The Role And Mechanism Of PKM2 In Pathological Cardiac Remodeling

Background:Heart failure（HF）,an end‐stage manifestation of many cardiac diseases,has gradually become a major public health challenge with the development of economy and society.The number of patients is now estimated more than 60 million globally.Due to the fast pace of modern lifestyle,high intensity of competitive pressure,as well as the large population of hypertension,diabetes and other diseases,the number of patients suffering from HF in China has exceeded 13 million,and this severe trend is difficult to alleviate in a short time.Although there has been some progress in lowering the mortality of HF patients with guideline-recommended medical therapy,the socio-economic burden of rehospitalization,incapacity and complications is still significant.Pathologic cardiac remodeling is the fundamental process underlying the development of HF.The pathogenesis is complex and diverse,involving cell dysfunction,inflammation,metabolic abnormalities and physical or chemical damage.Cardiomyocytes are the functional units of cardiac systolic function,and the weakening of cardiac function is essentially caused by the massive loss or impairment of cardiomyocytes.Previous studies have revealed various mechanisms of pathological cardiac remodeling and proposed corresponding therapeutic strategies.However,our understanding is still insufficient,and we still face great challenges in the prevention and treatment of HF.Therefore,revealing the novel key molecular mechanism is still an urgently need to protect heart against pathological cardiac remodeling.Pyruvate kinase（PK）is the key enzyme responsible for converting ADP and phosphoenolpyruvate（PEP）to ATP and pyruvate at the final step of glycolysis.In mammals,Pk is encoded by two genes（Pklr and Pkm）,and Pkm is expressed predominantly in the heart.PKM1 and PKM2,two alternative-splicing isoforms of the Pkm gene,differ significantly in their protein structure and biochemical functions.Different from the constitutive pyruvate kinase activity of PKM1,PKM2 may function as a protein kinase or transcription cofactor.It has been demonstrated that PKM2 is involved in the regulation of cell death,cell cycle,differentiation,autophagy and immune response independently of the pyruvate kinase activity.To date,PKM2 has been associated with various human diseases such as neurodegenerative diseases,cancer,dysplasia,inflammation,and infections.Therefore,PKM2 is regarded as a potential therapeutic target in related diseases.Although PKM2 expresses low in normal adult hearts,studies have shown that PKM2 expression was markedly upregulated in failing hearts.Research on the role of PKM2 in HF is still lacking.To elucidate the role and underlying mechanisms of PKM2 in the pathologic cardiac remodeling,we carried the study in vitro and in vivo.Downstream molecular targets were screened by mass spectrometry,and systematically verified by inhibitors and virus overexpression.Objectives:1 To determine the role of cardiomyocytic PKM2 in pathological cardiac remodeling.2 To identify the mechanism of pathological cardiac remodeling due to PKM2deficiency in cardiomyocytes.3 To establish the potential of PKM2 as an intervention target for pathological cardiac remodeling.Methods:1 The Pkm2 in isolated and purified primary neonatal rat cardiomyocytes（NRCMs）was knocked down by specific si RNA.Then,the NRCMs were treated with phenylephrine（PE）.The real-time fluorescence quantitative polymerase chain reaction（q RT-PCR）,immunofluorescence and western blot（WB）were applied to validate the effect of PKM2 deficiency on pathological cardiac remodeling.2 PKM2 cardiomyocyte specific knockout（Pkm2 c KO）mice were constructed by crossing Pkm2^f/fmice with Myh6-Cre mice.Embryonic development and survival were examined when loss of PKM2.3 Pkm2^f/f and Pkm2 c KO mice were divided into sham or transverse aortic constriction（TAC）operation groups randomly.Next,the effects due to PKM2deficiency on pathological cardiac remodeling and HF were assessed by