| With the improvement of people’s standard of living, cardiovasculardisease has now become a great threat to human life and health.Prolonged myocardial ischemia will make tissue damage even cell death.The key treatment of coronary heart disease is the blood reperfusion ofmyocardium. However, the damage of myocardial cell function andstructure becomes worse with the restoration of blood supply. Thepathophysiological state is called ischemia/reperfusion （I/R） injury, whichwas involved in the clinical practice such as thrombolysis, percutaneoustransluminal coronary angioplasty, coronary artery bypass grafting. Thereduction occurrence of ischemia/reperfusion injury has become a focusof current research.In1986, ischemic preconditioning （IPC）, first described by Murry, isan effective endogenous protective phenomenon whereby exposure to oneor more brief episodes of sub-lethal myocardial ischemia and reperfusionincreased the resistance of the myocardium to a subsequent sustainedischemic insult. However, due to the potential danger of IPC and thedifficulty of operation in clinical practice, the clinical application of IPCis limited. Subsequent studies have found that a variety of drugs cansimulate IPC to exert cardioprotective effect. These drugs include volatileanesthetics, opioid, statins and so on. Pharmacological preconditioning ismore suitable for clinical applications.Statins are the most important drugs for the treatment ofatherosclerosis. A previous study has shown that long-term use of statinsnot only improves the prognosis of patients, but also reduces the markersof myocardial injury after PCI surgery. A randomized controlled clinical trial showed that the pretreatment of a large dose of statins at24h beforePCI surgery significantly reduced the markers of myocardial injury afterPCI surgery. This real-time myocardial protection is likely to simulateischemic preconditioning. If this hypothesis is true, administration of asingle dose of statin should exert cardioprotective effect. In the presentstudy, we established three models: ischemia/reperfusion in vivo,ischemia/reperfusion in vitro and oxygen-glucose deprivation/reperfusion（OGD/R） in primary neonatal rat cardiac myocytes, which resemblesischemia/reperfusion in vivo. Our study aims to investigate whether thepretreatment of a single loading dose of atorvastatin （Ator） could exertmyocardial protective effect against I/R-induced injury in rats in vivo andto explore the mechanism of cardioprotection of Ator. This study willprovide the experimental evidence for the clinical application of Ator andnovel strategies for clinical cardioprotection against ischemia/reperfusioninjury.The present study included three parts as following:Part1The effect of single loading dose of atorvastatinpreconditioning on myocardial ischemia-reperfusioninjury in rats in vivoObjective: To investigate whether the pretreatment of a singleloading dose of atorvastatin could simulate ischemic preconditioning toexert myocardial protective effect against ischemia/reperfusion-inducedinjury in rats in vivo.Methods:The56healthy male Sprague-Dawley （SD） rats were randomlydivided into8groups, and in each group one rat was used for electronmicroscopy. For preparing suspension, Atorvastatin （Ator） was dissolvedin saline （20mg/ml）. The rats were orally treated with Ator （80mg/kg）,which was about1.0ml.（1） Sham group: SD rats were orally treated with the same amount ofsaline, then the heart was exposed by the thoracotomy, the left coronary artery （LCA）2to3mm away from the origin was threaded but withoutischemia/reperfusion （I/R）.（2） I/R group: SD rats were orally treated with the same amount ofsaline. Then the chest was opened, and the left coronary artery wasligated by tube method for30min and reopened for120min.（3） IPC （ischemic preconditioning） group: SD rats were orallytreated with the same amount of saline and randomized to an IPC group,which consisted of3cycles of5min of ischemia followed by5min ofreperfusion immediately prior to coronary occlusion.（4） Ator （atorvastatin） groups: SD rats were orally treated with Atorof80mg/kg at3h,6h,12h,24h or48h respectively before ischemia.Ultimately which group has the smallest infarct size from the abovegroups will be used as the Ator pretreatment group.The model of ischemia/reperfusion was produced by30min ofocclusion and120min of reperfusion of left coronary artery. Theischemic preconditioning model was produced by3cycles of5min ofischemia followed by5min of reperfusion immediately prior to coronaryocclusion. Myocardial ischemia and myocardial infracted area weremeasured using area calculation method by Evans blue and TTC doublestaining. Plasma lactate dehydrogenase （LDH） activity was measured atthe end of reperfusion. The myocardial ultrastructure and the damage ofmitochondria in different groups were observed by