Angiotensin Ⅱ Type 1a Receptor Knockout Ameliorates High-fat Diet Induced Cardiac Dysfunction By Regulating Glucose And Lipid Metabolism

Background:Obesity is considered to be an independent risk factor for cardiovascular disease and to some extent leads to changes in heart structure and function.Due to its complex pathogenesis,the prevalence of cardiovascular diseases caused by obesity continues to rise despite the continuous development of therapeutic drugs and methods.Recent studies have shown that renin-angiotensin system（RAS）is involved in the occurrence and development of obesity,diabetes and other related diseases.Experiments have shown that the activity of RAS is significantly increased in obesity.However,the underlying mechanisms of RAS in obesity-induced cardiac dysfunction remain unknown.The heart requires a large amount of energy to maintain its normal function.In obese patients,the imbalance in the oxidative utilization of substrate fatty acids and glucose by the heart leads to insufficient energy supply,resulting in cardiac dysfunction.Angiotensin Ⅱ（Ang Ⅱ）regulates cardiac function primarily through angiotensin Ⅱ type 1 receptor（AT1R）.It has been found that Ang Ⅱ elevation changes the degree of fatty acid oxidation and carbohydrate oxidation,and mediates mitochondrial respiratory enzyme damage.These studies suggest that RAS is involved in cardiac glucose and lipid metabolism and is closely related to mitochondrial energy supply.Therefore,this study intends to establish a rat obesity model and explore the role of RAS in energy metabolism in cardiac dysfunction.Objective:Obesity-induced cardiac dysfunction is accompanied by overactivation of RAS,but the underlying mechanism of both remains unclear.In this study,we investigate the role and mechanism of Ang Ⅱ in obesity-induced cardiac dysfunction by observing the changes of cardiac energy metabolism and cardiac function induced by AT1aR knockout in obesity induced by high-fat diet.Methods:1.The AT1aR gene knockout type(AT1aR^-/-)homozygous rats were established.Male wild type（WT）and AT1aR^-/-SD rats were respectively divided into two groups.They were fed a normal diet（ND）and a high fat diet（HFD）in an SPF laboratory animal facility for 12 weeks.2.The weight change of rats was recorded weekly during feeding.After 12 weeks,the fasting blood glucose of rats in the 4 groups was measured,and the oral glucose tolerance test and insulin tolerance test were conducted.At the end of the 12th week,echocardiography was used to detect the cardiac function.Finally,the rats were subjected to carotid artery intubation,and blood pressure was recorded by BL-420.3.Blood and heart tissue were collected from rats.Several kits measured serum lipid-related indicators,myocardial ATP content,and mitochondrial respiratory chain complex V activity.The contents of Ang Ⅱ in serum and myocardium were measured by ELISA.Cardiac structure was observed by H&E staining and myocardial lipid deposition was observed by oil red O staining.4.The protein expressions of angiotensin Ⅱ type 2 receptor（AT2R）,carnitine palmitoyl transferase 1B（CPT1B）,fatty acid translocase（CD36）,phosphorylated pyruvate dehydrogenase（P-PDH）,pyruvate dehydrogenase（PDH）,mitochondrial fusion protein 2（MFN2）,dynamin-related protein 1（DRP1）and mitochondrial fission protein 1（FIS1）were detected by Western blot.5.The m RNA levels of ATa1R,hexokinase 2（HK2）,phosphofructokinase 1（PFK1）,pyruvate kinase（PK）,CD36,medium-chain acyl Co A dehydrogenase（ACADM）,acyl Co A oxidase 1（ACOX1）,peroxisome proliferator activator receptor（PPARα/γ/δ）and PPARγcoactivator-1α（PGC-1α）were detected by RT-PCR.Results:1.AT1aR knockout improved obesity and metabolic disorders in rats fed high-fat dietThe body weight of AT1aR^-/--HFD rats was significantly lower than that of WT-HFD rats.Compared with WT-HFD rats,AT1aR knockout significantly reduced fasting blood glucose,blood pressure,total cholesterol（T-CHO）,triglycerides（TG）,non-esterified fatty acids（NEFAs）and low-density lipoprotein-cholesterol（LDL-C）,and improved glucose tolerance and insulin sensitivity.2.AT1aR knockout ameliorated cardiac dysfunction caused by high-fat dietCompared with WT-HFD rats,left ventricular ejection fraction（LVEF）and left ventricular fractional shortening（LVFS）were significantly increased in AT1aR^-/--HFD rats,while left ventricular internal diameter at end systole（LVIDs）and left ventricular end systolic volume（LVESV）were decreased.3.AT1aR knockout alleviated cardiomyocyte hypertrophy and lipid deposition induced by high-fat dietH&E staining showed obvious hypertrophy of cardiomyocytes in WT-HFD rats,but no hypertrophy in AT1aR^-/--HFD rats.Oil red O staining showed a large number of small lipid droplets in myocardial cells of WT-HFD rats,while AT1aR^-/--HFD rats significantly reduced lipid droplets.Compared with WT-HFD rats,the ratio of heart weight to body weight was increased in AT1aR^-/--HFD rats.4.AT1aR knockout attenuated RAS activity and cardiac energy supply deficiency in obese ratsThe Ang Ⅱ level and AT1aR gene level of WT-HFD rats were increased.Compared with WT-HFD rats,Ang Ⅱ level of AT1aR^-/--HFD rats was decreased,while myocardial ATP content was increased.5.AT1aR knockout improved impaired myocardial glucose oxidation in obese ratsThe gene levels of HK2,PFK1 and PK were significantly decreased in WT rats fed high-fat diet compared with those fed normal diet,and AT1aR knockout alleviated the adverse changes.Western blot results showed that P-PDH protein levels in AT1aR^-/--HFD rats decreased compared with WT-HFD rats.6.AT1aR knockout attenuated the imbalance of fatty acid oxidation metabolism in the myocardium of obese ratsWestern blot results indicated that the protein expressions of CD36 and CPT1B in AT1aR^-/--HFD rats were higher than those in WT-HFD rats,and the gene levels of CD36were consistent with this.RT-PCR showed that the gene levels of ACADM,ACOX1,PPARα,PPARδand PGC-1αin WT-HFD rats were decreased,while the gene levels of PPARγwere increased.However,AT1aR gene knockout alleviated these changes.7.AT1aR knockout alleviated the effects of high-fat diet on cardiac mitochondrial dynamic proteinsCompared with WT rats,the expression of mitochondrial fusion protein MFN2decreased,and the expressions of mitochondrial fission proteins FIS1 and DRP1increased significantly in WT-HFD rats,while AT1aR knockout attenuated the effect of high-fat diet on mitochondrial fusion and fission proteins.Moreover,the activity of mitochondrial respiratory chain complex V in AT1aR^-/--HFD rats was higher than that in WT-HFD rats.Conclusion:AT1aR knockout improved cardiac dysfunction induced by high-fat diet by enhancing glucose and fatty acid oxidation,regulating mitochondrial dynamic protein changes and promoting cardiac energy supply.