| BackgroundHeart failure（HF）is a globally prevalent disease,heart failure with preserved ejection fraction（HFpEF）accounted for more than 50%of heart failure.However,the pathogenesis of HFpEF is still unknown and no effective treatment has been found at present.Therefore,the research on HFpEF in this topic has significant clinical value and practical significance.HFpEF is caused by the interaction of aging,hypertension,obesity,diabetes and other co-existing diseases.Its clinical manifestations are complex,and most current HFpEF animal models cannot fully reflect the phenotypic characteristics of HFpEF.A study published in Nature in 2019 found that hypertension drugs+high-fat feed can successfully induce HFpEF formation in mice.This study intends to adopt the reference modification method to successfully construct an HFpEF animal model,laying a foundation for the study of HFpEF-related disease mechanism and treatment methods in this study.Adropin is a peptide hormone secreted by liver,which has been shown to reduce blood lipids,blood glucose and improve insulin resistance,as well as reduce blood pressure and vascular endothelial protection.How does Adropin affect HFpEF?No studies have been done.This study intends to explore the effects of Adropin on HFpEF and its related mechanisms,so as to provide some theoretical reference for the pathogenesis and treatment of HFpEF.Part I Construction of mouse heart failure with preserved ejection fraction modelObjectiveTo construct the heart failure with preserved ejection fraction model of mouse.MethodsMale C57/BL6 mice aged 8 weeks were randomly divided into Con group,HFD group,L-NAME group and HFD+L-NAME group,a total of 4 groups,10 in each group.（1）The mice in Con group ate ordinary feed and drank ordinary tap water.（2）HFD group:60%high fat diet+ordinary water.（3）L-NAME group:ordinary feed+0.5g/L L-NAME（nitric oxide synthase inhibitor）tap water feeding.（4）HFD+L-NAME group:60%fat diet+0.5g/L L-NAME tap water feeding.After 8 weeks of feeding according to the above regimen,the body weight,heart rate,blood pressure,cardiac ultrasonic indicators,blood lipid,blood glucose,NT-prob NP（amino terminal brain natriuretic peptide precursor）of the mice were measured.The left ventricular tissue was stained with HE,WGA,Masson and Oil red,and the lung tissue was stained with HE.The content of Adropin in serum and myocardial tissue of mice was detected.Results（1）Compared with Con group,body weight,heart weight,blood lipid,blood glucose,NT-prob NP,heart rate,systolic blood pressure,diastolic blood pressure,E/A,E/e’,IVS and LVPW in HFD+L-NAME group were significantly increased（P<0.05）,e’/a’and LVID were significantly decreased（P<0.05）,and there was no significant difference between EF and LVFS（P>0.05）.Myocardial histopathological injury score,myocardial cell area,myocardial fibrosis ratio and myocardial fat content increased significantly（P<0.05）,lung lesion score increased（P<0.05）,pulmonary edema and pulmonary congestion were observed.（2）Compared with Con group,only increased body weight,heart weight,blood pressure,blood lipids,LVPW and IVS（P<0.05）in HFD group,while there were no statistical differences in NT-proBNP,E/A,E/e’,e’/a’and LVID（P>0.05）,only mild myocardial injury and fat accumulation are seen（P<0.05）,no significant increase in myocardial fibrosis and no difference in lung lesion score（P>0.05）.（3）Compared with Con group,L-NAME group only increased blood pressure,heart weight,IVS,LVPW（P<0.05）,E/A and LVID were significantly decreased（P<0.05）,but there were no differences in body weight,blood lipid,blood glucose,NT-proBNP,E/e’,e’/a’（P>0.05）,myocardial pathological results showed hypertrophy of myocardial cells,mild myocardial injury,mild fibrosis,mild lung injury,no adipose accumulation in myocardial tissue.（4）Compared with HFD group,NT-proBNP,E/A,E/e’,LVPW and IVS of HFD+L-NAME group increased（P<0.05）,e’/a’and LVID were significantly decreased in HFD+L-NAME group（P<0.05）,HFD+L-NAME group,myocardial pathology also found that myocardial injury was significantly aggravated,myocardial fibrosis increased,myocardial cell area increased,lipid content in myocardial tissue increased,lung injury was aggravated,pulmonary edema and pulmonary congestion were obvious.