Study On Correlationship Between The Change Of Microtubules Of Myocardial Cytoskeleton And Cold Ischemic Injury In The Hypothermia

Heart transplantation is widely accepted as the only effective therapy for heart diseases in the end stage.But it is largely limited because the donor heart can only be preserved at low temperature for 4-6 hours.How to preserve the donor heart longer is the problem that need to be solved as quickly as possible.It is very important to elucidate,pathophysiological mechanism of hypothermal ischemic injury of myocardium and to find effective preventive method.Cytoskeleton is important conservative structure which plays very important role in maintenance of the orderly spatial structure and cell living activiyies.Because there is correlationship between the integrity of cell skeleton and the temperature it is necessary to study the effect of hypothermal cytoskeleton change of cardiocyte on the structure and function of myocardial cell.We studied the relationship between cytoskeleton changes and hypothmal ischemic injury of cardiomyocyte and the possibility to protect cytoskeleton as a new method in myocardium protection.Our study is of great significance in resolving the problem of preservation of donor heart in heart transplantation.Our study was divided into three parts.In the first part,intact hearts of adult rat were preserved hypothermaly for 6 hours while microtubule stabilizer and distabilizer were added in the preserving fluid.The myocardial enzyme leakage in coronary effluent were determined and the morphological observation was performed after rewarming reperfusion in order to approach the effect of microtubule stabilization and destabilization on the vitro rat hypothermal preservation.In the second part,separated rat cardiomyocytes were preserved hypothermally. Microtubular changes and myocardiac enzymic releasing rate were determined with immunoflorescence and colorimetric analysis to study the changes of microtubules and myocardial injury hypothermal preservation.In the third part,the effect of microtubule stabilizer on apoptosis of rat cardiomyocyte in hypothermal preservation and rewarming reperfusion was determined.The mechanism between microtubular change and cardiomyocyte injury were approached.Methods1.In vitro rat heat perfusion and hypothermal preservationSD rats were divided randomly into 3 group.Control group,the heart were arrested and stored in HTK solution.Taxol group,in which HTK solution contained taxol(10^-6M).Colchicine group,in which HTK solution contained colchicine(10^-6 M).Langendorff model perfusion apparatus of isolated rat heart was used.These isolated rat hearts were stored in 4℃different solutions of heart preservation for 6 hour,and then were reperfused.After 4 minutes reperfusion,the myocardial enzyme leakage in coronary effluent were determined.After 20 minutes reperfusion,the heart sample were obtained and observed under light and electron microscopes.2.Separation of cardiomyocytes of adult ratPerfusion was same as the mentioned above but with calciferous and noncalciferous Turode's fluids.The heart was digested by perfusion of noncalciferous Tyrode's fluid containing typeⅡcollagenase and calf serum albumin.The residual enzme was washed with KB fluid.During perfusion saturated oxygen was given continuously.Ventricular wall was cut into pieces,and slightly oscillated,the cardiomyocytes were isolated.After filtration,low-rate centrifugalization,the clear supernatant solution was decanted,the experimental cells were obtained and kept in KB fluid.3.Dealing with the separated cardiomyocytesThe separated cardiomyocytes were randomly divided into experimental and control groups,each of which was subdivided into 2 subgroups i.e.one was preserved at 4℃for 30 minutes the other for 3 hours.The experimental subgroups were given taxol(10^-6M).Little suspension was taken out and smeared for microtubule immunoflorescent detection and trypan blue staining.The left suspension was centrifugated,the supernantant was used for LDH activity analysis.The sediment cells were treated with 1%Triton X-100 at 37℃for 30min and then for LDH activity analysis The LDH activity was determined with photometer.LDH releasing was expressed with percentage of total LDH activity(releasing and remained).4.ImmunoflorescenceSeparated cardiomyocyte smear was dried at room temperature and fixed for 3 minutes in methanol at -20℃,saddened in cold acetone 3 times,kept at 4℃.Microtubule immunoflorescence was analyzed with Image-Pro Plus image analysis software.5.Trypan blue stainingCardiomyocyte smear was stained with 0.1%trypan blue.200 cardiomyocytes were counted under high magnification and antistained cell ratio was calculated.6.Apoptosis detectionApoptosis detection was performed with TUNEL method(Roche company). TUNEL positive nuclei were stained violet or violet-black in color while negative nuclei light pink.7.Enzymic leakage detectionEnzymic colorimetric analysis was detected in the coronary leakages of the rat hearts preserved hypothermally and reperfused after rewarming.Enzymes released after hypothermal preservation and those residual in the cardiomyocytes were also determined with the same method.8.Light and electron microscopic observationHypothermally preserved and reperfused cardiomyocytes were observed with routine light and electron microscopes. Results1.Microtubular change in the separate cardiomyocytes preserved hypothermallyMicrotubules in the cardiomyocytes were depolymerized after only 30 minutes hypothermal preservation,Immunpflorescence was weakened,continuity of the microtubules was lost,the microtubules turned rough with no clear structure. Microtubules changed more significantly in the control Subgroups.Mean optic density (MOD)of microtubules in the control subgroup preserved hypothermally for 30 minutes was 20.20±1.49,12.46±1.66 for 3 hours.While MOD of microtubules in the experimental subgroup preserved hypothermally for 30 minutes was 25.49±1.52, 20.41±1.52 for 3 hours.