| Background and aims: Dilated Cardiomyopathy(DCM)is one of the most common cardiac disease found worldwide,where the heart primarily displayed ventricular dilatation with deteriorated myocardial contraction.Clinically,patients with DCM often develop complications such as progressive congestive heart failure,thromboembolism,and arrhythmia,thus contribute heavily to high mortality rates.Although DCM has been heavily researched for the past century,there is still no effective treatments available to date.Current literature proposes DCM as the resultant consequences of known cardiac complications such as ischemic injury and familial genetic defects,while the exact cause of DCM remains a mystery due to unclear underlying regulatory pathways illustrated for heart development and functions.Among all the mammalian signaling pathways dissected thus far,Hippo signaling pathway is found as a relatively evolutionary conserved signaling pathway,with transcriptional enhancer factor TEF-1(TEAD1)as its crucial downstream transcription factor.When TEAD1 is combined with multiple transcriptional coactivators,different complexes can be formed to regulate expression of the genes such as those involved in embryonic development,heart development,and muscle formation.In parallel,current literature suggests that TEAD1 and its upstream regulatory factors can indeed play crucial roles in embryonic and perinatal cardiomyocyte proliferation,while some recent studies have highlighted the roles of Hippo-TEAD1 signaling pathways in cardiomyocyte regeneration after injury.Despite that,the roles of TEAD1 in adult mammalian heart and its specific underlying mechanism remains vague.Thus,this article aims to illustrate the regulatory roles of TEAD1 by identifying the downstream mechanisms implicated in adult heart,in order to provide novel targets and alternative therapeutic strategies for the prevention and the treatment of heart diseases.Methods: 1.Phenotypic and cellular mechanistic studies in TEAD1 inducible global knockout(TEAD1ig KO)and cardiomyocyte-specific knockout(TEAD1ic KO)adult mice(Part I and Part II): 1)Generation of TEAD1 ig KO and control genotypic mice through crossbreeding CAGG promoter driven Cre-ER TM+ mice with TEAD1 F/F mice;Generation of TEAD1 ic KO and control genotypic mice through crossbreeding ?-MHC promoter driven Mer Cre Mer +mice with TEAD1 F/F mice.2)After genotype verification,8-12 weeks old adult mice were given TAM(25mg TAM /Kg body weight)through intraperitoneal injection for 5 continuous days.3)VEVO770 to detect changes in echocardiographic parameters such as LVID;d,LVID;s,EF,FS etc.in TEAD knockout mice and control mice.4)Various organs from TEAD1 ig KO mice and control mice were also harvested.The weight of the organs were measured independently and compared against tibia length of each respective mouse.Analysis of differences in weight to tibia length ratio will indicate phenotypes triggered by TEAD1 knockout.5)Mouse hearts were collected,fixed,and paraffin embedded.Mouse heart slices were obtained at the thickness of 8?m.Histological staining such as HE staining was used to examine cardiac structure,while the degree of fibrosis was examined using Masson's Trichrome Staining.6)Mouse hearts were also collected,fixed and OCT embedded.Slices of mouse heart were obtained at the thickness of 7?m,and subjected to immunofluorescence(IF)staining to analyze MAC2 expression of mouse cardiac tissue.Additional staining using Evan's Blue Dye(EBD)and TUNELwere also used to detect cardiomyocyte membrane integrity and apoptosis respectively.7)Differences in m RNA expression of heart failure markers in adult TEAD1 ig KO / TEAD1 ic KO mice and control mice heart were detected by q RT-PCR.8)Changes in protein expression of heart failure markers,inflammatory markers,and cell death pathway-associated markers in adult TEAD1 ig KO / TEAD1 ic KO mice and control mice heart were detected by Western Blot.2.Identification of molecular mechanisms leading to cardiomyocyte necroptosis in TEAD1 ic KO adult mouse(Part III): 1)Generation of TEAD1 ic KO and control genotypic mice through crossbreeding ?-MHC promoter driven Mer Cre Mer +mice with TEAD1 F/F mice.After genotype verification,8-12 weeks old adult mice were given TAM(25mg TAM /Kg body weight)through intraperitoneal injection for 5 continuous days.2)Heart tissue from TEAD1 ic KO mice and control mice were harvested.RNAs were extracted from the heart tissues and RNA sequencing(RNA-seq)was performed to identify possible target genes of TEAD1.3)Chromatin immunoprecipitation(CHIP)assay in heart tissue of WT mice.Target gene DNA fragments that bind to TEAD1 antibody were identified(Ig G act as control).4)Changes in protein expression level of Ndufa5 and Ndufab1 in adult TEAD1 ic KO mice and control mice heart were detected by Western Blot.5)Changes in m RNA level of Ndufa5 and Ndufab1 in adult TEAD1 ic KO mice and control mice heart were detected by q RT-PCR.6)dual luciferase reporter assay to analyze the effect of TEAD1 on the transcriptional activity of Ndufa5 and Ndufab1 in 10T1/2 cells.7)TEAD1ic KO and control group cardiac tissues were also harvested for the detection and comparation of mitochondria complex I enzyme activity.8)Cardiac tissues from TEAD1 ic KO and control group mice were harvested for mitochondria extraction.The extracted mitochondria were then used to detect changes in mitochondria respiratory function using Sea Horse XF24.9)Primary cardiomyocytes were isolated from a MHC-Mer Cre Mer+/TEAD1 F/F;m Tm G+/+ adult mice and were cultured in vitro.The isolated cardiomyocytes were then treated with 4-Hydroxytamoxifen(4-OHT)to generate TEAD1 knockout cardiomyocyte.Western Blot was performed to confirm the deletion of TEAD1 after 4-OHT treatment.10)adult primary TEAD1 knockout cardiomyocytes were then subjected to staining such as PI staining,TMRM staining and mito SOX staining in order to quantify cell necrosis,to evaluate mitochondrial membrane integrity,and to detect superoxide production within mitochondria in the cardiomyocyte.3.Necrostatin-1(Nec-1)attenuated heart failure phenotypes in TEAD1 ic KO mouse through the inhibition of necroptosis in cardiomyocyte(Part IV): 1)TNF-? and Nec-1 were used to treat L929 cells,Nec-1 was verified to work by quantifying lactate dehydrogenase(LDH)released into the cell culture medium.2)Generation of TEAD1 ic KO and control genotypic mice through crossbreeding ?