The Role And Mechanism Of Mitochondrial Dynamics/PIKfyve In Mycardial Injury Caused By OSAHS Complicated With Obesity

Objective:Establishing the mouse model of CIH and HFD to simulate the physiological and pathological status of patients with OSAHS and obesity and investigate the effect of CIH and HFD on cardiac morphology,cardiac function,myocardial morphology,myocardial fibrosis,myocardial apoptosis,mitochondrial dynamics,mitochondrial function and expression of PIKfyve.Methods:1.Establishing of the CIH and HFD mouse model.Forty male C57/BL6 mice were randomly divided into four groups: control group(C),chronic intermittent hypoxia group(CIH),high fat diet group(HFD),chronic intermittent hypoxia + high fat diet group(CIH +HFD),n = 10.The model was established for 14 weeks.The mice in group C and CIH were fed with normal diet,and the mice in group HFD and CIH+HFD were fed with high fat diet.From the 9th week,the mice in CIH group and CIH+HFD group were treated with chronic intermittent hypoxia for 8 hours a day for 6 weeks.The general condition of mice in each treatment group was observed during model establishment.The body weight of mice in each group was recorded every week to evaluate the success of the establishment of the model.2.Exploring the effect of CIH and HFD on myocardial function and structure:pathological changes of myocardial tissue were measured by hematoxylin-eosin(HE)staining;cardiac function and structure of mice in each group was measured by echocardiography(Echo);myocardial fibrosis of mice in each group was observed by Masson staining and Sirius staining;the changes in mRNA levels of genes related to fibrosis were detected by RT-qPCR;the changes in protein levels of Casepase-3 in myocardium of mice in each group were detected by Western Blot.3.Observing the effect of CIH complicated with HFD on myocardial mitochondrial dynamics and the expression of PIKfyve: the mRNA and protein levels of key regulatory Vfactors of mitochondrial fission and fusion in control group and CIH+HFD group were detected by RT-qPCR and Western Blot;the mRNA levels of genes related to mitochondrial function in control group and CIH+HFD group were detected by RT-qPCR;the mRNA and protein levels of PIKfyve in control group and CIH+HFD group were detected by RT-qPCR and Western Blot.Results:1.The mouse model of CIH and HFD was successfully established: compared mice in the normoxia group,mice in the CIH group was less active,depressed,and had less food and water intake.The body weight of mice in the high-fat diet group was significantly higher than that in the normal diet group,and the body weight of mice in the CIH group was slightly lower than that in the normoxia group.2.Echo showed no significant change of cardiac contractile function(EF,FS)in both CIH and HFD group,while the contractile function(EF,FS)in CIH+HFD group decreased significantly(P<0.05).There were no significant changes in end-diastole interventricular septum(IVS;d),left ventricular mass corrected(LV Mass corrected),Left ventricular end-diastole posterior wall(LVPW;d)and left ventricular end-diastolic volume(LV Vol;d)between CIH group and control group;in HFD group,IVS;d was higher than that in control group(P<0.05),and LV Mass corrected and LV Vol;d in HFD group were higher than those in control group,while the difference was not statistically significant;In CIH+HFD group,IVS;d,LV Mass corrected,LVPW;d.increased considerably(P<0.05),but LV Vol;d didn't change statistically.HE staining showed that the morphology of cardiomyocytes was abnormal in CIH,HFD and CIH+HFD groups,especially in CIH+HFD group.Masson staining and Sirius staining showed that there was no myocardial fibrosis in neither CIH nor HFD group,while myocardial fibrosis occurred in CIH+HFD group.RT-qPCR showed that CIH+HFD up-regulated the mRNA level of fibrosis related gene(P<0.05),and there were no significant changes in the mRNA levels of fibrosis related genes in neither CIH nor HFD group.Western Blot showed that the level of Casepase-3 protein increased in CIH,HFD and CIH+HFD groups(P<0.05),especially in CIH+HFD group.3.RT-qPCR showed that the mRNA level of MTP18 was significantly increased,while the mRNA levels of mitofusin(Mfn)1 and Mfn2 were significantly decreased in CIH+HFD group(P<0.05).There was no significant difference in the mRNA level of dynamin-related protein1(DRP1)and optic atrophy1(OPA)between CIH+HFD and control group.Western Blot revealed that in CIH+HFD group,the level of mitochondrial regulatory protein MTP18 was up-regulated,the DRP1 phosphorylation at S637 was significantly reduced and phosphorylation at S616 increased.Mfn1 and Mfn2 protein levels were also significantly decreased in CIH+HFD group.Quantitative analysis showed that the difference was statistically significant compared with control group(P<0.05).RT-qPCR showed that the mRNA levels of genes related to mitochondrial function were significantly decreased in CIH+HFD group(P<0.05).RT-qPCR and Western Blot showed that the levels of PIKfyve mRNA and protein decreased significantly in CIH+HFD group(P<0.05).Conclusion:1.The mouse model of CIH and HFD was established successfully.2.CIH and HFD led to ventricular hypertrophy,abnormal cardiomyocyte morphology and myocardial apoptosis,while neither myocardial fibrosis nor cardiac contractile function decline was observed in the two groups.CIH complicated with HFD resulted in ventricular hypertrophy,significantly abnormal cardiomyocyte morphology,myocardial apoptosis,myocardial fibrosis and significantly impaired cardiac contractile function of mice.3.CIH complicated with HFD led to increased mRNA and protein expression of mitochondrial fission factors,decreased mRNA and protein expression of mitochondrial fusion factors,a disorder of mitochondrial dynamics,decreased mitochondrial function and decreased mRNA and protein expression of PIKfyve.