（5）Compared with L-NAME group,body weight,blood lipid,blood glucose,E/A,E/e’,LVPW and IVS of HFD+L-NAME group increased（P<0.05）,e’/a’decreased（P<0.05）and no difference in LVID（P>0.05）,myocardial injury,myocardial fibrosis,myocardial cell area,myocardial tissue fat content and lung injury were aggravated in mice with myocardial pathology（P<0.05）.According to the diagnostic criteria of HFpEF,we believed that HFpEF was formed in mice in HFD+L-NAME group,while HFpEF was not formed in mice in HFD and L-NAME group.In addition,we also found that the serum and myocardial Adropin content in HFD+L-NAME group was significantly decreased compared with that in Con,HFD and L-NAME groups（P<0.05）.ConclusionsHFpEF formation was induced by high-fat+L-NAME feeding for 8 weeks,while HFpEF formation was not induced by L-NAME alone or high-fat feeding for 8weeks.HFpEF mice in this study showed obesity,hyperlipidemia,hypertension,hyperglycemia,myocardial pathological remodeling,significantly decreased diastolic function,and pulmonary congestion and pulmonary edema.The decrease of Adropin content may be related to the formation of HFpEF.Part II Effect of Adropin on heart failure with preserved ejection fractionObjectiveTo study the effect of Adropin on heart failure with preserved ejection fraction.Methods8-week-old male Adropin gene knockout and normal C57/BL6 mice were randomly divided into Con group,HFD+L-NAME group,HFD+L-NAME+Ad group,HFD+L-NAME+Ad-/-group and HFD+L-NAME+Ad-/-group,with 10mice in each group.（1）Con group:normal mice were fed with normal die and the same volume of saline as Adropin was injected intraperitoneally,once a day.（2）HFD+L-NAME group:normal mice were fed with high-fat+L-NAME diet and intraperitoneal injection of equal normal saline,once a day.（3）HFD+L-NAME+Ad group:normal mice were fed with high-fat+L-NAME diet and intraperitoneal injection of Adropin 450 nmol/kg,once a day.（4）HFD+L-NAME+Ad+Ad-/-group:Ad-/-knockout mice were fed with high-fat+L-NAME diet and intraperitoneal injection of Adropin 450 nmol/kg,once a day.（5）HFD+L-NAME+Ad-/-group:Ad-/-knockout mice were fed with high-fat+L-NAME diet and intraperitoneal injection of equal normal saline,once a day.According to the above experimental scheme,after 8weeks,the heart rate,blood pressure,cardiac ultrasonic indicators,blood lipid,blood glucose,NT-proBNP,serum IL-6 and TNF-α,MDA,SOD,GSH in the myocardial tissue were measured.The left ventricular tissue stained with HE,WGA,Masson,Oil red,DHE and CD31 to observe the pathological changes of myocardium,the content of myocardial ROS and the density of myocardial microvessels.The effects of Adropin on HFpEF mice were comprehensively evaluated by the above indicators.Results（1）Compared with HFD+L-NAME group,in HFD+L-NAME+Ad group,body weight,heart weight,blood lipid,blood glucose,blood pressure,NT-proBNP,serum IL-6,MDA and ROS contents in cardiac tissue of mice were significantly decreased（P<0.05）,SOD and GSH in myocardium tissue and microvessel density were significantly increased（P<0.05）,myocardial hypertrophy,diastolic function,myocardial fibrosis and myocardial injury were significantly improved（P<0.05）,but there were no significant differences in serum TNF-α,EF and LVFS（P>0.05）.（2）Compared with HFD+L-NAME group,in HFD+L-NAME+Ad-/-group,body weight,heart weight,blood lipid,blood glucose,blood pressure,NT-proBNP,serum IL-6 and TNF-α,MDA and ROS contents in cardiac tissue were significantly increased（P<0.05）,SOD and GSH content in myocardium,microvascular density and ROS content in myocardium were significantly decreased（P<0.05）,myocardial hypertrophy,diastolic function,myocardial fibrosis and myocardial injury were significantly worsen（P<0.05）,there was no significant difference in EF and LVFS（P>0.05）.