Objective To evaluate the effects of ischemic postconditioning on mitochondrial cardiolipin synthesis during myocardial ischemia-reperfusion (Ⅰ/R) in rats in vitro.Methods Healthy male Sprague-Dawley rats,aged 16-20 weeks,weighing 250-350 g,were used in the study.The animals were anesthetized with intraperitoneal pentobarbital sodium 40 mg/kg and received intraperitoneal heparin 250 U/kg.Their hearts were excised and retrogradely perfused in a Langendorff apparatus.Sixty-four isolated rat hearts were randomly divided into 4 groups (n =16 each) using a random number table:control group (group C),Ⅰ/R group,ischemic postconditioning group (group IPO) and 5-hydroxydecanoate (5-HD) plus ischemic postconditioning group (group 5-HD + IPO).After 20 min of equilibration,the hearts were continuously perfused with K-H solution for 70 min in group C.In Ⅰ/R group,after 20 min of equilibration,the hearts were continuously perfused with 4 ℃ ST.Thomas cardioplegic solution 10 ml/kg,and exposed to 40 min of ischemia followed by reperfusion with oxygenated K-H solution at 37 ℃ for 30 min.In group IPO,after 20 min of equilibration,the hearts were subjected to 6 cycles of 10 s reperfusion followed by 10 s ischemia starting from 40 min of ischemia,and then were reperfused with oxygenated K-H solution at 37 ℃ for 28 min.In group 5-HD + IPO,after 20 min of equilibration,the hearts were perfused with K-H solution containing 100 μmol/L 5-HD (mitochondrial ATP-sensitive potassium channel blocker) for 5 min starting from 40 min of ischemia,and then the other procedures were similar to those previously described in group IPO.At 20 min of equilibration (T1) and 30 min of reperfusion (T2),HR,left ventricular developed pressure (LVDP),left ventricular end-diastolic pressure (LVEDP) and coronary flow (CF) were recorded.The coronary effluent 2 ml was collected for detection of lactic dehydrogenase (LDH) and creatine kinase (CK) activities.The mitochondria were extracted for determination of cardiolipin content.Results HR,LVDP,and CF were significantly lower,LVEDP was higher,and the LDH and CK activities in coronary effluent were higher at T2 than at T1 in the four groups.Compared with group C,HR,LVDP and CF were significantly decreased,LVEDP was increased,and the LDH and CK activities in coronary effluent were increased at T2 in the other three groups.Compared with Ⅰ/R group,HR,LVDP and CF were significantly increased,LVEDP was decreased,and the LDH and CK activities in coronary effluent were decreased at T2 in IPO group.Compared with IPO group,HR,LVDP and CF were significantly decreased,LVEDP was increased,and the LDH and CK activities in coronary effluent were increased at T2 in 5-HD+IPO group.Conclusion The mechanism by which ischemic postconditioning reduces myocardial Ⅰ/R injury is related to opening of mitochondrial ATP sensitive potassium channels and increasing mitochondrial cardiolipin synthesis in rats.

