The Role And Mechanism Of PGAM2 In Promoting The Transition Of Cardiac Hypertrophy To Heart Failure

Purpose:Cardiovascular disease is the world’s largest cause of death.Heart failure is the final stage of many cardiovascular diseases.Myocardial hypertrophy is an important cause of heart failure,heart disease and death.Under pathological conditions,the metabolism of the heart changes.In the process of myocardial remodeling,the metabolic pathway changes from preferring fatty acid oxidation to glucose oxidation.At this time,the rate of glucose uptake and glycolysis increases.On the basis of our previous study,After screening PGAM2 through gene sequencing,we carried out some preliminary experiments and found that the expression of PGAM2 was related to cardiac hypertrophy.Therefore,we successfully applied for the bid of National Natural Science Foundation of China Project,but we are not clear about its detailed function.We speculate whether PGAM2 can induce heart failure by affecting fatty acid metabolism according to its functional site?Phosphoglycerate mutase(PGAM)2 is a key enzyme in the glycolysis pathway,and plays an important role in coordinating the production of energy and reducing power,as well as the synthesis of nucleotide precursors and amino acids.Overexpression of PGAM2 induced a decrease in cardiac tolerance in mice.This study is intended to combine with clinical practice,using cell and rat models of heart failure,virus transfection,RNA interference and a variety of molecular biological methods were used to clarify the role and specific mechanism of PGAM2 in heart failure,so as to provide a potential new target for the prevention and control of heart failure in clinical science.This experiment is divided into three parts.The first part is the study of PGAM2 gene knockdown to prevent heart failure and improve myocardial energy in model mice.Objective:To detect the effect of PGAM2 knockdown on left ventricular function,cardiac muscle tissue morphology,mitochondrial and energy metabolism in mice with transverse aortic constriction(TAC).Part Ⅱ:PGAM2 induced cardiomyocyte hypertrophy,myocardial metabolic disorder and effects on AMPK/PPARα/GLUT4 pathway.Objective:To explore the effects of knockdown and overexpression of PGAM2 on the morphology and function of cardiac myocytes at the cellular level,and further explore the potential mechanism of PGAM2 affecting cardiac hypertrophy.Part Ⅲ:Study on the relationship between PGAM2 and heart failure patients and diseases.Objective:To analyze the expression of PGAM2 in heart failure patients and determine whether it can be used as a marker to evaluate the severity of heart failure.Method:Part Ⅰ:Study on PGAM2 gene knockdown preventing heart failure and improving myocardial energy in model mice1.The animal experiment strictly complies with the relevant regulations of the Jinan Central Hospital Ethics Committee.The healthy male C57BL/6J mice used in the experiment are from Beijing Weitong Lihua and are raised under standard conditions.All mice were randomly divided into 4 groups,6 in each group:sham operation(Sham)group,TAC group,TAC+sh-NC group,TAC+sh-PGAM2 group.TAC model mice were constructed.Sham group received the same operation,but the aorta was not removed.2.Detect the left ventricular function of mice by echocardiography,measure the length of the tibia,the weight of the heart,the weight of the left ventricle,the weight of the lung and the weight of the body,and calculate the ratio,as an index to evaluate the myocardial hypertrophy of mice.3.Take the heart tissue of mice,and then use it for histological observation and biochemical index detection.The morphology of myocardial tissue and the degree of fibrosis were observed by HE staining and Masson staining.The changes of ANP and BNP levels in myocardial tissue RNA in each group were detected by fluorescence quantitative polymerase chain reaction(RT-PCR).Mitochondria