transmission electronmicroscope.Results:1Electrocardiogram （ECG） changeAfter the ligation of left descending artery of the rats in I/R group,the ST segment elevated and Merged with T-wave, looking liketombstone. However, the ECG had no significant change in sham groupbefore and after surgery.2The comparison of the infarct area among I/R group and Atorgroups Compared with I/R group, the infarct area of Ator6group, Ator12group, Ator24group, Ator48group were all significantly reduced（P<0.05）. Compared with Ator48group, the infarct area of Ator12group was significantly reduced （P<0.05）, but compared with Ator6group and Ator24group, there was a decreasing trend in Ator12group,but no statistically significant difference（P>0.05）. There was no statisticaldifference between Ator3group and I/R group （P>0.05）. Ultimately Ator12group was selected as the Ator pretreatment group.3The comparison of the area at risk among the four groups.Myocardial area at risk was expressed by area ratio （area at risk/leftventricle, AAR/LV）.Compared with I/R group, the myocardial area at risk of IPC groupand Ator group were all significantly reduced （P<0.05）. There was nostatistical difference between Ator group and IPC group （P>0.05）.4The comparison of the infarction area in the four groupsMyocardial infract area was expressed by area ratio （IA/AAR）.Compared with I/R group, the myocardial infract area （IA/AAR） ofIPC group and Ator group were all significantly reduced （P<0.05）. Therewas no statistical difference between Ator group and IPC group （P>0.05）.5The comparison of LDH in the four groupsCompared with I/R group, the LDH levels of IPC group and Atorgroup were all significantly reduced （P<0.05）. However, the LDH levelof Ator group compared with IPC group was significantly increased（P<0.05）.6The comparison of the myocardial ultrastructure in the four groupsSham group showed normal ultrastructures. Muscle fibrils andcristae were arranged in order. Mitochondria were complete and nuclear.There were a large number of glycogen granules. The myocardialultrastructures of I/R group were damaged. Cardiac muscle fibrils andcristae were arranged in disorder. Intracellular edema and damagedmitochondria were significant, the number of glycogen granules was reduced. Compared with I/R group, the injury of myocardial tissue in IPCgroup was significantly reduced. Muscle fibrils were mild swollen.Mitochondria were densely distributed. Cristae were partly disorganized.There were amount of glycogen granules. Similar to IPC group, the injuryof myocardial tissue in Ator group was significantly decreased. Musclefibrils and sarcolemma were morderately swollen. The muscle fibrils andmyofilament were partly irregular arrangement. Mitochondria were mildedema. Cristae were disorderly arranged, but the number of glycogengranules was not significantly reduced.Conclusions:1The study found that the pretreatment of a single loading dose ofAtor （80mg/kg） reduced myocardial I/R injury in rats in vivo. And, themost significant protective effect appeared at about12h after dosing.2The pretreatment of Ator is not better than IPC in the protection ofmyocardial I/R injury.Part2The effects of atorvastatin preconditioning on myocardialischemia/reperfusion injury in isolate rat heartsObjective: To assess whether the pretreatment of atorvastatin （Ator）could exert cardioprotective effect against ischemia/reperfusion （I/R）injury in rats in vitro and to explore the role of mitochondrialATP-sensitive potassium channels （mitoKATPchannels） and mitochondrialpermeability transition pore （mPTP） in cardioprotection of Atorpreconditioning.Methods:Sprague-Dawley rat hearts were Langendorff-perfused withKrebs-Henseleit （K-H） buffer and subjected to30min global ischemiafollowed by120min of reperfusion. In this study, the mitoKATPchannelsinhibitor5-hydroxydecanoate （5-HD） and the mPTP opener lonidamine（LND） were used to analyze the underlying mechanisms.Rats were randomly divided into7groups:（1） control group: the hearts was perfused without I/R. （2） I/R group: the hearts underwent30min global ischemia followedby120min of reperfusion.（3） Ator group: time-related effects of Ator on myocardial infarctarea, Ator （1μM） was added to the K-H buffer for15min,30min,60min or120min before global ischemia. The optimal duration of treatmentwas used in subsequent experiments. Concentration-related effects ofAtor on myocardial infarct area. Ator was diluted for use at finalconcentrations ranging from0.01μM to10μM for30min （according tothe optimal treatment time） before global ischemia. The optimalconcentration was used in subsequent experiments.（4） Ator+5-HD group: the hearts were first perfused for20min with100μM5-HD, and then treated with1μM Ator for30min before globalischemia.（5） Ator+LND group: the hearts were treated with1μM Ator for30min before global ischemia, and exposed to30μM LND for20min at thebeginning of reperfusion.