Delayed Cardioprotective Effects By Sevoflurane Preconditioning And Its Mitochondrial Mechanisms In Rats

Objective:1. To determine whether sevoflurane preconditioning could induce a late phase of cardioprotection on rats.2. To investigate the roles of mitochondrial ATP sensitive potassium channel (mito-KATP) and mitochondrial permeability transition pore (MPTP) in the delayed cardioprotective effects by sevoflurane preconditioning.3. To detect the changes of myocardial mitochondrial proteome24h after sevoflurane preconditioning on the ischemia-reperfused rat hearts.4. To test whether voltage dependent anion channel1(VDAC1) and nicotinamide adenine dinucleotide dehydrogenase (NADH-DH) are regulated in the delayed preconditioning by sevoflurane.Methods:1. The late phase of cardioprotection induced by sevoflurane. Adult male Sprague-Dawley rats (250-300g) were randomly assigned into four groups:①Sham operation group (SHAM), rats were opened chest without coronary occlusion;②Ischemia-reperfusion group (IR), the myocardial ischemia-reperfusion model was induced by ligation of the left anterior descending (LAD) coronary artery for30min and followed by120min of reperfusion.③sevoflurane preconditioning24h group (SEVO-24H), rats were exposed to2.5%sevoflurane mixed with oxygen (for60min)24h before LAD occlusion;④sevoflurane preconditioning48h group (SEVO-48H), rats were exposed to2.5%sevoflurane mixed with oxygen(for60min)48h before LAD ligation. Hemodynamics, including heart rate (HR), mean arterial blood pressure (MAP), and rate pressure product (RPP) were recorded through the operation. At the end of reperfusion, the arterial blood sample was taken to detect cardiac troponin Ⅰ (cTnl) levels. Myocardial infarct size was determined by Evans Blue and2,3,5-triphenyltetrazolium chloride (TTC) staining and the morphological inspection of myocardial injury was performed by the electronic microscopy. For the mitochondrial injury assessment, we examined mitochondrial energy exhausting, oxidative stress and apoptosis with2-3items, respectively. The respiratory control rate (RCR) of isolated mitochondria and the myocardial ATP levels were used to assess the ability to produce ATP in the mitochondria; myocardial malondialdehyde (MDA) and superoxide dismutase (SOD) activity were measured for the oxidative stress to the mitochondria; cytoplastic cytochrome C (Cyto C) release, Bcl-2/Bax expression and MPTP opening were contributed to evaluating apoptosis induced by mitochondria.2. The role of mitochondrial channels in delayed cardioprotection by sevoflurane preconditioning. Experimental rats were randomly divided into eleven groups:sham operation group (SHAM), ischemia-reperfusion group (IR), sevoflurane preconditioning group (SEVO), the selective mito-KATP blocker5-hydoxydecanoate (Trigger)+sevoflurane group [5-HD(T)+SEVO],5-HD (Trigger) group [5-HD(T)], sevoflurane+5-HD (mediator) group [SEVO+5-HD(M)],5-HD (mediator) group [5-HD(M)], MPTP opener atractyloside (trigger)+sevoflurane group [ATR(T)+SEVO], atractyloside (trigger) group [ATR(T)],sevoflurane+atractyloside (mediator) group [SEVO+ATR(M)], atractyloside (mediator) group [ATR(M)]. Rats were subjected to30min of LAD occlusion followed by120min of reperfusion except for the SHAM group. Rats in the SEVO group were set in a chamber breathing2.5%sevoflurane mixed with oxygen (60min)24h before ischemia was induced.5-HD (5mg/kg) or ATR (5mg/kg) was intravenous injected10min prior to sevoflurane (or oxygen) inhalation in the trigger groups including5-HD(T)+SEVO group,5-HD(T) group, ATR(T)+SEVO group and ATR(T) group; in contrast,5-HD or ATR was administered15min before myocardial ischemia induction in the mediator groups including SEVO+5-HD(M) group,5-HD(M) group, SEVO+ATR(M) group and ATR(M) group. Myocardial infarct size, serum cTnl levels and electronic microscopy of the myocardium were measured at the end of reperfusion in each group.3. The changes of myocardial mitochondrial proteome induced by sevoflurane preconditioning on the ischemia-reperfused rat hearts. Rats were randomly assigned to one of three groups. Rats in the sevoflurane preconditioning group (SEVO) were exposed to sevoflurane (2.5%for60min)24h before the ischemia was induced. Myocardial ischemia in the ischemia-reperfusion group (IR) and SEVO group was induced by LAD occlusion for30min, followed by120min of reperfusion. The SHAM group (SHAM) received a sham operation. After reperfusion, the hearts were harvested and the mitochondria fractions from exposed hearts were prepared by differential centrifugation with density gradient isolation for proteomic analysis. Two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF-MS) was used to identify differences in the protein expression from mitochondria of rat hearts.4. Voltage dependent anion channe (VDAC1) and nicotinamide adenine dinucleotide dehydrogenase (NADH-DH) were changed after sevoflurane preconditioning. Rats were randomly assigned to one of the seven groups:The sham operation group (SHAM); the ischemia reperfusion group (IR) underwent30min of LAD ligation followed by120min of reperfusion; rats in the sevoflurane group (SEVO) were subjected to a sevoflueane preconditioning (2.5%for60min)24h before LAD occlusion; rats in the the5-hydoxydecanoate+sevoflurane group (5-HD+SEVO) and2-mercaptopropionyl glycine+sevoflurane group (MPG+SEVO) were given5-HD (a mito-KATP blocker,5mg/kg) and 2-mercaptopropionyl glycine (a reactive oxygen species scavenge,20mg/kg)10min before