Underlying Mechanism Of Attenuated Myocardial Endogenous Protection From Ischemia/Reperfusion Injury In Diet-induced Obesity Rat

Part I: experiment in vivoStudy on susceptibility of myocardial ischemia/reperfusion injury inhigh-fat diet induced obesity rat and the underlying mechanismsObjective：As a major risk factor of developing coronary artery heart disease, obesitycontributes to be a growing public health problem all over the world. Traditionalobservations showed that obesity individuals with established coronary artery diseaseappeared to confer a survival benefit, which was termed with “obesity paradox”. Is it realthat obesity protective against cardiovascular outcomes? This study investigated whetherobesity rats were liable to more severe acute myocardial ischemia reperfusion injury andexplored the underlying mechanisms.Methods:4to6week-old male wistar rats (n=64) were fed with a normal diet for oneweek to adapt to the environment. Then they were separated into two groups at random:control group(n=32) fed with normal chow and obesity group(n=32) fed with a high-fatdiet with1mass percent cholesterol and15mass percent lard oil for24weeks to establishobesity model. Obesity-prone rats of the control group (n=6) and diet-resistant rats(n=7) ofthe obesity group were eliminated from the study at the end of12thweek. Body weight andbody length were measured during24-week follow-up. Glycemic parameter and lipidprofile were examined at the end of12thweek and24thweek, while the same time, thecardiac geometry and function were assessed by echocardiography. Then a rat model ofmyocardium ischemia-reperfusion injury (MIRI) was developed by ligating left anteriordescending branch of coronary artery which subjected to ischemia30minutes andreperfusion120minutes (control group, n=12but successed with8; obesity group, n=15but successed with12). Hemodynamic parameters and arrythmias were measured orrecorded. Ischemic and infarct areas were tested with Evans blue and TTC respectively. Animals that didn’t suffer from MIRI were sacrificed for structure observing with HEstaining and ultra-structure examination by electron microscope. TUNEL kit was used toidentify apoptosis. Total RNA was extracted from cardiac tissue and transcription wasperformed. The mRNA of caveolin-3and different subunit of adenosine triphosphatesensitive potassium channel (KATP) were compared between control group and obesitygroup with quantitative polymerase chain reaction (q-PCR). The membrane and cytoplasmexpressions of caveolin-3and KATPsubunits were measured by western-blot. Thedistribution of caveolin-3in cardiac myocyte was measured by immunohistochemicaltechnique both control and obesity rats.Results: In comparison with control group, body weight, body length and BMI wereincreased significantly in obesity group. There were significances in fasting blood glucose(FBG), total cholesterol (TC), LDL-C between control group and obesity group. Althoughleft atrial diastolic diameter and left ventricular posterior wall diastolic were increased inobesity rats at the end of24thweek, there were no differences about EF and FS betweenthese two groups. Dramatic increase in stroke volume and cardiac output accompaniedwith body weight in obesity rats. Also fatty deposition and apoptosis could be observed inobesity heart. Mitochondrial morphology and distribution changed accompanied withpartial caveolae destroyed. Obesity rats showed enhanced susceptibility to MIRImanifested with improved mortality, worsened hemodynamics and much more ventricalarrythmias. While the same time, ischemic and infarct areas were extened in obesity group.Though mRNA transcription levels of caveolin-3and KATPsubunits were dramaticincreased in obesity group compared with control group by qPCR technique, disorder ofcaveolin-3and Kir6.2translocation to plasma membrane were found by western-blot andimmunohistochemical technique.Conclusion: The cardiac remodeling and metabolic disorder induced by high-fat dietmay impact normal tolerance to MIRI via lower translocation of caveolin-3and kir6.2toplasma membrane. Part II: experiment in vitroThe impact and potential mechanisms of palmitate onH9C2myoblast tolerance to hypoxia/reoxygenation injuryObjective: It was estimated that cardiac remodeling associated with obesityattenuates endogenou protection to MIRI in vivo experiments. The mechanisms linkingobesity and cardiac remodeling were too complicated to clarify and which approach wasmost responsible for MIRI. We had hypothized that metabolic abnormalities of the obesityheart should be responsible for MIRI.Methods: As palmitate induces H9C2cell apoptosis due to ROS productionincreasing had been confirmed recently, H9C2myoblast was pretreatmented with a lowerdose (0.25mmol/L), after that physical hypoxia/reoxygenation（2hours/24hours）intervention was committed. The cell proliferations was measured by cell counting kit-8(CCK-8) and mitochondrial transmembrane potential (MMP)△Ψm evaluated using5，5’，6，6’-tet rachloro-1，1’，3，3’-tetrethyl benzimidalyl carbocyanine iodide (JC-1) detection.Annexin V/PI Assay was used to assess apoptosis in different conditions. mRNA level ofcaveolin-3and KATPsubunit were compared between palmitate-potassium hydroxide（KOH）group and KOH group with quantitative polymerase chain reaction (q-PCR).The membrane and cytoplasm expressions of caveolin-3and kir6.2subunits weremeasured by western-blot. Besides, expression of bax and bcl-2in mitochondrial wasdetected to verify if the apoptosis induced by mitochondrial pathway. The mito-KATPandsarc-KATPactivator diazoxide (200umol/L) was used to verify if KATPdysfunction existed.Results: Palmitate suppress H9C2cell proliferation and induced H9C2cell apoptosisafter MIRI, accompanied with△Ψm depletion. The mRNA transcription levels ofcaveolin-3and KATPsubunits were increased in palmitate group compared with KOHgroup. But only caveolin-3translocation disorder was found. The diazoxide pretreatmentwas benefit for palmitate group cell proliferation and△Ψm sustain. But there was no usefor KOH group. However, statistical significance still existed between palmitate-KOH-DZand KOH-DZ. The bax/bcl-2was elevated in mitochondrial protein from palmitate-KOHgroup compared with KOH group. Conclusions: The experiment replicated mechanism of obesity impacting cardiactolerance to MIRI in vivo mostly. So the saturated fatty acid palmitate induced caveolin-3translocation in cytoplasm by decreasing△Ψm and H9C2cell apoptosis viamitochondrial pathway. This could be responsible for it. And mechanisms via activatingboth mito-KATPand sarc-KATPmay be limited useful.