（3）Compared with HFD+L-NAME+Ad-/-group,in HFD+L-NAME+Ad+Ad-/-group,body weight,heart weight,blood lipid,blood glucose,blood pressure,NT-proBNP,serum IL-6,MDA and ROS contents in cardiac tissue of mice were significantly decreased（P<0.05）,SOD and GSH in myocardium tissue and microvessel density were significantly increased（P<0.05）,myocardial hypertrophy,diastolic function,myocardial fibrosis and myocardial injury were significantly improved（P<0.05）,but there were no significant differences in serum TNF-α,EF and LVFS（P>0.05）.ConclusionsExogenous Adropin supplementation can significantly improve the HFpEF of mice,which may be related to improving of blood lipid,blood glucose,blood pressure,inflammatory response,oxidative stress and microvascular injury.Endogenous Adropin deficiency can significantly aggravate the progression of HFpEF,and Adropin protein plays an important regulatory role in the progression of HFpEF.Part III Mechanisms of Adropin in improving heart failure with preserved ejection fractionObjectiveTo study the mechanisms of Adropin in imprving heart failure with preserved ejection fraction.MethodsLentivirus was used to knock down the expression of GPR19 in the myocardium of mice.After successful construction of GPR19 knockdown mice,male 8-week-old C57/BL6 mice with GPR19 knockdown and normal C57/BL6 mice were selected.The rats were randomly divided into control group（Con group）,HFD+L-NAME group,HFD+L-NAME+Ad group,HFD+L-NAME+GPR19 sh RNA group and HFD+L-NAME+GPR19 sh RNA group,with 10 mice in each group.（1）Con group:normal mice were fed with normal die and the same volume of saline as Adropin was injected intraperitoneally,once a day.（2）HFD+L-NAME group:normal mice were fed with high-fat+L-NAME diet and intraperitoneal injection of equal normal saline,once a day.（3）HFD+L-NAME+Ad group:normal mice were fed with high-fat+L-NAME diet and intraperitoneal injection of Adropin 450 nmol/kg,once a day.（4）HFD+L-NAME+Ad+GPR19 sh RNA group:GPR19 knockdown mice,high-fat+L-NAME diet,intraperitoneal injection of Adropin 450 nmol/kg,once a day.（5）HFD+L-NAME+GPR19 sh RNA group:GPR19 knockdown mice,high-fat+L-NAME diet feeding,intraperitoneal injection of normal saline,once a day.According to the above experimental scheme,after 8 weeks,the heart rate,blood pressure,cardiac ultrasonic indicators,blood lipid,blood glucose,NT-proBNP,MDA,SOD,GSH,IL-6,TNF-αcontents in the myocardial tissue of the mice were measured.Left ventricular tissue was stained with HE,WGA,Masson,Oil red,DHE and CD31.Ultrathin sections of some myocardium were made to observe the mitochondrial structure and morphology of myocardium cells by transmission electron microscopy.Partial myocardial tissue was isolated and extracted for myocardial cells.Mitochondrial membrane potential（MMP）of myocardial cells and mitochondrial permeability transition pore（m PTP）opening were detected.Finally,the expression of GPR19-MAPK-PDK4 signaling pathway was detected by PCR and western blotting.Results（1）Comparison between HFD+L-NAME+GPR19 sh RNA group and HFD+L-NAME group,in HFD+L-NAME+GPR19 sh RNA group,body weight,heart weight,blood lipid,blood glucose,blood pressure,NT-proBNP,myocardial MDA,myocardial ROS,TNF-αin myocardial tissue,m PTP openness were significantly increased（P<0.05）,SOD and GSH contents,microvascular density and myocardial MMP in myocardial tissue were significantly decreased（P<0.05）,myocardial hypertrophy,diastolic function,myocardial fibrosis,myocardial pathological injury,and myocardial mitochondrial morphology and structure damage were significantly worsen（P<0.05）,GPR19-MAPK-PDK4 signaling pathway was significantly inhibited.（2）Compared with HFD+L-NAME+Ad group,in HFD+L-NAME+Ad+GPR19 sh RNA group,body weight,heart weight,blood lipid,blood glucose,blood pressure,NT-proBNP,myocardial MDA,myocardial ROS,TNF-αin myocardial tissue,m PTP openness were significantly increased（P<0.05）,SOD and GSH contents,microvascular density and MMP in myocardial tissue were significantly decreased（P<0.05）,myocardial hypertrophy,diastolic function,myocardial fibrosis,myocardial pathological injury,and myocardial mitochondrial morphology and structure damage were significantly worsen（P<0.05）,GPR19-MAPK-PDK4 signaling pathway was significantly inhibited.ConclusionsGPR19 is an important receptor protein of Adropin on cardiomyocytes.Adropin may activate intracellular MAPK-PDK4 signaling pathway by acting on myocardial cell membrane GPR19,thereby improving heart failure with preserved ejection fraction. |
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