Effects And Mechanism Of HIPK2 In Cardiac Remodeling

Objectives:Cardiovascular disease(CVD)is a global epidemic that threatens human health and the leading cause of human disease-associated death worldwide.Heart failure(HF)is a common endstage of many CVDs.Pathological ventricular remodeling is the pathological basis of the occurrence and development of HF.Therefore,exploring the key molecular mechanisms of pathological ventricular remodeling is of great significance to find novel threapeutic targets for preventing and treating heart failure.Homologous domain interacting protein kinase 2(HIPK2),which belongs to the serine/threonine kinase family,contains a conserved protein kinase domain.HIPK2 is involved in the regulation of multiple biological processes,including cell proliferation,apoptosis and DNA damage response through its kinase,transcription co-factor and signal transducer activity.HIPK2 was previously reported to be a target of micro RNA-222,which is a key molecule that mediates exercise-induced cardiac protection effect.Recent studies showed that HIPK2 maintains essential cardiac function through phosphorylating extracellular signal–regulated kinase 1/2(ERK1/2).More recently,HIPK2 was found to be upregulated by exercise training and downregulated by myocardial infarction.HIPK2 inhibiton is able to protect cardiomyocytes against apoptosis and alleviate MI-induced cardiac injury.However,the function and mechanism of HIPK2 in pathological ventricular remodeling and heart failure are still not fully understood.This thesis aimed to explore the role and molecular mechanism of HIPK2 in pathological ventricular remodeling and heart failure at both the cellular level and animal level.This study was designed to examine the expression of HIPK2 in two main cell types in the heart including cardiomyocytes and cardiac fibroblasts and to investigate the function and molecular mechanism of HIPK2 in phatholgocical cardiac hypertrophy and myocardial fibrosis,to study the protective effect of HIPK2 inhibiton in treating pathological ventricular remodeling and cardiac dysfunction,and finally to explore the efficacy of HIPK2 inhibitor PKI1 H delivered by platelet biomimetic liposomes for targeting and repairing cardiac injury in vivo,in order to find the key target of inhibiting ventricular remodeling,explore the new strategy of preventing and treating ventricular remodeling,so as to provide ideas for the prevention and treatment of cardiovascular diseases.Methods:(1)Reverse transcription-quantitative PCR(RT-q PCR)was used to measure HIPK2 expression in phenylephrine(PE)-induced cardiomyocyte pathological hypertrophy model,transforming growth factor beta 1(TGF-β1)-induced cardiac fibroblast fibrosis model,and transverse aortic constriction(TAC)-induced murine cardiac hypertrophy model.(2)The role of HIPK2 in pathological cardiac hypertrophy and fibrosis was investigated in PE-induced neonatal rat cardiomyocytes(NRCMs)hypertrophy model,PE or isoprenaline(ISO)-stressed human embryonic stem cell-derived cardiomyocyte hypertrophy model,and TGF-β1-induced neonatal rat cardiac fibroblast(NRCFs)fibrosis model,through downregulating HIPK2 by short hairpin(sh RNA),overexpressing HIPK2,and inhibiting HIPK2 kinase activity by HIPK2-specific inhibitor.The size of cardiomyocytes was detected by α-actinin fluorescente staining.RT-q PCR was used to detect the expression of pathological hypertrophy markers ANP and BNP.Flow cytometry was used to analyze α-SMA labelled myofirbroblasts.Costaining of α-SMA and Ed U was used to elvaluate cardiac fibroblast activation and proliferation.(3)To verify the role of HIPK2 in vivo,experiments of gene knockout mice(HIPK2 KO)were performed to measure the loss of corresponding functions in TACinduced pathological ventricular remodeling model.Echocardiography was used to determine cardiac function.Masson’s trichrome staining was used to analyze the effect of HIPK2 deficiency in TAC-induced cardiac fibrosis.Hematoxylin-eosin(HE)staining and wheat germ agglutinin(WGA)staining were used to analyze the effect of HIPK2 deficiency in TAC-induced cardiomyocyte hypertrophy.Furthermore,whether HIPK2 affects cardiomyocyte hypertrophy through the ERK1/2-c AMP-response element(CREB)signaling pathway and downstream C-type lectin domain family 4Member D(CLEC4D)and early growth response 3(EGR3)in NRCMs,and whether HIPK2 affects fibrosis through the TGF-β1/Smad3 signaling pathway were analyzed in NRCMs and NRCFs,respectively.(4)HIPK2 inhibitor PKI1 H was encapsulated in lipsome nanoparticle,which was further fused to platelets-derived membranes.This platelet biomimetic liposome delivery system encapsulated PKI1 H could target and repair cardiac injury.Results:(1)The expression of HIPK2 was increased in NRCMs hypertrophic model,NRCFs fibrosis model,and murine pathological ventricular remodeling model.(2)HIPK2 inhibition by either sh RNA or HIPK2 inhibitors t BID and PKI1 H alleviated PE-induced cardiomyocyte hypertrophy in NRCMs and reduced PE/ISOinduced cardiomyocyte hypertrophy in h ESC-derived cardiomyocytes and protected against cardiac fibrosis induced by TGF-β1 in NRCFs.(3)HIPK2 depletion attenuated the decline of cardiac function in TAC-induced pathological ventricular remodeling,and reduced cardiac hypertrophy,cardiac fibrosis,the expression of hypertrophy as well as fibrosis marker genes.Mechanism study found that in PE-induced NRCMs hypertrophy model,HIPK2 regulated cardiomyocyte hypertrophy by affecting downstream CLEC4 D and EGR3 through ERK1/2-CREB signaling pathway;CLEC4D overexpression or EGR3 overexpression reversed the protective effect of HIPK2 inhibition.In TGF-β1 induced NRCFs fibrosis model,exogenous overexpression of HIPK2 increased while inhibition of HIPK2 reduced the phosphorylation level of Smad3;Smad3 agonist(Alantolactone)reversed the protective effect of HIPK2 inhibiton against cardiac fibrosis,indicating that inhibition of HIPK2 protected against cardiac fibrosis through TGF-β1/Smad3 pathway.(4)Finally,at the animal level,platelet biomimetic liposomes delivery system packaging PKI1 H was efficient to target and repair the damaged heart.Conclusion:This thesis study demonstrates that inhibition of HIPK2 protects against pathological vetricular remodeling and heart failure.It also reveals the mechanism of action: HIPK2 enhances cardiomyocyte pathological hypertrophy by activating downstream CLEC4 D and EGR3 through the ERK1/2-CREB signaling pathway and then induces cardiac fibrosis through the TGF-β1/Smad3 pathway.Furthermore,we developed a therapeutic strategy to treat cardiac injury: platelet biomimetic liposome encapsulated PKI1 H can target and repair the damaged heart.In summary,inhibition of HIPK2 is a potential strategy for preventing and treating pathological ventricular remodeling and heart failure.