Experimental Research On Protective Effect Of Lactate Shuttle Against Ischemia-reperfusion Injury In Cardiac Myocytes

The research results prove that lactic acid is one of the important energy. Lactate shuttle is existed widely in the organism. Myocardium has strong oxidative characteristic. Lactic acid can be utilized initiatively by heart. We have found that Lactic acid can be utilized preferentially by heart when the lactic acid passes through heart by means of blood circulation. It is mainly due to lactic acid can be utilized by about60%substrate in myocardium. Lactate shuttle need be verified further.Ischemia-reperfusion injury is found mainly in high oxygen demand organ such as heart and brain. So far the mechanism of ischemia-reperfusion injury have not been overall clarified. Energy metabolism participates in ischemia reperfusion injury of myocardial cell. The changes of blood flow lead to imbalance of oxygen demand and metabolism. The lack of energy leads to the accumulation of metabolites such as lactic acid. Then ischemia reperfusion injury occurs. The lack of energy because of anoxia results in injury of myocardial cell essentially. Thus the obstacle of ionic channel and oxygen radicals come into being. Thereby we want to know whether lactate shuttle exist and whether lactate shuttle protects against the cardiac ischemia-reperfusion injury. Lactate shuttle means that lactic acid is rolled out to help glycolysis during ischemia and that lactic acid is rolled in to help oxidation during reperfusion. The study is divided into three parts. Firstly, the variation of monocarboxylate transporter MCT1and MCT4in myocardial ischemia-reperfusion injury will be studied. Secondly, the variation of cell viability and apoptosis in myocardial ischemia-reperfusion injury will also be studied when these cells were transfected by siRNA. Thirdly, the variation of lactate shuttle will be explored when Trimetazidine acted on myocardial cells during ischemia-reperfusion injury. It contributes to previous clinical study about lactate shuttle applied in clinic.PARTⅠChanges in the expression of MCT during the process of ischemia-reperfusion in cardiac myocytesPurpose:This study was to investigate the variation of monocarboxylate transporter and lactic acid in myocardial ischemia-reperfusion injury.Methods:Embryonic rat’s cardiocytes H9C2were cultivated at95%N2+5%CO2incubator.After four hours5ml DMEM containing10%fetal bovine serum was added to cell culture fluid. Then these cells were cultivated in95%air+5%CO2incubator at37℃. After ischemia cells had been cultivated for four hours, apoptotic rate was detected with flow cytometer. It was to verify whether the ischemia-reperfusion model conformed to the requirement of this experiment. H9C2cells were Immunofluorescence histochemistry stained. MCT1antibodies, MCT4antibodies and antibodies labeled with FITC were added. Nucleus were coloured with DAPI dye. Anti-quench agent was added to every glass slide.37℃for30minutes, these were photographed with fluorescence microscopy. The total RNA of H9C2cells was extracted. Reverse transcription was done. Real-time quantitative PCR was also done with SYBRGreen Fluorescent Dyes. Cell protein was extracted and then Western blot was done. Protein level was determined with BCA. After SDS-PAGE electrophoresis protein was trarsmembraned, closed and hybridized, relative level of protein was determined with Chemiluminescence, development, fixation and gelatin image analysis. The concentration of lactic acid clear from supernatant liquid was measured. Above experiments were repeated at least three times.Results:Apoptotic rate was detected with flow cytometer. The results showed that the ischemia-reperfusion model conformed to the requirement of this experiment.1. Normally, the following is the results of MCT1and MCT4expressed in H9C2cells:1) The results of immunohistochemisty told us MCT1was expressed abundantly while MCT4sparing.2) The results of Western-blot told us MCT1was expressed abundantly while MCT4deficiently. These were in accordance with former study.2. The following is the results of MCT1mRNA and MCT4mRNA expressed in H9C2cells when ischemia occurs:The results of real-time quantitative RT-PCR showed that MCT4mRNA was expressed increasingly with the prolongation of ischemia time. After four hours increase of the expression was obvious (P<0.05) while MCT1mRNA was expressed decreasingly.3. The following is the results of MCT1mRNA and MCT4mRNA expressed in H9C2cells when reperfusion occurs:The results of real-time quantitative RT-PCR showed that MCT1mRNA was expressed increasingly with the prolongation of reperfusion time. After eight hours increase of the expression was obvious. The expression reached the highest peak after twelve hour (P<0.05). MCT4mRNA was expressed decreasingly with the prolongation of reperfusion time. After eight hours decrease of the expression was obvious.