The Effect Of PKM1 In Pressure Overload Induced Cardiac Remodeling And Heart Failure,and Its Mechanism Study

Heart failure,a life threatening chronic disease with yearly increasing morbidity,is a leading cause of death worldwide and has posed a tremendous burden on the healthcare and social economic systems.Heart failure is a clinical chronic syndrome caused by different etiologies,in which the heart’s pump or fill function is impaired and can not pump enough blood to meet the body’s physiological requirements.Despite modern biotechnology improvements and increasing new drugs as well as advanced patient care,our understanding of the disease remains incomplete and heart failure is still one of the major reasons of mortality globally.As a result,to further explore the underlying mechanism of the disease and search for a new effective therapeutic target is of great urgency.Cardiac remodeling,including myocardial structure remodeling,metabolic remodeling and electrophysiological remodeling,is one of the most important features of heart failure.Growing evidence indicates that metabolic remodeling precedes most,if not all,other pathological alterations and likely plays an essential role in heart failure.Metabolic remodeling is characterized by myocardial metabolic substrate preference switch from fatty acid to glucose under pathological conditions like pressure overload,where glycolysis and glucose utilization is increased while fatty acid usage remains unchanged or decreased.Although mounting evidence implies metabolic remodeling exerts an important role in heart failure,the exact mechanism of metabolic remodeling,especially glycolysis,in the regulation of heart failure remains to be fully elucidated.As the last physiologically irreversible step in glycolysis,PKM1(Pyruvate kinase isozyme M1)converts phosphoenolpyruvate(PEP)to pyruvate by transferring a phosphate group to ADP.Pyruvate then can either be reduced to lactate by LDHA in the cytosol or enter the mitochondria through MPC(mitochondria pyruvate carrier)to be further oxidized for ATP production,which makes pyruvate a key intermediate metabolite that couples glycolysis and glucose oxidative phosphorylation.Therefore,PKM1 is a crucial regulator of both glycolysis and glucose oxidation.Here,we set to dissect the role of PKM1 and its potential mechanism in the development of cardiac remodeling and heart failure under pressure overload.Firstly,heart failure in C57BL/6 mice is induced by TAC(transverse aortic constriction)surgery,and metabolic changes of these failing hearts are measured by unbiased metabolomics.The result shows that glucose metabolism related pathways like Warburg effect and glycolysis are among the most significantly modulated pathways by heart failure compared to the control condition.Given its critical role in glycolysis,we then focus on the expression and enzymatic activity changes of PKM1 in these failing hearts.We find that both expression level and enzymatic activity of PKM1 are reduced in heart failure samples compared with control ones.Next,cardiac specific PKM1 knockout mice are applied to investigate the role of PKM1 in the development of heart failure.In response to TAC surgery,the c KO mice show significant decrease of heart function and progress to heart failure within one week.To further explore the underlying mechanisms,we implement ex vivo heart perfusion 13C-NMR isotopomer analysis and find that cardiac deficiency of PKM1 results in substantially lower glycolysis flux rate and decreased utilization of glucose,pyruvate as well as lactate after TAC.Also,OCR(oxygen consumption rate)and ECT(electron transport chain)related proteins,including NDUFB8 and SDHB,are shown to be reduced in c KO mice after pressure overload.These findings suggest that PKM1 plays an essential role in maintaining a homeostatic response to cardiac hemodynamic stress by governing cardiac glucose metabolism,and might be a potential therapeutic target for heart failure.Part one: Characterization of metabolic remodeling and change of PKM1’s expression and enzymatic activity in mouse heart failureObjective: To observe the metabolic changes,and to detect the expression levels and enzymatic activity of key glycolytic enzyme PKM1 in pressure overload induced heart failure.Methods and results: Heart failure was achieved by TAC surgery in C57BL/6 mice.After confirmation of cardiac dysfunction by echo,failing heart samples were collected and heart weight,lung weight,body weight along with tibia length were recorded.Compared with the sham mice,TAC mice showed remarkably decreased heart function,and significantly increased heart weight body weight ratio,heart weight tibia length ratio and lung weight body weight ratio.Through unbiased metabolomics screening,we found that glucose metabolism related metabolic pathways such as warburg effect and glycolysis are among the most significantly modulated pathways by heart failure compared to the control condition.Results from real-time PCR,western blot together with immunofluorescence staining showed glycolytic enzyme PKM1 were substantially reduced in mice failing heart.Constantly,pyruvate kinase activity measured by lactate dehydrogenase-coupled assay was critically downregulated in mouse heart failure samples.Conclusions: In hemodynamic stress induced heart failure,the heart underwent tremendous metabolic remodeling,with warburg effect and glycolysis being the top modulated metabolic pathways.In addition,the expression and enzymatic activity of glycolysis ratelimiting enzyme PKM1 were decreased.Part two: The regulation of pressure overload induced heart failure by PKM1Objective: To investigate the role of PKM1 in the development of hemodynamic stress caused heart failure.Methods and results: Cardiac specific deletion of PKM1 was achieved by crossing the foxed PKM1 mouse model with the cardiomyocyte-restricted α-MHC Cre transgenic mice.Mouse genotype and PKM1’s knockout efficiency and specificity were confirmed by agarose gel electrophoresis and western blot,respectively.No developmental defects or baseline cardiac dysfunction was observed in PKM1 c KO mice.One week after TAC,echo results revealed that cardiac function was remarkably decreased in the c KO group compared with the control group.Heart tissues were harvested 10 days after TAC,and heart weight,lung weight,body weight along with tibia length were recorded.Our results displayed a significant increase of heart weight body weight ratio,lung weight body weight ratio in c KO mice.The expression of heart failure markers and cardiac fibrosis markers were highly increased in c KO hearts,confirmed by real-time PCR and western blot.Constantly,masson’s trichrome staining and H&E staining showed that the c KO hearts underwent dramatic cardiac remodeling with more cardiac fibrosis compared to the control hearts.Conclusions: Cardiac specific loss of PKM1 does not cause developmental defects or baseline cardiac dysfunction,however,it will deteriorate cardiac dysfunction and result in heart failure in response to hemodynamic stress.Part three: Mechanism study of cardiac specific PKM1 deficiency induced heart failure under pressure overloadObjective: To explore the underlying mechanism of cardiac specific PKM1 deficiency induced heart failure under pressure overload.Methods and results: TAC surgery was applied to provoke hemodynamic stress in mice.3 days after TAC,the hearts were isolated and perfused with isotopomer labeled substrates for 30 mins at 37℃.The perfusate before and after perfusion was collected for OCR measurement,and the heart tissues were freeze-clamped after perfusion and stored in-80℃ for further 13 CNMR analysis.Our results showed that cardiac PKM1 deficiency resulted in greatly lower OCR and utilization of glucose,pyruvate and lactate after TAC.Another cohort that underwent 3 days of TAC was used for cardiac PDH activity measurement.The analysis result revealed that cardiac PDH activity was significantly lower in c KO hearts after TAC.Western blot was used to detect the inhibitory phosphorylation levels of PDH and mitochondrial ETC related proteins,and found that PDH inhibitory phosphorylation levels were highly upregulated while complex I component NDUFB8 along with complex II component SDHB were strikingly reduced in PKM1 deficient hearts.Conclusions: Cardiac specific loss of PKM1 results in suppression of glycolysis and glucose oxidation in response to pressure overload.PKM1 is required to maintain a homeostatic response to hemodynamic stress in the heart by regulating glucose metabolism.