sevoflurane exposure, respectively; rats in the5-hydoxydecanoate group(5-HD) and2-mercaptopropionyl glycine group (MPG) were given5-HD and2-MPG before oxygen inhalation instead of sevoflurane, respectively. The mRNA levels of VDAC1and NADH-DH were detected4h after sevoflurane (or oxygen) exposure. At the end of reperfusion, myocardial infarct size, serum cTnI levels and electronic microscopy were measured. Western blot was used to detect the relative expression of VDAC1, NADH-DH, Bcl-2and Bax.Results:1. All the ischemic groups had a decrease in HR, MAP and RPP after ischemia and reperfusion, but there were not distinguished differences between groups at every time point (P>0.05). Compared with the SHAM group, there was a significant increase in infarct size and cTnl levels in the IR group (P<0.05), paralleled with the myocardial injury severity under the electron microscope. Meanwhile, the mitochondrial injury in the ischemic group was significant, including the decreased RCR, deficiency in ATP production, an increase in MDA and a decrease in SOD, cytosolic Cyto C release, Bcl-2/Bax ratio inversion and MPTP opening (compared with SHAM group,P<0.05).Both the SEVO-24H group and SEVO-48H group could attenuate the mitochondrial dysfunction, they improved the energy production reflected by the increased RCR and ATP levels, alleviated oxidative stress (an decrease in MDA and an increase in SOD), reduced pro-apoptotic factors including decreased cytosolic Cyto C and MPTP opening, and increased Bcl-2/Bax (compared with IR group, P<0.05).However, we did not detect any differences between the SEVO-24H group and the SEVO-48H group both on cardiomyocyte injury and mitochondrial injury (P>0.05).2. Preconditioning by2.5%sevoflurane had a delayed cardioprotection against ischemia-reperfusion injury, including reducing infarct size and serum cTnI levels (compared with IR group, P<0.05). The mito-KATP blocker5-HD given before sevoflurane preconditioning or before LAD occlusion could abolished the effects of sevoflurane (compared with SEVO group, P<0.05). However, the MPTP opener ATR only given before LAD ligation but not before sevoflutane inhalation could abrogated the effects of SEVO group (P<0.05). The5-HD and ATR alone groups given in the trigger phase or in the mediator phase had no effects on infarct size and cTnI levels compared with IR group (P>0.05).3. Based on the Image Master software, we detected an average number615±8,634±13, and611±9protein spots from the SHAM, IR, and SEVO groups, respectively. The results showed that twenty-one protein spots had more than a1.5-fold difference in quantity, and these spots were isolated from the gels and subjected to mass spectrometry analysis. Of these,15spots were identified and categorized into the following four function groups:(ⅰ) substrate metabolism:E1and E2of pyruvate dehydrogenase complex, branched-chain alpha-keto acids dehydrogenase, and Isocitrate dehydrogenase;(ⅱ) respiratory chain:two subunits of nicotinamide adenine dinucleotide dehydrogenase, Cytochrome C oxidase subunit5A, two subunits of ATP synthase, and Electron transfer flavoprotein subunit;(ⅲ) transport and translocation: voltage-dependent anion-selective channel1and mitochondrial import inner membrane translocase9B; and (iv) protein synthesis factors: mitochondrial elongation factor Tu. These proteins were associated with mitochondrial substrate metabolism, respiration, and ATP/ADP transport. The modifications of the mitochondrial proteome suggest an enhanced capacity of mitochondria to maintain myocardial ATP levels after ischemia-reperfusion injury.4. Sevoflurane elicited a delayed protection against myocardial ischemia reperfusion injury, reducing infarct size and serum cTnl levels (P<0.05). There was a dramatic increase in mRNA levels of VDAC1and NADH-DH4h after sevoflurane inhalation, accompanied with an increase in protein levels of VDAC1and NADH-DH, and Bcl-2/Bax24h after sevoflurane preconditioning (compared with the IR group, P<0.05). Both the mito-KATP blocker5-HD and reactive oxygen species scavenger2-MPG intravenous injection in the trigger phase could abolished the effects of sevoflurane, increasing infarct size and serum cTnl levels, decreasing the mRNA and the proteins levels of VDAC1and NADH-DH, and the protein levels of Bcl-2/Bax (compared with the SEVO group, P<0.05). However, the5-HD or MPG alone group had no effects compared with the IR group (P>0.05).Conclusion:1. Sevoflurane preconditioning (2.5%for60min) can induce a delayed protection against myocardial ischemia-reperfusion injury, on both attenuating the cardiomyocyte necrosis and mitochondrial injury.2. Mito-KATP can serve as both the trigger and mediator in the delayed cardioprotection induced by sevoflurane; MPTP can only act as the mediator but not the trigger role.3. Using two-dimensional electrophoresis in combination with mass spectrometry, this comparative proteomics study revealed a remodeling of the mitochondrial proteome by sevoflurane in rat hearts exposed to IR. In addition, the proteins that we found to be altered after sevoflurane pretreatment were related to improvements in ATP generation and transport and included those involved in the mitochondrial Krebs cycle, oxidative phosphorylation, and adenine nucleotide exchange.4. The mitochondrial protein VDAC1and NADH-DH were up-regulated after sevoflurane preconditioning, mito-KATP and ROS may participate in the gene transcription in the trigger phase of delayed preconditioning.