echocardiography,stereomicroscopy,picrosirius red（PSR）,hematoxylin-eosin（HE）and fluorescent wheat germ agglutinin（WGA）staining.4 After NRCMs were transfected with Pkm2 si RNA,the pyruvate kinase activity was measured by detecting pyruvate production.5 By employing an anti-PKM2 antibody-conjugated agarose assistant immunoprecipitation mass spectrometry（IP-MS）,we tentatively selected the potential target of PKM2 in regulating pathological cardiac remodeling for further analysis.Immunoprecipitation（IP）and immunofluorescence were carried out to validate the relationship between PKM2 and Rac family small GTPase 1（RAC1）.6 By in vitro kinase activity assay and cycloheximide（CHX）chase assay,the regulation mechanism of PKM2 on its target RAC1 was identified.7 After NRCMs were transfected with Pkm2 si RNA,WB was used to investigate whether the RAC1 and the downstream MAPKs were activated.8 After TAC,WB was used to detect whether RAC1 and the downstream mitogen-activated protein kinases（MAPKs）were activated.9 After specific RAC1 inhibitor NSC23766 or si-Rac1 sequence were applied to Pkm2 knockdown NRCMs,WB was used to test the protein expression ofβ-MHC,PKM2 and RAC1.10 After TAC,the effects of NSC23766 on pathological cardiac remodeling due to PKM2 deficiency were investigated by the echocardiography,stereomicroscopy,PSR,HE and WGA staining.11 An adeno-associated virus overexpressing PKM2（AAV9-c Tn T-m Pkm2）was administered to the mice via tail vein injection.AAV9-c Tn T-null was used as a control.After TAC,the protective effects of PKM2 overexpression on pathological cardiac remodeling were assessed by the echocardiography,stereomicroscopy,PSR,HE and WGA staining.Results:1 Knockdown of Pkm2 resulted in a potent cardiohypertrophic response,as reflected by elevated expression of cardiac hypertrophic markers,which was further exacerbated after PE treatment.Concomitantly,Pkm2 deficient cardiomyocytes displayed larger cell surface area and were further enlarged upon PE stimulation.2 Pkm2 c KO mice did not manifest overt developmental and morphological defects in hearts at baseline.3 Cardiomyocyte specific knockout of PKM2 exacerbated pathological cardiac remodeling and HF after TAC.4 In NRCMs,specific si RNA targeting Pkm2 did not affect the expression of PKM1.Meanwhile,pyruvate kinase activity remained unchanged between control and Pkm2 knockdown groups.5 By combining our IP-MS data with related studies,we selected RAC1 as a potential target of PKM2 in regulating pathological cardiac remodeling for further analysis.6 Kinase activity assay and coomassie brilliant blue staining suggested that PKM2was able to phosphorylate RAC1.Moreover,Pkm2 knockdown caused a significant decrease in phosphorylated RAC1 at Serine 71（p-RAC1（S71））in NRCMs.The decrease of RAC1 phosphorylation level enhanced the stability of RAC1 protein and delayed its degradation,which was confirmed by the CHX chase assay showing that the half-life of the RAC1 protein in Pkm2 knockdown NRCMs was significantly increased.7 In NRCMs,WB results revealed that phosphorylation MAPKs were activated in response to Pkm2 knockdown.8 The RAC1 and phosphorylation MAPKs displayed similar expression level in Pkm2^f/f and Pkm2 c KO mice at baseline,while significantly increased in Pkm2 c KO mice compared with controls after TAC.9 In NRCMs,NSC23766 significantly reduced Pkm2 knockdown-inducedβ-MHC expression.Similarly,Rac1 knockdown also inhibitedβ-MHC expression in Pkm2deficient NRCMs.10 NSC23766 drastically ameliorated pressure overload-induced pathological cardiac remodeling in Pkm2 c KO-TAC mice.11 Maintaining a high PKM2 expression can protect the heart against pressure overload-induced cardiac remodeling.Conclusion:Here,we have proved that PKM2 exerted cardioprotective effects against pressure overload-induced cardiac remodeling through RAC1-MAPKs axis both in vivo and in vitro.PKM2 deficiency exacerbated the progression of cardiac remodeling.Conversely,maintaining a high PKM2 expression can delay the process,which highlighted PKM2as a prospective target in cardiac remodeling and HF intervention.