Fasting Alleviates Myocardial Ischemia Reperfusion Injury In Rats

As the altitude rises,the oxygen partial pressure(PO2)in the air continues to decrease.When the human body rapidly enters the high-altitude environment,it will cause different degrees of symptoms and signs,and even damage due to hypoxia.Our country has a long plateau border and the Sino-Indian border is more than 2,000 kilometers long.The average altitude of the border is over 4,500 meters.When a sudden military conflict,the troops need to be quickly sent to combat.How to reduce the incidence of altitude sickness and reduce non-war attrition when troops rush into the plateau to perform military tasks is a difficult problem to be solved urgently.However,the mechanism of adaptation to acute hypoxia has not been fully elucidated.We had established a rat model of enhancing hypoxia tolerance.Fasting for 72 hours effectively improve the survival rate of Sprague-Dawley(SD)rats in a simulated 7620-meter altitude environment.The heart is one of the organs that are extremely sensitive to hypoxia in the human body.Fasting improves the acute hypoxia tolerance in rats,and whether fasting also improve the hypoxia tolerance of myocardial tissue and has a protective effect on ischemia-reperfusion myocardium is worthwhile to elucidate.Myocardial ischemia reperfusion(IR)injury refers to the pathological process that after partial or complete acute block of the coronary artery,even if the blood vessels are recanalized in a certain period of time and the ischemic myocardium can be restored to normal perfusion,the myocardial tissue damage is progressively aggravated.A series of changes in myocardial ultrastructure,energy metabolism,cardiac function and electrophysiology are caused by ischemia.Myocardial damage in the early stage of reperfusion,such as myocardial remodeling,inflammatory response,oxidative stress,energy metabolic disorders and apoptosis is more prominent.Pyroptosis initiated by NOD-like receptor family pyrin domain-containing 3(NLRP3)inflammasome assembly is an important part of reperfusion injury.In the late stage of reperfusion,the myocardium begins to repair,which can partially restore myocardial function.Early sdudy in the laboratory have demonstrated that fasting inhibits protein and fat anabolism by inhibiting the mammalian target of rapamycin(mTOR)signal transduction pathway,thereby reducing the consumption of non-essential adenosine triphosphate(ATP)in tissue cells and activating mitophagy.Removing aged or micro-damaged mitochondria in advance may prevent excessive increase of reactive oxygen species(ROS)under hypoxic conditions.Mitochondrial oxygen utilization efficiency was enhanced,thereby the body’s hypoxia tolerance was improved.These mechanisms may ensure the necessary ATP supply in a limited time during myocardial ischemia and prevent myocardial damage.During reperfusion,enhanced autophagy may inhibit cardiomyocyte pyroptosis as a protective role.Therefore,this study aimed to observe the protective effect and mechanisms of fasting on ischemia-reperfusion myocardium in SD rats with molecular imaging methods,and to provide experimental data and new ideas for the establishment of a multi-target cardiac protection strategy.Objective:1.To investigate the effect of fasting on myocardial glucose uptake in rats.2.To investigate the effect of fasting on the myocardial infarction(MI)area after ischemia-reperfusion injury in rats.3.To explore the mechanism of fasting protecting myocardium from ischemia-reperfusion injury in rats.Method:1.Blood was collected from tail vein of SD rats and the blood glucose and blood ketone levels in different fasting time groups were detected by blood glucose and ketone meter.2.The left anterior descending branch(LAD)of the rat coronary artery was ligated for 30 minutes,and then the ligation was released to prepare a myocardial IR injury model.The changes of different indexes were observed at 3h,4d and 7d after the reperfusion.3.Three groups were set up:the rats underwent thoracotomy without LAD ligation,which was regarded as the sham-operated group(Sham group);the rats in the synchronous control group underwent IR surgery(CON-IR group),and the rats were fasted for 72 h without food.Drinking water was restricted,then IR surgery was performed(F72h-IR