（6）5-HD group: the hearts were first perfused for20min with100μM5-HD, and then perfused with K-H buffer for30min before globalischemia.（7） LND group: the hearts were exposed to30μM LND for20minat the beginning of reperfusion.Hemodynamics, myocardial infarct area, lactate dehydrogenase（LDH） in the coronary effluent and ATP concent in myocardium weremeasured. After reperfusion for20min, the nicotinamide adeninedinucleotide （NAD+） content in myocardium was measured to evaluatethe mPTP opening.Results:1The effects of Ator on I/R-induced infarct area in isolated rathearts.Compared with I/R group, the infarct area was significantly reducedin Ator15, Ator30and Ator60groups （P<0.05）. There was no statistical difference between Ator120group and I/R group （P>0.05）. Comparedwith Ator30group, the infarct area was significantly increased in Ator15and Ator60groups （P<0.05）. The exposure of hearts to Ator for30mincaused a significant cardioprotective effect.Compared with I/R group, Ator exerted its cardioprotective effects atall four concentrations （P<0.05）, but its protective effect was optimal atthe concentration of1μM.5-HD or LND significantly abolished theeffect of Ator on the infarct area （P<0.05）.5-HD or LND alone had noapparent effect on the infarct area （P>0.05）.2The effects of Ator on hemodynamicsBaseline hemodynamics among all experimental groups were similar（P>0.05）. Compared with control group, the values of LVSP, dp/dtmax,-dp/dtmaxand HR decreased, while LVEDP increased significantly inother six groups. Compared with I/R group, all hemodynamic variableswere better in Ator group （P<0.05）. However, Ator-induced preservationof cardiac function was completely abolished by either5-HD or LND.3The effects of Ator on the levels of LDH in the perfusateWhen compared to control group, LDH levels was significantlyincreased in I/R group （P<0.05）. The pretreatment of Ator significantlydecreased the LDH level. However, Ator-induced reduction of LDH levelwas completely abolished by either5-HD or LND, both of which did notinfluence post-ischemic recovery when given alone （P>0.05）.4The effects of Ator on the ATP content in myocardial tissueCompared to control group, the ATP content was significantlydecreased in I/R group （P<0.05）. The pretreatment of Ator significantlyincreased the ATP content （P<0.05）. However, Ator-induced increase ofATP level was completely abolished by either5-HD or LND.5The effects of Ator on the mPTP openingThe control hearts had the highest myocardial NAD+content amongall group （P<0.05）. Ator-preconditioned hearts retained higher contentsof NAD+than other hearts exposed to I/R （P<0.05）, which suggested that Ator prevented NAD+release upon reperfusion by inhibiting the mPTPopening.5-HD or LND had no effect on mPTP opening when given alone（P>0.05）. However, when coadministered with Ator, either5-HD or LNDabolished the inhibitory effect of Ator on the mPTP opening.6The comparison of the myocardial ultrastructure in the sevengroupsMyocardial mitochondria structure of I/R group was greatly alteredcomparing with the control group. The myocardial fibers were in disorder,myocomma structure was unclear, and part of myofilament was broken ordissolved. Mitochondria were swollen, even ruptured, cristae weredisorganized or had disappeared, and vacuolar degeneration and matrixloss could be seen in some mitochondria. The number of glycogengranules was reduced. These ultrastructural lesions were ameliorated inAtor group.5-HD or LND reduced the protection by the pretreatment ofAtor.Conclusion:1Ator has the direct cardioprotective effect on isolated rat hearts,without neurohumoral factors.2Ator protected isolated rat hearts from I/R-induced injury, and thiseffect was the most evident when hearts were pretreated with1μM Atorfor30min.3The effects of Ator may be mediated by activation of mitoKATPchannels and inhibition of mPTP opening. Furthermore, mitoKATPchannels may act upstream from mPTP.Part3The effects of atorvastatin preconditioning on oxygen-glucosedeprivation/reperfusion injury in primary neonatal ratcardiomyocytesObjective: To investigate whether the pretreatment of atorvastatin（Ator） could exert cardioprotective effect against oxygen-glucosedeprivation/reperfusion （OGD/R） injury in primary neonatal ratcardiomyocytes and to explore the role of mitochondria in cardioprotection of Ator preconditioning.Methods:Primary cardiomyocytes were isolated from neonatalSprague-Dawley rats. To establish an in vitro model of OGD/R, whichresembles ischemia/reperfusion in vivo, primary cardiomyocytes wereexposed to OGD for10h followed by a3h reperfusion.Neonatal rat cardiac myocytes in primary culture were randomlyassigned to one of the following7groups:（1） control group: cells were cultured under normal conditions.