AIM: To investigate the effect of diazoxide (D) postconditioning on Cardiac function and mito-chondrial cardiolipin in isolated rat heart and to explore the protective effect of ATP sensitive potassium channel on diazo-xide postconditioning myocardium.METHODS: The myocardial ischemia/reperfusion injury model in isolated rat hearts was established by Langendorff apparatus.The isolated rat hearts were randomized into 4 groups ( n=8): control group ( control) , myocardial ischemia/reperfusion injury group ( I/R) , diazoxide postconditioning group ( I/R+D) , 5-hydroxy decanoic acid (5-HD) plus diazoxide postconditioning group (I/R+5-HD+D).The hearts in each group were started with 20 min perfusion for equilibration.The hearts in control group perfused for 70 min;The hearts in I/R group was global ischemia for 40 min after ischemia reperfusion at 4℃ST.Thomas cardioplegia, then reperfusion for 30 min;The hearts in I/R+D group were treated with diazoxide (50μmol/L) in K-H perfusion for 5 min after global ischemia for 40 min, then reperfusion for 25 min;The hearts in I/R+5-HD+D group were treated with 5-HD (100μmol/L) in K-H perfusion for 5 min before diazoxide postconditioning, then reperfusion for 20 min.The heart rate, coronary outflow volume, heart func-tion, myocardial enzymes and myocardial mitochondrial cardiolipin at the end of perfusion in each group were determined. RESULTS:Compared with control group and I/R+D group, the heart rate, the concentration of heart phospholipid and the coronary outflow volume were reduced, the heart function was significantly impaired the contents of myocardial enzymes were increased in I/R group.However, no significant difference between I/R group and I/R+5-HD+D group was ob-served.CONCLUSION:The diazoxide postconditioning protects the myocardium by increasing mitochondrial cardiolipin content, reducing the release of myocardial enzymes, improving heart function and reducing myocardial reperfusion injury. The myocardial protective effect of diazoxide is completely blocked by 5-hydroxy decanoic acid.

Objective To investigate the effects of diazoxide postconditioning on myocardial ischemiareperfusion (I/R) injury in isolated rat hearts. Methods Male SD rats weighing 250-300 g were anesthetized with intraperitoneal pentobarbital 40 mg/kg. Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95% O2-5% CO2 at 37 ℃. Sixty-four isolated rat hearts were randomized into 4 groups after 20 min of equilibration (n = 16 each): group control (group C), group I/R, group diazoxide postconditioning (group D) and group mito-KATP channel blocker 5-hydroxydecanoate (5-HD) + diazoxide postconditioning (group 5-HD + D). Group C received continuous perfusion for another 70 min. In group I/R, D and 5-HD + D, the hearts underwent 40 min of iscbemia followed by 30 min of reperfusion. 4 ℃ ST. Thomas cardioplegic solution was administered prior to ischemia in group I/R. Group D received 5 min of perfusion with K-H solution containing 50μ mol/L diazoxide at 5 min of reperfusion. Group 5-HD + D received 5 min of perfusion with K-H solution containing 50 μmol/L 5-HD before reperfusion with diazoxide. Eight hearts were taken at the end of equilibration and reperfusion and the indexes of cardiac function were recorded. Then the mitochondria were extracted for determination of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) release, and respiratory function indexes. Results Compared with group C, MMP was significantly decreased, ROS release was significantly increased, mitochondrial respiratory function and cardiac function declined at the end of reperfusion in the other three groups ( P ＜ 0.05 or 0.01 ). MMP was significantly increased, ROS release was significantly decreased, mitochondrial respiratory function and cardiac function were improved in group D compared with group I/R and 5-HD + D.Conclusion Diazoxide postconditioning can reduce myocardial I/R injury in rats via the opening of mito-KATPchannels and improving the mitochondrial function.

Objective To evaluate the myocardial protective effect of hyperpolarized heart arrest at different temperature during cardiopulmonary bypass CPB.Methods Eighteen dogs were randomly allocated to be infused with St.Thomas solution containing 50?mol/L pinacidil and 5mmol/L K + at 37℃ (group A) or 4℃ (group B), or the standard St.Thomas solution containing K + 16mmol/L at 4℃ (group C) respectively, through aortic root after aortic clamping . The global surgical ischemia lasted 60min with the declamping of 20min .The activities of serum myocardial enzymes, and levels of lipid peroxide and adenine nucleotide of myocardium were measured.Results All paramaters showed to occur serious ischemic and reperfusion damages during the whole procedures in group C, while there were the mild damages in two hyperpolarized groups, especially in group B.Conclusions Myocardial protective effect of hyperpolarized arrest induced with pinacidil, an ATP sensitive potassium channel opener, is superior to that of traditional hyperkalemic cardioplegia , which is much more prominent in hypothermic state.

