUNEL staining was using In situ Cell Death Detection Kit(POD,Roche,Germany).The percentage of TUNEL positive nuclei to all nuclei counted was used as apoptotic index(AI).Cororiary sinus effluent was colected on baseline and reperfusion 60 min for biochemical anaylysis including lactate dehydrogenase(LDH),Creatine Kinase-MB(CK-MB) and cardiac troponin 1 (cTnI).Anesthetic postconditioning was induced by sevoflurane administered for 15 min at 1.0 minimum alveolar concentration(MAC;2.0 vol%) at the onset of reperfusion.The buffer was equilibrated with sevoflurane using a Vapor 2000(Dr(a|¨)ger Medical AG & Co.kGaA,Lubeck,Germany) with an air bubbler.Sevoflurane concentrations were also measured in the buffer before entering the aortic cannula using a Datex Volatile Anesthetic Gas Monitor(Copenhagen,Denmark) to guarantee 1.0 MAC sevoflurane delivered.All spontaneously beating hearts underwent a 20-min stabilization period as equilibration succeeded by 40 min of global ischemia except the control group whose hearts underwent no intervention for the whole duration of experiment.Subsequently,all hearts underwent 60 min of perfusion.Infarct size was verified by 2,3,5-triphenyltetrazolium chloride(TrC) staining after 60 min of reperfusion(n=5 for each experimental group).Shortly,hearts were frozen at -20℃for 2 h at the termination of the reperfusion and sliced into five 2-mm cross sections along the short axis.The slices were hatched for 15 min in buffered 1%TTC adjusted to pH 7.4 at 37℃and subsequently incubated overnight in 10%formaldehyde. Normal myocardium was then recognized as red stained by TIC,while necrotic one demonstrates pale gray.The 5 continuous sections were digitally photographed and analyzed by Image J 1.37 software.As a result of that the heart is global ischemia, infarct size was identified by dividing the total necrotic area of the left ventricle by the total left ventricular slice area(percent necrotic area).Separate experiments served to determine the density of protein(Western blot analysis) after 60 min of reperfusion(n = 5 in each group).The following antibodies were used for Western blot analysis: monoclonal antibody specific for B-cell lymphoma/leukemia 2(Bcl-2)(Cell Signaling Technology,Beverly,CA),monoclonal antibody specific for Cytochrome C(Cyt C) (Cell Signaling Technology,Beverly,CA),monoclonal antibody specific for protein kinase B(PKB/Akt)(Santa Cruz,Biotechnology,Inc.,Santa Cruz,CA);monoclonal antibodies specific for p-PKB/Akt(Ser-473),GSK3β,p-GSK3β(Ser-9);monoclonal anti-β-actin(Santa Cruz,Biotechnology,Inc.,Santa Cruz,CA).Mitochondria and cytosoi of left ventricular muscle were separated by using Mitochondria Isolalion Kit (Applygen.Beijing,China).Protein concentration was determined using Protein Concentration Measurement Kit(Applygen.Beijing,China) with Bicinchoninic acid (BCA) method.Result When compared with unprotected hearts,15 min of 1.0 MAC sevoflurane postconditioning at the onset of reperfusion after 40 min of global ischemia significantly reduced infarct size and enhanced recovery of heart function.The effects of cardioprotection by sevoflurane postconditioning were entirely eliminated by co-administration of the mPTP opener atractyloside or mK_ATP channel blocker 5-hydroxydecanoate during early reperfusion.Atractyloside or 5-hydroxydecanoate alone administered in the experiment did not further worsen functional recovery or enlarge infarct size during reperfusion period.The infarct size and the AI of Control and IPO groups were much lower during reperfusion period than those in other groups (p＜0.05).Compared with Control group,values of LDH,CK-MB and cTnI from coronary sinus effluent in ISCH and IPO groups are increased significantly(p＜0.05 ). Compared with ISCH group,values of LDH,CK-MB and cTnI in IPO group were decreased significantly(p＜0.05).Compared with Control group,the densities of Bcl-2 in experimental groups are increased significantly(P＜0.05) except in Vehicle and IPO groups.Sevoflurane-postconditioning inhibits mPTP opening in a manner of reducing ischemia/reperfusion-induced cytochrome C release from the mitochondria.Meanwhile,in the presence of 5-HD postischemic cytochrome C release from mitochondria were Sevoflurane-postconditioning modulates mKATP channel opening taking the same result of decreasing cytochrome C release from the mitochondria as well.The results showed the amounts of cytosolic cytochrome C significantly decreased(P＜0.05 versus ISCH group) in the sevoflurane postconditioning group while the amounts of mitochondrial cytochrome C markedly increased(P＜0.05 VS ISCH group).The phosphorylation state of protein kinase B (PKB)/Akt(60 kd) and glycogen synthase kinase 3β(GSK3β)(47 kd) were illustrated by a representative Western blot.Total Akt and GSK3βexpression were comparable in all samples.The densities of phosphorylated Akt and GSK3βwere normalized against these total Akt and GSK3βconcentrations respectively.1.0 MAC sevoflurane postconditioning during early reperfusion significantly(P＜0.05) increased the phosphorylation of Akt Ser 473 and GSK3β(Ser 9) compared with Control group and Ischemia group.While sevoflurane was administered during the early reperfusion phase,a considerable raise in phosphorylation of PKB/Akt and GSK3βwere viewed. Phosphorylation of PKB/Akt and GSK3βwas increased to a certain extent in even unprotected hearts when compared with time-matched perfused heart,indicating that a partial or delayed phosphorylation is insufficient to prevent myocardial damage. However,atractyloside,the direct mPTP opener,or 5-HD,the specific mKATP channel blocker,did not suppress phosphorylation of PKB/Akt and GSK3βadequately,they eliminated sevoflurane-induced cytoprotection.Conclusion These results propose that inhibition of the mPTP or blockage of the mK_ATP channel are directly engaged in the cardioprotection observed after sevoflurane postcondtioning.Sevoflurane-induced cardioprotection is mediated by activation of PKB/Akt signaling and successive inhibition of GSK3β.These results suggest that there is a connection between inhibition of mPTP and protection of mK_ATP channels in sevoflurane-induced cardioprotection.