（2） OGD/R group: cells were subjected to10h OGD treatmentfollowed by a3h reperfusion only.（3） Ator group: time-related effects of Ator on myocardial cellviability, Ator （1μM） was added to the medium for1h,3h,6h,12h or24h before OGD treatment. The optimal duration of treatment was usedin subsequent experiments. Concentration-related effects of Ator onmyocardial cell viability. Ator was diluted for use at final concentrationsranging from0.01μM to10μM for3h （according to the optimaltreatment time） before OGD treatment. The optimal concentration wasused in subsequent experiments.（4） Ator+5-HD group: cells were treated with100μM5-HD for20min, washed with medium to remove5-HD and then exposed to1μMAtor for3h before OGD treatment.（5） Ator+LND group: cells were preconditioned with1μM Ator for3h before OGD treatment and then exposed to30μM LND for20minafter the onset of reperfusion.（6）5-HD group: cells treated with100μM5-HD for20min, washedwith medium to remove5-HD and allowed a5-HD-free period of3hbefore OGD treatment.（7） LND group: cells exposed to30μM LND for20min at thebeginning of reperfusion.After reperfusion for3h, the myocardial cell viability was evaluated using the MTT and lactate dehydrogenase （LDH） release assays. Afterreperfusion for20min, the opening of the mitochondrial permeabilitytransition pore （mPTP）, Ca2+concentration and mitochondrial membranepotential in cardiomyocytes were measured using confocal laser scanningmicroscopy （CLSM）. Reactive oxygen species （ROS） generation wasexamined using flow cytometry and confocal laser scanning microscopy.Results:1The effects of Ator on OGD/R-induced cell death in culturedventricular myocytesThere was significant difference among the OGD/R group and Atorgroups （P<0.05）. The exposure of cardiomyocytes to Ator for3h causeda significant cardioprotective effect. Thus, we chose this condition forsubsequent experiments.Compared with OGD/R group, Ator exerted its cardioprotectiveeffects at all four concentrations （P<0.05）, but its protective effect wasoptimal at the concentration of1μM.5-HD or LND significantlyabolished the effect of Ator on the cell viability （P<0.05）.5-HD or LNDalone had no apparent effect on the cell viability （P>0.05）.2The effects of Ator on LDH release from cultured ventricularmyocytesLDH release was significantly increased in OGD/R group （P<0.05vs. control group）. The treatment with1μM Ator for3h before OGDtreatment significantly reduced LDH levels in culture medium （P<0.05）,and this effect was completely reversed by concomitant treatment with5-HD or LND.3The effects of Ator on OGD/R-induced mPTP openingCompared to control group, OGD treatment for10h followed by a3h reperfusion significantly reduced the fluorescence intensity ofmitochondrial calcein （P<0.05）. Pretreatment with1μM Ator for3hresulted in a significant increase in fluorescence intensity （P<0.05vs.OGD/R group）, suggesting that Ator inhibits mPTP opening in cardiomyocytes. Cotreatment with5-HD or LND completely inhibitedthe Ator-induced increase in fluorescence intensity. Compared to OGD/Rgroup, treatment with5-HD or LND alone in the absence of Ator afterOGD/R treatment did not affect mPTP opening.4The effects of Ator on OGD/R-induced ROS levelsOGD treatment for10h followed by a3h reperfusion significantlyincreased cellular ROS levels compared to control group （P<0.05）.However, the pretreatment with Ator significantly reduced the ROSproduction （P<0.05vs. OGD/R group）. This Ator-induced reduction inROS levels was reversed by the co-administration of5-HD or LND.5The effects of Ator on OGD/R-induced calcium overloadThe fluorescence intensity of OGD/R group was significantlyincreased compared to control group （P<0.05）. The pretreatment of Ator（1μM） for3h before OGD treatment significantly attenuated thefluorescence intensity, suggesting the reduction of OGD/R-induced Ca2+overload. This effect was abolished by co-administration of5-HD orLND （P<0.05）.6The effects of Ator on OGD/R-induced mitochondrial membranepotential dissipationThe OGD/R group significantly decreased TMRE fluorescenceintensity compared to control group, suggesting the depolarization ofmitochondrial membrane potential. The decrease in TMRE fluorescenceintensity was significantly suppressed by pretreatment with Ator,indicative of the maintenance of mitochondrial membrane potential.However, Ator-induced increase in TMRE fluorescence was completelyabolished by exposure to5-HD or LND.Conclusions:1Ator has the direct cardioprotective effect on primary neonatal ratventricular myocytes.2The effects of Ator may be mediated by the activation of themitoKATPchannels, a reduction in both ROS generation and Ca2+overload, the inhibition of mPTP opening and the attenuation of mitochondrialmembrane potential collapse. Furthermore, mitoKATPchannels may actupstream of mPTP. |
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