The Role And Mechanism Of PFKFB3-mediated Glycolysis In Myocardial Fibrosis

Background: Heart failure is still the main cause of morbidity and mortality.There are nearly 24 million patients worldwide,and the mortality rate in the past five years has reached about 50%.Myocardial fibrosis plays an important role in the pathogenesis and progression of heart failure.Glycolysis has been well studied in heart failure and myocardial infarction,but its role in myocardial fibrosis is rarely studied.Recent studies have shown that glycolysis can promote the activation of cardiac fibroblasts and myocardial fibrosis,but the role of key proteases regulating glycolysis in myocardial fibrosis has not been reported.Fructose-2,6-bisphosphatase(Fru-2,6-BP),as a metabolite of Fructose-2-kinase 6-phosphate/fructose-2,6-bisphosphatase 3(PFKFB3),is known to regulate the expression of phosphofructokinase-1(PFK-1)during glycolysis,which is an important rate-limiting enzyme.At present,studies have shown that PFKFB3 promoted the activation of liver and lung fibroblasts and the proliferation of pulmonary artery smooth muscle cells,but there is no study about the role of PFKFB3 in myocardial fibrosis.Therefore,this study aims to clarify the role and mechanism of PFKFB3 in myocardial fibrosis from the perspective of glycolysis,so as to provide new strategies and methods for the treatment of myocardial fibrosis and heart failure in clinical practice.Objective: 1.To analyze the changes of glycolytic metabolism in cardiac fibroblasts during differentiation from the perspective of single cell RNA sequencing.2.To clarify the role of PFKFB3 in myocardial fibrosis;3.To determine the effect of PFKFB3 on the proliferation,differentiation and migration of cardiac fibroblasts;To explore the effect of PFKFB3 protein on macrophage inflammation and migration.4.To explore the correlation between plasma PFKFB3 level and myocardial fibrosis in patients with chronic heart failure(CHF),and to predict the relationship between plasma PFKFB3 level and adverse cardiovascular composite end point events in patients with CHF.Methods:1.Single-cell RNA sequencing bioinformatics analysis: Annotating cardiac fibroblast subclusters and analyzing changes in glycolytic pathways at the metabolic level in cardiac fibroblast subsets(see Part I).2.Animal experiments:(1)Construct animal models;(2)To clarify the role of PFKFB3 in myocardial fibrosis(see Part II).3.Cell experiments:(1)To clarify the effect of PFKFB3 on the differentiation,proliferation and migration of cardiac fibroblasts,and to confirm the mechanism of 3-(3-pyridyl)-1-(4-pyridyl)-2-propen-1-ketone(3PO)regulating the function of cardiac fibroblasts;(2)The apoptosis of cardiac fibroblasts and the extracellular release of PFKFB3 protein were detected.(3)To observe the effect of PFKFB3 recombinant protein on cardiac fibroblasts differentiation and the activation and migration of macrophages(see Part II).4.Clinical experiment: patients with heart failure and control group were enrolled in our hospital.The difference of plasma PFKFB3 concentration between the two groups was compared.We evaluated the diagnostic efficacy of PFKFB3 for myocardial fibrosis and the value in predicting the occurrence of cardiovascular composite end point events(see Part III).Results:1.Results of single-cell RNA sequencing bioinformatics analysis: Metabolic pathway analysis showed that glycolytic pathway was mainly concentrated in new rat cardiac myofibroblasts(myo CF).2.Results of animal experiments: PFKFB3 protein was significantly increased in fibrotic myocardial tissues,and was mainly expressed in fibrotic regions.Inhibition of PFKFB3 expression by 3PO or genetic knockout could improve cardiac function and fibrosis in mice induced by ISO and LAD ligation.3PO attenuated macrophage infiltration and inflammation response in fibrotic myocardial tissue areas.3.Results of cell experiments: PFKFB3 promoted the differentiation,proliferation and migration of cardiac fibroblasts.3PO could regulate the function of cardiac fibroblasts through TGF-β1/ HIF-1α /PFKFB3/AKT signaling pathway.PFKFB3 protein could be released from intracellular to extracellular in the form of apoptosis.PFKFB3 recombinant protein induced the activation of cardiac fibroblasts,promoted the secretion of inflammatory cytokines by M0 and M1 macrophages,and promoted the migration of M0 macrophages.4.Results of clinical experiment: Compared with the control group,the plasma PFKFB3 level in the HF group was significantly increased,which was positively correlated with the ratio of myocardial fibrosis.PFKFB3 was a risk factor for cardiovascular composite end point events in patients with CHF.Conclusions:1.From the perspective of single-cell RNA sequencing,it was found for the first time that the metabolic pathway of cardiac myofibroblasts mainly concentrated at the glycolysis level.Transforming growth factor-β1(TGF-β1)up-regulated Pfkfb3 m RNA in new rat cardiac fibrobalsts.2.PFKFB3 protein was up-regulated in fibrotic myocardial tissues of mice,and mainly expressed in fibrotic regions.The level of PFKFB3 protein in new rat myofibroblasts was significantly higher than that in cardiomyocytes.3.Inhibition of PFKFB3 by 3PO or genetic method could both improve the degree of myocardial fibrosis and left ventricular function in mice,indicating that PFKFB3 played an important role in myocardial fibrosis and could be used as a target for the treatment of myocardial fibrosis.4.Inhibition of PFKFB3 by 3PO or si RNA attenuated the ability of differentiation,proliferation and migration of cardiac fibroblasts,and significantly reduced the level of lactate produced in glycolysis.3PO inhibited extracellular glucose uptake by cardiac fibroblasts.Overexpression of Pfkfb3 by adenovirus transfection promoted the differentiation of cardiac fibroblasts.3PO regulated the function of cardiac fibroblasts through TGF-β1/ HIF-1α /PFKFB3/AKT signaling pathway.PFKFB3-mediated glycolysis plays a vital role in the differentiation,proliferation and migration of cardiac fibroblasts.5.TGF-β1 induced the release of PFKFB3 protein from myofibroblasts.PFKFB3 mediated cardiac fibroblasts differentiation and promoted the activation and migration of macrophages.3PO reduced the infiltration of macrophages and inflammatory response in fibrotic myocardial tissue by inhibiting PFKFB3.6.PFKFB3 was significantly increased in plasma of CHF patients,and its level was positively correlated with the myocardial fibrosis ratio measured by cardiac nuclear magnetic resonance(CMR).PFKFB3 is a predictor of cardiovascular composite end point events in CHF patients.