(P<0.05)4. The following is the results of MCT1proteins and MCT4proteins expressed in H9C2cells when ischemia occurs:The results of Western-blot told us MCT4proteins were expressed increasingly with the prolongation of ischemia time. After four hours increase of the expression was obvious (P<0.05). Then the expression was maintained at that level. After four hours MCT1proteins were expressed decreasingly, but it was not statistically significant.5. The following is the results of MCT1proteins and MCT4proteins expressed in H9C2cells when reperfusion occurs:The results of Western-blot indicated that MCT1proteins were expressed increasingly with the prolongation of reperfusion time. After eight hours increase of the expressionwas obvious (P<0.05) while MCT4proteins was expressed decreasingly. After about eight hours decrease of the expression was obvious.6. The following is the variation of the concentration of lactic acid clear from supernatant liquid. The concentration of lactic acid from supernatant liquid was increasingly with the prolongation of ischemia time. After four hours this increase reached the highest peak. The concentration of lactic acid from supernatant liquid was decreasingly with the prolongation of reperfusion time. After eight hours this decrease reached the highest peak. These were in accordance with the results of MCT1proteins and MCT4proteins expressed in H9C2cells.Conclusions:When ischemia occurred, MCT4was expressed abundantly and the concentration of lactic acid from supernatant liquid was increasingly. When reperfusion occurred, MCT1was expressed abundantly and the concentration of lactic acid from supernatant liquid was decreasingly. From the resultes it suggested that lactate shuttle occurred in myocardial ischemia-reperfusion injury. PART ⅡThe function of lactate shuttle during the process of ischemia-reperfusion in cardiac myocytesPurpose:This study was to observe the variation of cell viability and apoptosis in order to verify whether lactate shuttle occurred in myocardial ischemia-reperfusion injury.Methods:After cardiocytes H9C2had been cultivated and passaged MCT siRNA transfected cardiocytes H9C2.8μg synthetic MCT-RNAi fragment and20μl liposomes were added to serumfree DMEM. Then these cells were cultivated in5%CO2incubator at37℃. After24hours these cells were collected. Transfection efficiency was evaluated through immunofluorescence. Western-blot analysed the effect of RNA interference. H9C2cells were Immunofluorescence histochemistry stained. MCT1antibodies, MCT4antibodies and antibodies labeled with FITC were added. Nucleus were coloured with DAPI dye. Anti-quench agent was added to every glass slide.37℃for30minutes, these were photographed with fluorescence microscopy. Cell protein was extracted and then Western blot was carried out. Protein level was determined with BCA. After SDS-PAGE electrophoresis protein was trarsmembraned, closed and hybridized, relative level of protein was determined with Chemiluminescence, development, fixation and gelatin image analysis. Cardiomyocyte apoptosis rate was detected by AnnexinV-FITC apoptosis kit. Flow cytometer assayed apoptosis. MTT colorimetry assayed cell survival rate. Cells were collected and then these were splited. Proteins from cell lysate supernatant were detected by Bradford.50μl2xReaction Buffer and5μl4nM DEVD-PNA substrate were added to the partial supernatant. DEVD-PNA substrate was not added to the control group.37℃for1hour, optical density value was measured by spectrophotometer. Caspase activity was calculated.Results:1. MCT siRNA transfection efficiency was evaluated:After cells had being transfected48hours3typical visual fields were chosed in every hole.100cells were observed in every hole. MCT siRNA transfection efficiency was93%、86%、91%respectively. That achieved the requirement of this experiment.2. The following is the results about transfected cells:Cell survival rate was more than85%after60pmol siRNA had been transfected by LipofectamineTM2000with MTT colorimetry. That conformed to the requirement of this experiment.3. The following is the variation of MCT1proteins and MCT4proteins which were acted by MCT siRNA with Western blotting:The results told us MCT1was expressed abundantly while MCT4deficiently. After H9C2cells were being transfected48hours by Chemosynthesis MCT siRNA proteins strap