AIM: To study the protective effect of hyperpolarized cardioplegic arrest on reperfused rat heart performance and to investigate the role of mitochondrial ATP-sensitive K+ channels(mitoKATP) opening in the protection of hyperpolarized cardioplegia against ischemia/reperfusion damage.METHODS: Forty Sprague-Dawley rats were randomized into five groups(n=8 in each group): control group(Con);depolarized arrest group(D);hyperpolarized arrest group(H);depolarized cardioplegia with 5-hydroxydecanoate(5-HD) group(5HD+D);hyperpolarized cardioplegia with 5-HD group(5HD+H).The rat hearts were quickly removed to Langendorff apparatus.The heart perfusion was performed for 20 min with 37 ℃ Krebs-Henseleit buffer balanced with gas mixture(O2∶CO2=95%∶5%) at 5.8 kPa perfusion pressure,then cardial arrest was induced by different cardioplegic solution.Hearts were subjected to ischemia at 37 ℃ for 40 min followed by 30 min reperfusion.(1) The hemodynamics was detected at recovery after 30 min reperfusion.(2) Before ischemia and at the end-reperfusion,tissue was harvested for mitochondrial isolation and ultrastructure was observed by transmission electron microscopy(TEM).(3) Production of reactive oxygen species(ROS) was also determined at different time points.RESULTS:(1) Compared with end-equilibration,30 min reperfusion caused significant differences in left ventricular developed pressure(LADP),left ventricular end-diastolic pressure(LVEDP),double product(DP),heart rate (HR),coronary flow(CF)(P

Objective To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on plasma concentrations of proinflammatory cytokines and myocardial energy metabolism induced by cardiopulmonary bypass (CPB) .Methods Twelve healthy mongrel dogs of both sexes weighing 13.5-17.5 kg were randomly divided into 2 groups (n = 6 in each group) : PDTC group and control group. The animals were anesthetized with intraperitoneal 2.5% pentobarbital sodium 25 mg?kg-1, intubated and mechanically ventilated. In PDTC group PDTC 30 mg?kg-1 was given i.v. after tracheal intubation while in control group normal saline was given instead of PDTC. Aorta was clamped for 60 min and then undamped for 60 min reperfusion during CPB. Blood samples were taken before (baseline), 30 and 60 min after aortic clamping and 30 and 60 min after aortic unclamping for determination of plasma concentrations of TNF-? and IL-1?. Myocardial tissue was obtained before and 60 min after aortic clamping and 60 min after aortic unclamping for determination of myocardial content of adenine nucleotide (ATP, ADP, AMP, TAN, EC) and expression of ICAM-1 protein.Results Plasma TNF-? concentration was increased after aortic cross-clamping as compared to the baseline value before clamping in both groups but the TNF-? concentration was significantly lower in PDTC group than in control group (P

Objective To evaluate the role of phosphoinositide 3 kinase/protein kinase B/glycogen synthase kinase 3β (PI3K/Akt/GSK-3β) signaling pathway in mitigation of ischemia-reperfusion (I/R) injury by diazoxide postconditioning in isolated rat hearts.Methods Pathogen-free Sprague-Dawley rats were used in the study.Thirty hearts were excised and passively perfused in a Langendorff apparatus with oxygenated K-H solution at 37 ℃.The hearts were randomly divided into 5 groups (n =6 each) using a random number table:control group (group C),I/R group,diazoxide postconditioning group (group DZ),PI3K inhibitor LY294002 group (group LY),and diazoxide postconditioning + LY294002 group (group DZ + LY).In group C,the hearts were continuously perfused with K-H solution for 70 min.In group I/R,the hearts were perfused with cardioplegic solution 4 ℃ ST-Thomas 10 ml/kg,the perfusion pump was then stopped to induce global ischemia,and 40 min later the hearts were perfused with K-H solution for 30 min.In DZ group,5 min of retrograde perfusion with diazoxide 50μmol/L was performed through the aorta starting from the onset of reperfusion.In LY group,5 min of retrograde