The Study Of "Subthreshold Dose" Calcium Channel Blockers Attenuate The Injury Of Mouse Myocardial Ischemia/Reperfusion By Regulating MIF

Myocardial Ischemia/Reperfusion（I/R）injury refers to the phenomenon that myocardial blood flow returns to normal after Ischemia for a period of time,while the degree of tissue damage and organ function is further aggravated and worsened than that during Ischemia.Calcium overload is one of the important causes of injury.Calcium channel blockers can protect the myocardium by blocking calcium ion channels,inhibiting the intracellular flow of calcium ions,and reducing calcium overload.In addition to blocking L-type voltage dependent calcium channel（L-VDCC）,Calcium channel blockers may also have a non-L-VDCC-dependent protection mechanism.Our previous study showed that the Calcium channel blockers Nifedipine（Nif）;Verapamil（Ver）;Diltiazem（Dil）and our laboratory’s new Calcium channel blocker N-n-butyl haloperidol iodide（F₂）attenuated myocardial Ischemia/Reperfusion（I/R）and myocardial Hypoxia/Reoxygenation（H/R）injury in mice through a non-calcium channel dependent pathway.Macrophage Migration Inhibitory Factor（MIF）plays an important role in myocardial I/R injury,mainly involved in inflammatory response,energy metabolism,cell apoptosis and necrosis,and oxidative stress.In the early stage,we found through the whole gene expression profile chip that under the condition of calcium ions in chelated cell culture medium,H/R could significantly improve the expression level of MIF in H9c2 cells,while F₂ could inhibit the high expression of MIF,indicating that F₂ may participate in the regulation of MIF expression in a non-calcium channel dependent form.Our previous work has shown that the dose of calcium antagonist given to mice affects hemodynamic changes without blocking L-VDCC（It is called"subthreshold dose"）and can still attenuated the myocardial I/R injury in mice.Therefore,this paper explores the important relationship between the non-calcium channel-dependent myocardial protection of Calcium channel blockers and the regulation of MIF expression.Methods:1.Western blot detection of proteins:MIF,Cleaved caspase-3,Bcl-2,Bax,p-AMPK,AMPK,GLUT4.2.In vivo imaging system of small animals was used to detect the cardiac function of mice.3.Apoptosis of myocardial tissue was detected by Tunel.4.The inflammatory factors TNF-a,IL-6,IL-1βand enzymatic factors LDH,CK and CK-MB were detected by ELISA.5.TTC/Evans blue was used to detect the size of myocardial infarction in mice.6.Immunofluorescence detection of inflammatory cells in myocardial tissue.7.ROS leakage in myocardial tissue was detected by DHE staining.Results:Part Ⅰ:In the myocardial ischemia experiment,the expression of MIF in myocardium was the highest at I45min.In the myocardial I/R experiment,the expression of MIF in the myocardium from I45min/R0.5h showed a trend of gradually upregulation compared with Sham group,and reached its peak at I45min/R2h,and gradually decreased at I45min/R3h.The expression of MIF in I45min/R4h and I45min/R5h was lower than that in Sham group.I45min/R0.5h was determined as the time point of short-term myocardial I/R in mice,and I45min/R4h as the time point of long-term myocardial I/R in mice.Part Ⅱ:Mice with MIF gene knockout reduced short-term myocardial I/R injury.The MIF inhibitor ISO-1 aggravated long-term myocardial I/R injury in mice,and AMPK agonist AICAR attenuated long-term myocardial I/R injury in mice.Part Ⅲ:Subthreshold calcium channel blockers was given 20min before MI/R can inhibit the high expression of short-term myocardial I/R MIF and attenuate myocardial injury in mice.Subthreshold calcium channel blockers was given 20min before MI/R can up-regulate the low expression of long-term myocardial I/R MIF and attenuate myocardial injury in mice.The subthreshold calcium channel blockers was administered at I45min/R3h,which had no effect on the expression of MIF and had no effect on the myocardial injury in mice.Conclusion:Subthreshold doses of calcium channel blockers F₂,Ver,Nif and Dil can attenuate myocardial I/R injury in mice by regulating the expression of MIF,which may be one of the mechanisms by which calcium channel blockers protect myocardial I/R injury through non-calcium channel-dependent pathways.