weakened obviously. It was obvious that MCT1and MCT4proteins were expressed decreasingly.4. After H9C2cells had being transfected48hours by MCT siRNA cell vitality weakened and apoptosis increased during ischemia-reperfusion in contrast with normal H9C2cells.5. After H9C2cells had being transfected48hours by MCT siRNA Caspase-3activity increased gradually and MCT siRNA enhanced this effects further.Conclusions:This technique was excellent that cells were transfected by siRNA. It had higher efficiency and smaller cytotoxicity. This was a kind of good gene knockout means. We found that cell vitality weakened and apoptosis increased during ischemia-reperfusion after cells had been transfected by siRNA. These results indicate that lactate shuttle protects against ischemia-reperfusion injury in Cardiac Myocytes PARTIIIThe Effect of trimetazidine on lactate shuttle during the process of ischemia-reperfusion in cardiac myocytesPurpose:This study was to investigate the variation of monocarboxylate transporter when Trimetazidine acted on rat’s myocardial cells during ischemia-reperfusion injury.Methods:After cardiocytes H9C2had been cultivated and passaged embryonic rat’s cardiocytes H9C2were cultivated at95%N2+5%CO2incubator. After four hours5ml DMEM containing10%fetal bovine serum was added to cell culture fluid. Then these cells were cultivated in95%air+5%CO2incubator at37℃. After ischemia cells had been cultivated for four hours, apoptotic rate was detected with flow cytometer.lt was to verify whether the ischemia-reperfusion model conformed to the requirement of this experiment. H9C2cells were Immunofluorescence histochemistry stained. MCT1antibodies, MCT4 antibodies and antibodies labeled with FITC were added. Nucleus were coloured with DAPI dye. Anti-quench agent was added to every glass slide.37℃for30minutes, these were photographed with fluorescence microscopy. The total RNA of H9C2cells was extracted. Reverse transcription was done. Real-time quantitative PCR was also done with SYBRGreen Fluorescent Dyes. Cell protein was extracted and then Western blot was done. Protein level was determined with BCA. After SDS-PAGE electrophoresis protein was trarsmembraned, closed and hybridized, relative level of protein was determined with Chemiluminescence, development, fixation and gelatin image analysis. Flow cytometer assayed apoptosis. MTT colorimetry assayed cell survival rate. Cells were collected and then these were splited. Proteins from cell lysate supernatant were detected by Bradford. The value of A595was calculated.50μl2×Reaction Buffer and5μl4nM DEVD-PNA substrate were added to the partial supernatant. DEVD-PNA substrate was not added to the control group.37℃for1hour, optical density value was measured by spectrophotometer. Caspase activity was calculated.Results:1. The following is morphologic variations:After treated with trimetazidine, the cell growth state of experimental group was superior to control group. The number of dead cells and apoptotic cells in experimental group was lower than that in control group cells.2. The following is the variation of MCT mRNA expression:The results of real-time quantitative RT-PCR showed that the number of MCT4mRNA expression in experimental group was larger than control group during ischemia. The number of MCT1mRNA expression was not statistically significant. The number of MCT1mRNA expression in experimental group was larger than that in control group during reperfusion. The number of MCT4mRNA expression was not statistically significant.3. The result of Western-blot is as follows:The number of MCT4mRNA expression in experimental group was larger than control group during ischemia. The number of MCT1mRNA expression was not statistically significant. The number of MCT1mRNA expression in experimental group was larger than control group during reperfusion. The numberof MCT4mRNA expression was not statistically significant. These were in accordance with the second results.4. MTT results is as follows:cell vitality in experimental group was more increasing than that in control group during ischemia-reperfusion.5. Flow cytometry result is as follows:apoptosis in experimental group was decreased more than control group during ischemia-reperfusion.6. The results of Caspase-3activity is as follows:Caspase-3activity was higher in experimental group than control group during the process of ischemia-reperfusion.Conclusions:Trimetazidine can promote lactate shuttle during the process of ischemia-reperfusion in Cardiac Myocytes. The lactate shuttle activation by trimetazidine protects against ischemia-reperfusion injury in Cardiac Myocytes...