perfusion with LY294002 15 μnol/L was performed through the aorta starting from the onset of reperfusion.In LY + DZ group,5 min of retrograde perfusion with LY294002 15 μmol/L was performed through the aorta starting from the onset of reperfusion,followed by 5 min of retrograde perfusion with diazoxide 50 μmol/L.At 20 min of stabilization (T1) and 30 min of reperfusion (T2),heart rate (HR),coronary flow (CF),left ventricular developed pressure (LVDP),left ventricular developed pressure (LVEDP) and ± dp/dtmax were measured.The expression of total Akt (t-Akt) and total GSK-3β (t-GSK-3β) and phosphorylation of Akt and GSK-3β in myocardial tissues were determined by Western blot.Results Compared with C group,HR,LVDP and ± dp/dtmax were significantly decreased,and LVEDP was increased at T2 in the other four groups,CF was decreased in I/R,LY and DZ + LY groups,and the phosphorylation of Akt and GSK-3β in myocardial tissues was increased in DZ group.Compared with I/R group,HR,CF,LVDP and ± dp/dtmax were significantly increased,and LVEDP was decreased at T2,and the phosphorylation of Akt and GSK-3β in myocardial tissues was increased in DZ group,and no significant changes were found in the phosphorylation of Akt and GSK-3 β in LY and DZ + LY groups.Compared with DZ group,HR,CF,LVDP and ± dp/dtmax were significantly decreased,LVEDP was increased,and the phosphorylation of Akt and GSK-3β in myocardial tissues was decreased in LY and DZ + LY groups.Conclusion PI3K/Akt/GSK-3β signaling pathway is involved in the mechanism by which diazoxide postconditioning mitigates I/R injury in isolated rat hearts.

Objective To investigate the relationship between the mechanism of ischemic postconditioning-induced activation of nuclear factor-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway during myocardial ischemia-reperfusion (I/R) and reactive oxygen species (ROS).Methods Healthy male Sprague-Dawley rats, aged 16-20 weeks, weighing 250-300 g, were heparinized and anesthetized with intraperitoneal 1％ pentobarbital sodium 40 mg/kg.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution.Thirty-two isolated rat hearts were randomly divided into 4 groups (n=8 each) using a random number table: control group (group C) , group I/R,ischemic postconditioning group (group IPO) , and N-(2-mercaptopropionyl)-glycine (a ROS scavenger) + IPO group (group M + IPO).After 20 min of equilibration, group C was continuously perfused with K-H solution for 100 min, and the isolated hearts received the drugs via the perfusion system in the other groups.Group I/R was perfused with cardioplegic solution 4 ℃ St.Thomas, and then was subjected to 40 min of ischemia at 32 ℃ followed by 60 min of reperfusion.In group IPO, ischemic postconditioning was induced by 6 cycles of 10 s reperfusion followed by 10 s limb ischemia starting from the onset of reperfusion, and the hearts were then perfused for 58 min.In group M + IPO, the hearts were perfused with K-H solution containing N-(2-mercaptopropionyl)-glycine 2 m mol/L for 3 min starting from the onset of reperfusion,underwent 2 min of ischemic postconditioning, and then was perfused for 55 min.Heart rate (HR), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP),and positive maximal pressure of left ventricular increase (+dp/dtmax) were recorded at the end of equilibration and of reperfusion.At 5 min of reperfusion and the end of reperfusion, myocardial specimens were obtained from the left ventricle for determination of ROS content by enzyme-linked immunosorbent assay.At the end of reperfusion, myocardial specimens were obtained from the left ventricle for examination of the ultrastructure of myocardial cells and for determination of Nrf2, heme oxygenase-1 (HO-1) , quinone oxidoreductase 1 (NQO1), and superoxide dismutase 1 (SOD1) mRNA and protein expression (by using Western blot and real-time polymerase chain reaction).The damage to myocardial mitochondria was assessed using Flameng scoring.Results Compared with group