The Effects Of Cardiac-specific CD36 Inhibition In Obesity-induced Cardiac Remodeling And Its Underlying Mechanism

Background: Cardiac remodeling is divided into physiological cardiac remodeling and pathological cardiac remodeling.The former is caused by non-pathological factors such as exercise and growth.Although the heart is enlarged but the cardiac function is normal.Physiological cardiac remodeling is characterized by decreased ventricular wall stress,increased pumping function and increased microvascular formation,which are beneficial to fine-tuning and coordination of cardiac function.There are many reasons for pathological cardiac remodeling,including myocardial cell energy metabolism disorder,excessive activation of RAAS system,increased pre-and post-load,myocardial ischemia,oxidative stress,endoplasmic reticulum stress,and inflammation.Pathological cardiac remodeling is characterized by structural and functional changes in various heart chambers,especially the left ventricle.Structural changes of left ventricle caused by internal or external pathological factors can ultimately lead to heart failure.At present,drug therapy for cardiac remodeling mainly focuses on inhibiting excessive activation of the RASS system,reducing the anterior and posterior load of the heart,ameliorate myocardial ischemia,and increasing myocardial contractility.Patients with obesity or diabetes are often accompanied by disorders of glycolipid metabolism,myocardial energy metabolism substrate of which is changed,which can lead to metabolic heart remodeling.For metabolic heart remodeling,the energy metabolism pathway of the myocardium may be an effective therapeutic target.Fatty acid(FA)and glucose are the most important energy metabolism substrates for cardiomyocytes.In pathological obesity,the uptake and oxidation rate of FA by cardiomyocytes are significantly increased,which will lead to an increase in the production of various toxic metabolic intermediates.As one of the key enzymes of fatty acid metabolism,CD36 is distributed in the cytoplasm and membrane of cardiomyocytes.Down-regulation of CD36 by RNA interference(RNAi)may reduce the uptake of FA by cardiomyocytes in obese patients,thereby reducing the oxidation rate of FA and ameliorating obese cardiac remodeling.In this study,we gave mice high fat feeding and induced obesity.By intramyocardial injection of CD36 lentivirus,the expression of CD36 was down regulated.We examined the effects of down-regulating CD36 expression on obese cardiac remodeling and investigated its underlying mechanisms,so as to provide new therapeutic strategies for the treatment of metabolic cardiac remodeling.Part I: To study the safety and efficacy of RNAi in mice,and to evaluate the effect of RNAi on cardiac structure and function in obese miceObjective: To study the safety and efficacy of RNAi in mice,and to evaluate the effect of RNAi on cardiac structure and function in obese mice.Methods: Mice were randomly divided into 4 groups: N-CD36,N-mock,O-CD36,O-mock.At 4 weeks of age,mice(O-CD36 group and O-mock group)were fed with high fat diet(HFD)for 12 weeks to induce obesity,mice(N-CD36 group and N-mock group)were fed with control normal diet(CND);at 6 weeks of age,mice were given intramyocardial injection of lentivirus.The body weight,blood glucose,blood lipids,serum biochemical parameters,cardiac function and morphological changes of each group were evaluated at 16 weeks of age.Results:1.In hearts injected with CD36 lentivirus,the m RNA and protein levels of CD36 decreased.2.At 16 weeks of age,the weight of obese mice was significantly higher than that of the control group,and the obese mice had significant cardiac hypertrophy,cardiac mass increased,and remained increased in mass/weight ratio.Echocardiography showed that LVEF and LVFS of obese mice was lower than N-mock group.In the cardiac structure,the LVEDd and LVESd were significantly increased in the O-mock group,and the down-regulation of CD36 ameliorated this change.3.Compared with N-mock group,obese mice had higher levels of ALT,AST,ALP,BUN,creatinine,FSG,TC and triglycerides,and CD36 gene silencing decreaced the levels of serum BUN and creatinine,but did not affect ALT,AST,ALP,FSG,TC or triglyceride concentrations.Conclusion: Injection of lentivirus to cardiac can significantly down-regulate the expression of CD36.and this inhibition does not cause obvious abnormal function on liver or kidney.Cardiac systolic function in obese mice was lower than normal group,but inhibition of CD36 expression attenuated obesity-induced the decrease of left ventricular systolic function.RNAi had no significant adverse effects on the biochemical parameters of mice.Part II: Effects of CD36 gene silencing on myocardial FA accumulation,ROS content and cardiomyocyte apoptosis in obese miceObjective: To investigate the effects of CD36 gene silencing on myocardial FA accumulation,ROS content and cardiomyocyte apoptosis in obese mice.Methods: Mice were grouped and treated as same as Part I.The following studies were performed at 16 weeks of age: 1.Paraffin sections were taken from left ventricle,and neutral lipid content in cardiomyocytes was detected by oil red O staining;2.Left ventricular tissue was taken and HPLC-MS/MS was used to detect the content of ceramide and diglyceride;3.Three different methods were used to detect the content of ROS in cardiomyocytes;4.Paraffin sections were taken from left ventricle tissue,and the apoptosis rate of cardiomyocytes was detected by TUNEL method.Results: Compared with N-mock group,the neutral lipid content and ceramide in the myocardium of the O-mock group were significantly increased,while there was no significant difference in diglyceride.The ROS content was also significantly increased in the O-mock group.The apoptotic rate of O-mock group was higher than the N-mock group.Down-regulation of CD36 expression ameliorated and partially reversed the above abnormalities.Conclusion: CD36 gene silencing reduced the deposition of neutral lipids and ceramide in cardiomyocytes,reduced the ROS content and reduced the number of cardiomyocyte apoptosis.Part III: Effect of CD36 gene silencing on myocardial ultrastructure,endoplasmic reticulum stress,and calcium regulation in cardiomyocytes in obese miceObjective: To investigate the effect of CD36 gene silencing on myocardial ultrastructure,endoplasmic reticulum stress,and calcium regulation in cardiomyocytes in obese miceMethods: Mice were grouped and treated as same as Part I.The following studies were performed at 16 weeks of age: 1.Transmission electron microscopy to observe the ultrastructural changes of myocardial cells,compare the changes of mitochondria number and mitochondrial area ratio;2.Western blot to detect the level of endoplasmic reticulum stress-related protein;3.Compare the changes of myocardial cell calcium regulation related indicators 4.Western blot to detect the expression level of IRS-PI3K-m TOR signaling pathway.Results: Compared with the N-mock group,the number of mitochondria and mitochondrial area in the O-mock group increased.The expression level of endoplasmic reticulum stress-related protein in myocardial tissue of O-mock group was significantly up-regulated.In the O-mock group,the calcium transient amplitude was lower than that in the control group,and the calcium transient 50% recovery time was also prolonged.Down-regulation of CD36 ameliorated and even reversed the above abnormalities.Activation of the IRS-PI3K-m TOR signaling pathway plays an important role in metabolic cardiac remodeling,and down-regulation of CD36 inhibitd activation of this signaling pathway.Conclusion: CD36 gene silencing can ameliorate myocardial ultrastructure,endoplasmic reticulum stress,cardiomyocyte calcium regulation and high expression of IRS-PI3K-m TOR pathway in obese mice.