group).4.M-mode echocardiography was used to measure the cardiac pumping function of rats during fasting and at different time points after ischemia-reperfusion.5.TTC(triphenyltetrazolium chloride)staining method was used to measure myocardial infarct size in rats with ischemia-reperfusion in different groups.6.Fluoro-18-deoxyglucose positron emission tomography/computed tomography(18F-FDG PET/CT)imaging was used to observe the myocardial metabolic transformation process,FDG uptake and myocardial defect after ischemia-reperfusion in vivo.7.Gallium-68 fibroblast activation protein inhibitor(68Ga-FAPI)04 PET/CT imaging was used for observation of myocardial fibroblast activation level in vivo after ischemia-reperfusion in rats.8.Rat myocardial H9C2 cell line was cultured,and the effects of different glucose concentrations on the expression and activity of mTOR and Adenosine 5’-monophosphate-activated protein kinase(AMPK)in myocardial cells were detected by Western blotting and immunofluorescence methods.9.The effects of 72h-fasting on the expression and activity of mTOR,AMPK,autophagy flux,inflammatory response and apoptosis rate in ischemia-reperfusion myocardium were observed by Western blotting and immunofluorescence.Result:1.The 24h-fasting caused a significant decrease in body weight and blood glucose,but an increase in blood ketones.Fasting for 48h-72h,further decreases body weight,blood glucose and increases blood ketones,which reached a certain balance state,then after 24h of diet recovery they all returned to normal level.Heart rate(HR),left ventricular ejection fraction and short-axis shortening rate of fasted rats had no significant difference in Control,F24h,F48h and F72h groups.Myocardial 18F-FDG uptake and SUV levels of fasted rats decreased continuously with the prolongation of fasting time and returned to normal after 24 hours of diet recovery.2.The myocardial cell line H9C2 was cultured in different concentrations of glucose medium for 72 hours.With the decrease of glucose concentration,the activity of mTOR in cardiomyocytes decreased and the activity of AMPK increased with Western blotting and immunofluorescence methods.3.Compared with the Sham group,the left ventricular ejection fraction(EF)and short-axis shortening rate(FS)were significantly decreased in each synchronous control group(CON-IR-3h,CON-IR-4d and CON-IR-7d),and the EF and FS in each fasting group(F72h-IR-3h,F72h-IR-4d and F72h-IR-7d)also decreased,but the EF and FS in the fasting groups were significantly higher than those in their synchronous control group.The myocardial defect area under 18F-FDG uptake in the CON-IR-4d and CON-IR-7d groups was significantly larger than that in the F72h-IR-4d and F72h-IR-7d,respectively.The myocardial infarct size by TTC staining in the CON-IR-4d and CON-IR-7d groups was significantly larger than that in the F72h-IR-4d and F72h-IR-7d groups.The SUV levels of 68Ga-FAPI uptake in the CON-IR-4d and CON-IR-7d groups were significantly higher than those in the F72h-IR-4d and F72h-IR-7d.4.Myocardial mTOR activity decreased and AMPK activity increased in the early stage of ischemia-reperfusion in fasting rats.Fasting increased the autophagy flux of myocardial cells during ischemia-reperfusion,and inhibited the NLRP3 inflammasome in myocardial ischemia-reperfusion tissue.Fasting also reduced the activation levels of Caspase 9 and Caspase 3,which are the execution proteins of apoptosis in ischemia-reperfusion cardiomyocytes.Conclusion:1.Fasting reduces myocardial infarct size in acute and convalescent stages of ischemia-reperfusion in rats.2.The mechanism of the protective effect by fasting on acute myocardial ischemia-reperfusion in rats may be to reduce unnecessary ATP consumption and relatively increase ATP production.3.Fasting reverses myocardial remodeling during recovery from ischemia-reperfusion in rats and the mechanism may be inhibition of inflammatory response.In summary,fasting has a significant protective effect on ischemia-reperfusion myocardial injury in both the acute stages and convalescent stages.The mechanism may be to inhibit mTOR then reduce the consumption of ATP by anabolism,thereby reducing the injury in the acute phase of ischemia.It inhibits the inflammatory response induced by pyroptosis and promotes the repair of myocardium.