C, HR, +dp/dtmax and LVDP were significantly decreased, and LVEDP was increased at the end of reperfusion in I/R and M+IPO groups, HR and LVDP were decreased, LVEDP was increased, and no significant changes were found in +dp/dtmax at the end of reperfusion in IPO group, Flameng score was increased in I/R, IPO and M+IPO groups , the ROS content was increased at the end of reperfusion in I/R, IPO and M+IPO groups, and Nrf2, HO-1,NQO1 and SOD1 mRNA and protein expression was down-regulated at the end of reperfusion in I/R, IPO and M+IPO groups.Compared with group I/R, HR, +dp/dtmax and LVDP were significantly increased, and LVEDP and ROS content were decreased at the end of reperfusion, Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was up-regulated at the end of reperfusion in IPO and M+IPO groups, Flameng score was decreased in IPO group, there was no significant change in Flameng score in M+IPO group.Compared with group IPO, HR, +dp/dtmax and LVDP were significantly decreased, LVEDP and ROS content were increased at the end of reperfusion, Flameng score was increased, and Nrf2, HO-1, NQO1 and SOD1 mRNA and protein expression was down-regulated in M+IPO group.Conclusion Ischemic postconditioning can regulate ROS level and activate Nrf2-ARE signaling pathway, thus attenuating myocardial I/R injury in rats.

Objective To observe the activation mechanism of Nrf2-ARE pathway in protective effect of ischemia and pinacidil postconditioning on isolated rat hearts.Methods The hearts of adult male Sprague Dawley rats were established ischemia-reperfusion injury model,and devided into six groups(n =8,each group),i.e.Normal group(Group N),ischemiareperfusion group (Group Con,I/R),ischemic postconditioning group (Group IPO),pinacidil postconditioning group (Group P50),N-(2-mercaptopropionyl)-glycine(MPG,2mmol/L) + IPO group(Group M + IPO),MPG + P50 group(Group M + P50).Rat hearts were perfused with Krebs-Henseleit(K-H) buffer for 20 minutes for equilibration.Subsequently,Group N was perfused with K-H buffer for 100 minutes after equilibration,Group Con was perfused with 4℃ ST.Thomas solution to stop the heart beating after equilibration,then the hearts were underwent 40 minutes global ischemia under 32℃,and followed by the K-H solution for 60 minutes.Group IPO after global ischemia period,the hearts were subjected to six 10-seconds cycles of ischemia/reperfusion at the beginning of reperfusion,then were reperfused for 58 minutes.Group P50 after global ischemia,rat hearts were perfused with K-H buffer containing pinacidil(50.μmol/L) for 2 minutes before reperfusion.Group M + IPO after global ischemia,the hearts were subjected to perfuse with K-H buffer containing MPG(2 mmol/L) for 3 minutes,and then underwent six 10-seconds cycles of ischemia/reperfusion before reperfusion.Group M + P50 after global ischemia,the hearts were perfused with K-H buffer containing MPG(2 mmol/L) for 3 minutes,and then subjected to perfuse with K-H buffer containing pinacidil(50 μmol/L) for 2 minutes before reperfusion.Cardiac function indexes(such as HR,LVDP,LVEDP,and the Max dp/dt) at the end point of equilibration and repeffusion were observed and recorded.The ultrastructure of myocardial tissue was observed by electron microscopy and the mitochondrial Flameng score was calculated.RT-PCR and western-blot were applied to detect the gene transcription and protein expression of HO-1,NQO1,SOD1,and Nrf2 in left ventricular myocardial tissue after reperfusion.Results The HR,LVDP and + dp/dtmax at the end of reperfusion:the cardiac function indexes are lower among each group compared with group N,group 1PO and group P50 are better than group Con (P ＜ 0.05).Compared with group IPO,there is no significant difference in group group P50,but group M + IPO is obviously decreased(P ＜ 0.05).Compared with group P50,group M + P50 index is decreased significantly(P ＜ 0.05).The LVEDP at the end of reperfusion is lower than that among each group as compared with group Con,which is significantly