were isolated,the changes of mitochondrial membrane potential were detected by JC-1 method,and the mitochondrial structure was observed by transmission electron microscopy(TEM).ATP,LDH,SDH,NADH in myocardial tissue and serum,.Part Ⅱ:PGAM2 induced cardiomyocyte hypertrophy,myocardial metabolic disorder and its effect on AMPK/PPARα/GLUT4 pathway.1.H9C2 myocardial cells and 293T cells were purchased from Shanghai Institute of Cell Biology,Chinese Academy of Sciences.SiRNA and Ad-PGAM2 were used to inhibit and overexpress PGAM2.Angiotensin(Ang Ⅱ)treatment induced hypertrophy of H9C2 cardiomyocytes.The experiment was divided into interference group and overexpression group,each group was divided into four groups,interference group:A si-control group B:si-PGAM2 group C:si-control+Ang Ⅱ group D:si-PGAM2+AngⅡ group;Overexpression group:A:GFP group B:Ad-PGAM2 group C:GFP+Ang Ⅱgroup D:Ad-PGAM2+Ang Ⅱ group2.Detection of PGAM2 expression in different groups;CCK-8 was used to detect cell viability and determine the effect of PGAM2 on cell viability;The effects of PGAM2 on apoptosis and ROS were further tested to determine the effects of PGAM2 on the level of oxidative stress;The mitochondrial membrane potential and the structure of mitochondria were observed by TEM;Protein extraction,Western blot detection of fatty acid transporter(PPAR)in myocardial cells α)The expression of AMPK,pAMPK,glucose transporter(GLUT4),and the effect of PGAM2 on energy metabolism.Clarify the relationship between PGAM2 and AMPK/PPAR α/The influence of GLUT4 pathway.Part Ⅲ Research on the relationship between PGAM2 and heart failure patients and diseases1.Select heart failure 153 patients from Jinan Central Hospital from October 2016 to February 2018,and select 40 healthy adults as control.The experiment was approved by the Ethics Review Committee of our hospital,and all signed informed consent.Relevant clinical data of patients were collected,including left ventricular ejection fraction,left ventricular end-diastolic diameter,left atrial diameter and other indicators measured by color Doppler ultrasound.2.Collect venous blood and detect PGAM2,NT-proBNP,and Cys-C in serum.3.The correlations of PGAM2 with NT-proBNP,Cys-C and EF values were analyzedResults:PartⅠ:1.PGAM2 is highly expressed in mice with heart failure;After transfection of PGAM2 interfering adenovirus,the expression of PGAM2 decreased,indicating that PGAM2 interfering adenovirus was successfully transfected into mice.2.The results showed that knockdown of PGAM2 expression could reduce the heart weight and body weight of TAC mice,improve the degree of myocardial fibrosis,and restore the left ventricular function.3.RT-qPCR results showed that the contents of ANP、BNP indicators in mice after TAC were significantly increased;After knockdown of PGAM2 gene,its content decreased significantly.4.ELISA results showed that knockdown of PGAM2 expression could reduce the content of LDH in TAC mice.At the same time,it can increase the content of ATP in myocardial tissue and improve myocardial energy.5.knockdown of PGAM2 expression can increase mitochondrial membrane potential,increase the contents of NADH and SDH,improve myocardial fibrosis,and restore mitochondrial structure and function in mice with cardiac hypertrophy.PartⅡ:1.Using Ang Ⅱ to stimulate H9C2 to establish an in vitro model of myocardial hypertrophy,The cell area of Ang Ⅱ group was larger than that of the control group,and the difference was statistically significant.Compared with the control group,the expression of ANF and BNP in myocardial hypertrophy index detected by RT-PCR was up-regulated,and the difference was statistically significant.2.WB and RT-qPCR detected the expression of PGAM2 in cardiac myocytes,and determined the transfection efficiency of si-PGAM2 and Ad-PGAM2.First,the expression of PGAM2 in cardiac myocytes was detected,.Consistent with animal experiments,PGAM2 expression