(vs control,P＜0.01).As the preserving period prolonged the microtubular immunoflorescence in both groups were weakened.And the mirotubular network lost continuously.2.LDH releasing rate in separated cardiomyocytes preserved hypothermallyLDH releasing in the separated cardiomyocytes was detected at different time of the experiment.The result showed that LDH releasing rates in the experimental subgroups were significantly lower than those in the control.LDH releasing rates in the cardiomyocytes of the controls subgroups preserved hypothermally for 30minutes and 3 hour were 56.44%±1.45 and 65.68%±1.37 respectively.While in the experimental 51.88%±1.06 and 58.87%±1.09(vs control,P＜0.01).3.Survival rate of the separated cardiomyocytes preserved hypothermallySurvival rate of the isolated cardiomyocytes was detected with trypan blue stained. The rate in the control group was 27.20%±5.05,and 38.80%±2.66 in the experimental. There was significant difference between the rates in the two groups(P＜0.01).4.Light microscopic changes in the rat myocardium preserved hypothermally and reperfused after rewarming Edema can be observed in the subendocardium,,myocardium,and epicardium, intercellular space was widened,perivascular space enlarged,cardiomyocytes turned thinner and shrinked but with a smooth and regular edge in the control group. Compared to the structure change in the control group less edema,,less intercellular space and nearly normal myocardial structure were observed in the taxol group.While in the colchicine group we found significant edema in different layers of the ventricular wall,irregular edge of the cardiomyocytes,obviously enlarged intercellular space,even some dissolved cardiomyocytes in nodules form.5.Ultrastructure change of cardiocyte following hypothermy conserved and rewarming/reperfusion in SD ratsIn control group,mitochondrium swollen,crista of mitochondrium partly lysed and Sarcoplasm tube distended.But construction of myocomma is still clear,and construction of myofilament is distinct.In taxol group,swelling was not found in mitochondrium,crista of mitochondrium was normal,myofibrilla arrange orderly,each band of myocomma was lampros,and Sarcoplasm tube partly distended.In colchicines group,the construction of myofibrilla is not integrated,mitochondrium obviously broaden,cristas of mitochondrium were lysed.Cavitation phenomenon is obvious, Sarcoplasm tube distended obviously,tunica muscularis swollen and appeared digitatus.6.Myocard enzyme Assay from the transudate following heart hypothermy conserve/rewarming reperfusionThe amount of transudate of myocard enzymes(LDH,GOT,CK)is maximal in colchicines group,is more than that in control group,and is at least in taxol group.There is significant difference between groups.7.Apoptotic happening in cadiocytes of rats after heart hypothermy conservation or/and rewarming/reperfusionNucleus images of apoptosis were found in all control groups and experimental groups under electron microscope,Chromatin were convergenced toward karyotheca in partly apoptotic cells,or accumulated with difuse distribution in partly.The apoptotic cells with TUNEL staining were found in cadiocytes,meanwhile the little amount of infiltrative lymphocytes and vascular endothelial cells were also found.The comparative results as following;(1)Apoptotic index of cadiocytes was in proportion with time of hypothermy conservation of heart in control group and experimental group,the longer the time of heart with hypothermy conservation was, the more the number of apoptotic cells was(P＜0.01).The number of apoptotic cells with hypothermy conservation was more at 6h than that at 4h.(2)The longer the time of heart with hypothermy conservation was,the more the number of apoptotic cells with hypothermy conservation rewarming/reperfusion was,namely the number of apoptotic cells with hypothermy conservation rewarming/reperfusion is more at 6h than that at 4h(P＜0.01).(3)The apoptotic index was more in hypothermy conservation / rewarming reperfusion group than that only in hypothermy conservation group(P＜0.01).Which indicated that rewarming reperfusion brought severely cellar damage,and increased the number of apoptotic ceils.(4)The apoptotic index is less than in experiment group than that in control group in each test timepoint(P＜0.01).Preservation solution with taxol can obviously protect the heart with hypothermy conservation.Conclusions1.Microtubule of cardiocyte is depolymerized under preservation for 0.5h at4℃, and the depolymerization is severe at 3h.The degree of depolymerization is in proportion with ischemic time on the hypothermia.Taxol can stabilize microtubules and alleviate the depolymerization of microtubules.2.Cadiocytes damage and release of myocard enzyme(LDH,GOT,CK)resulting from hypothermy are in proportion with microtubule depolymerization.Stabilizing microtubule may relieve the release of LDH,GOT,CK from heart.3.Hypothermy for 4℃can lead to damage of mitochondrial construction.The distabilize agent of microtubule aggravate damage of mitochondrium,and the stabilizing agent can relieve damage of mitochondrium.4.Cardiocyte apoptosis occurs under condition of low temperature as 4℃,and the number of apoptotic cardiocytes is more in hypotherrny conservation / rewarming reperfusion than that only in hypothermy conservation.The longer the time of heart with hypothermy conservation is,the more the numbeir of apoptotic cells with hypothermy conservation / rewarming reperfusion is.The stabilzing agent can decrease the number of apoptotic cardiocytes in hypothery conservation.