increased in group Con (P ＜ 0.05).Compared with group IPO,there is no significant difference in group P50,but group M + IPO is significantly increased(P ＜ 0.05).Compared with group P50,the group M + P50 is obviously decreased(P ＜ 0.05).The ultrastructure of myocardial tissue in group N is mostly normal,group Con presence serious damage.The ultrastructure damage of myocardial tissue is improved in group IPO and group P50 as compared with that in group Con,while group M + IPO is more serious than group IPO,group M + P50 is more serious group P50.The mitochondrial Flameng score is higher among each group as compared with group N (P ＜ 0.05),the score is lower in group IPO and group P50 as compared with group Con and corresponding nonblocking group (M + IPO,M + P50,P ＜0.05).The mRNA and the protein expressions of HO-1,NQO1,SOD1 and Nrf2 among each group are lower as compared with group N(P ＜0.05).Compared with those in group Con,the mRNA and the protein expressions in group IPO and group P50 are obviously increased(P ＜ 0.05),group IPO and group P50 are higher than those in group adding active oxygen scavenger(MPG) (P ＜ 0.05).Conclusion Ischemic postconditioning and pinacidil postconditioning have protective effect of myocardial tissue from ischemia reperfusion injury,while improve the cardiac function index.The cardiac protective effect of Ischemic and Pinacidil postconditioning methods may be involved the ROS in early reperfusion,which activate the Nrf2-ARE pathway,and up-regulate the expression downstream antioxidant protein and phase Ⅱ detoxifying enzyme,ultimately improve the cardiac function index during the reperfusion period.

ObjectiveTo investigate the effect ofpinacidil hyperpolarizaed arrest on p38 mitogen-activited protein kinase (p38MAPK) during myocardial ischemia-reperfusion (I/R) in isolated rat hearts.MethodsFortyeight male SD rats weighting 250-300 g were randomly divided into 6 groups( n =8 each): natural arrest group (group A) ; St.Thomas group (group B) ; pinacidil hyperpolarization arrest group (group C) ;5-hydroxydecanoate (5-HD) group (group D);HMR-1098 group(group E) and 5-HD + HMR-1098 group(group F).Langendorff reperfusion model was established and K-H solution was retrogradely perfused for 15 min.In group A the hearts were arrested naturally afar perfusion was stopped; in group B St.Thomas solution was perfused; in group C pinacidil hyperpolarization solution was perfused; in the other three groups,K-H solution was perfused to isolated rat hearts for 10 min followed by 5 min 5-HD (group D) or HMR-1098(group E) or 5-HD and HMR-1098(group F) perfusion,then hyperpolarization arrest solution was given in each group.Each hearts suffered 60 min ischemia after arrest followed by 30 min K-H solution reperfusion.Coronary flow(CF),HR,left ventricular developed pressure( LVDP),left ventricular systolic pressure(LVSP) and the maximum rate of pressure rise (dp/dtmax) were measured at the end of 15 min K-H solution perfusion and at 20 min of reperfusion.Myocardial phosphatic and nonphosphatic p38MAPK expression was determined by Western blot at 30 min of reperfusion.ResultsCompared with group C,CF,HR,LVDP,LVSP and dp/dtmax were significantly decreased at 20 min of reperfusion and phosphatic p38MAPK expression was down-regulated,non-phosphatic p38MAPK expression was up-regulated at 30 min of reperfusion in groups A,B,D,E and F (P ＜ 0.05).Compared with group E,CF,HR,LVDP,LVSP and dp/ dtmax were significantly decreased at 20 min of reperfusion and phosphatic p38MAPK expression was down-regulated,non-phosphatic p38MAPK expression was up-regulated at 30 min of reperfusion in groups D and F ( P ＜0.05).ConclusionHyperpolarized arrest induced by pinacidil can improve cardiac function following myocardial I/R injury by up-regulating phosphatic p38MAPK expression,down-regulating non-phosphatic p38 MAPK expression and mitochondrial potassium channel is more important than membranous one during the regulation of phosphatic p38MAPK expression.