was up-regulated in hypertrophic cardiomyocytes;After transfection of PGAM2 interfering adenovirus,the expression of PGAM2 decreased significantly,indicating that PGAM2 interfering adenovirus was successfully transfected into cardiac myocytes and inhibited the expression of PGAM2;After overexpression of adenovirus,the expression of PGAM2 increased,indicating that the transfection was successful;3.CCK-8 detected cell viability,and PGAM2 knockdown increased the viability of myocardial cells;On the contrary,the overexpression of PGAM2 significantly decreased the viability of myocardial cells.These results showed that PGAM2 could lead to the decrease of myocardial cell activity and damage.4.Effect of PGAM2 on Ang Ⅱ stimulated cardiomyocyte apoptosis.TUNEL was used to detect cardiomyocyte apoptosis.PGAM2 knockdown improved cardiomyocyte apoptosis;On the contrary,the overexpression of PGAM2 increased the degree of cardiomyocyte apoptosis.In conclusion,PGAM2 can induce cardiomyocyte apoptosis and cause cardiomyocyte damage.5.Effect of PGAM2 on Ang Ⅱ stimulated ROS in cardiac myocytes.The expression of ROS in cardiac myocytes was detected.The knockdown of PGAM2 improved ROS expression in cardiac myocytes;On the contrary,the overexpression of PGAM2 significantly increased ROS.In conclusion,excessive PGAM2 can induce oxidative stress of myocardial cells and cause myocardial cell damage.6.Effects of PGAM2 on cardiomyocyte mitochondria:knockdown of PGAM2 increased the red/green fluorescence ratio and mitochondrial membrane potential.Mitochondrial vacuolization was reduced,mitochondrial inner membrane swelling was less,and mitochondrial structure was recovered.Taken together,the expression of PGAM2 destroys mitochondrial structure,affects mitochondrial function,alters mitochondrial energy metabolism,and ultimately leads to cardiomyocyte injury.7.Effect of PGAM2 on the AMPK/PPARα/GLUT4 pathway,:The expressions of AMPK and PPARa in H9C2 cardiomyocytes were decreased,and the expression of GLUT4 was increased after Ang Ⅱ treatment;Compared with Ang Ⅱ+sh-NC,PGAM2 knockdown increased the expression of AMPK and PPARα,and decreased the expression of GLUT4;On the contrary,compared with Ang Ⅱ+GFP,PGAM2 overexpression decreased the expression of AMPK and PPARα,and increased the expression of GLUT4;In conclusion,PGAM2 may regulate AMPK/PPAR α/GLUT4 pathway,which affects the energy metabolism of myocardial cells,promotes apoptosis and oxidative stress.PartⅢ:1.The expression of PGAM2 in patients with heart failure was higher than that in the control group.The ROC area of PGAM2 in the diagnosis of heart failure was 0.8688(P<0.0001).There was significant difference between class Ⅱ and class Ⅳ,and between class Ⅲ and class Ⅳ,but there was no significant difference between class Ⅱ and class Ⅲ.2.Patients with heart failure had lower left ventricular ejection fraction and higher left ventricular diameter and left atrial diameter.The levels of NT-proBNP and Cys-C increased in patients with heart failure.The ROC area of NT-proBNP and CysC in the diagnosis of heart failure was 0.9789 and 0.9002,respectively(P<0.0001);There were significant differences between class Ⅱ and class Ⅲ,class Ⅲ and classⅣ,class Ⅱ and class Ⅳ.3.PGAM2 expression was positively correlated with NTproBNP.Conclusion:Based on all the experimental results,we found for the first time that PGAM2 is involved in cell apoptosis,oxidative stress,mitochondrial metabolism,energy metabolism and fatty acid metabolism,and passed AMPK/PPAR α/GLUT4 pathway can induce cardiomyocyte hypertrophy and heart failure.At the same time,we collected clinical samples to prove again that PGAM2 is involved in the process of heart failure.The above research results will further improve the theoretical system of the molecular mechanism of heart failure,provide new ideas for the clinical treatment of heart failure,and find new therapeutic targets and biomarkers.