-MHC promoter driven Mer Cre Mer+ mice with TEAD1F/F mice.After genotype verification,8-12 weeks old adult mice were given TAM(25mg TAM /Kg body weight)through intraperitoneal injection for 5 continuous days.Simultaneously,Nec-1 and PBS were also given through intraperitoneal injection to establish both Nec-1 treatment group and PBS group in TEAD1 ic KO and control group mice respectively.3)VEVO770 to detect changes in echocardiographic parameters such as LVID;d,LVID;s,EF,FS etc.in Nec-1 treatment group and PBS group of TEAD1 ic KO and control mice respectively 4)mice hearts weight,and tibia length from both Nec-1 treatment group and control group were also recorded.Analysis were performed by comparing heart weight to tibia length ratio respectively for each group mice.5)Masson's Trichrome Staining was also performed on collected mouse heart slices from both Nec-1treatment group and PBS control group to examine the degree of fibrosis.6)Mouse hearts were also collected,fixed and OCT embedded.Slices of mouse heart were obtained at the thickness of 7?m,and were subjected to IF staining to analyze MAC2 expression of mouse cardiac tissue.Additional staining using EBD was also used to detect cardiomyocyte membrane integrity.7)Changes in the protein expression level of cell death pathway-associated markers in adult mice heart after treatments were verified by Western Blot.Results:Part I: TEAD1 ig KO mice developed acute dilated cardiomyopathy with severe heart failure phenotype.Mechanistic studies performed at cellular level suggested that deletion of TEAD1 can lead to the activation of necroptosis in adult cardiomyocyte.1)TEAD1ig KO mice were successfully generated through intraperitoneal injection of TAM.TAM injection did not affect TEAD1 expression in the control group mice.2)TEAD1ig KO mouse developed acute dilated cardiomyopathy,where dilatation of the ventricular cavity,thinning of the ventricular wall,and impaired cardiac function,were found to be progressively worsen with time.no significant changes in the echocardiographic parameters were observed for the control group mice.3)Mouse survival curve analysis suggested that TEAD1 ig KO mice exhibited high mortality rates and cannot survive past 27 days from the first TAM injection day.However,mice death was not observed in control group.4)Besides exhibiting mouse heart ventricular wall thinning and the dilatation of ventricular cavity,MT staining also revealed significant deposition of collagen fiber in TEAD1 ig KO mouse heart,which indicated that TEAD1 deletion in adult mouse can induce cardiac remodeling.5)IF results suggested that TUNEL-positive cardiomyocytes in TEAD1 ig KO mouse were not significantly different from control group,although both were found at a very low level.However,TEAD1 ig KO group had significantly higher number of EBD-positive and MAC-2 positive cardiomyocytes when compared to the control group,which were found to be statistically different.6)q RT-PCR results revealed that TEAD1 ig KO can upregulate expression of heart failure markers such as connective tissue growth factor(CTGF),atrial natriuretic peptide(ANP),and myosin heavy chain 7(MYH7)as well as inflammatory markers such as MAC2,Cluster of differentiation 68(CD68),and interleukin-6(IL-6),while at the same time downregulated myosin heavy chain 6(MYH6)expression.7)Western Blot results revealed that TEAD1 ig KO also induced upregulation of heart failure markers such as CTGF,upregulation of inflammatory markers such as MAC2 and CD45,and necroptosis pathway components such as receptorinteracting serine/threonine-protein kinase 1(RIP1),receptor-interacting serine/threonine-protein kinase 3(RIP3),p-RIP3,mixed lineage kinase domain like pseudokinase(MLKL),and PGAM family member 5,mitochondrial serine/threonine protein phosphatase(PGAM5).Part II: TEAD1 ic KO in adult mouse lead to the development of acute dilated cardiomyopathy with severe heart failure phenotypes.Mechanistic studies performed at cellular level suggest that cardiomyocytes specific deletion of TEAD1 can lead to the activation of necroptosis in adult cardiomyocyte.1)TEAD1ic KO mice were successfully generated through intraperitoneal injection of TAM.TAM injection did not affect TEAD1 expression in the control group mice.2)TEAD1ic KO mouse developed acute dilated cardiomyopathy,where dilatation of the ventricular cavity,thinning of the ventricular wall,and impaired cardiac function,were found to be progressively worsen with time.no significant changes in the echocardiographic parameters were observed for the control group mice.3)Mouse survival curve analysis suggested that TEAD1 ic KO mice exhibited high mortality rates and cannot survive past 25 days from the first TAM injection day.However,mice death was not observed in control group.4)Besides exhibiting mouse heart ventricular wall thinning and the dilatation of ventricular cavity,MT staining also revealed significant deposition of collagen fiber in TEAD1 ic KO mouse heart,which indicated that TEAD1 deletion in adult mouse can induce cardiac remodeling.5)IF results suggested that TUNEL-positive cardiomyocytes in TEAD1 ic KO mouse were not significantly different from control group,although both were found at a very low level.However,TEAD1 ic KO group had significantly higher number of EBD-positive and MAC-2 positive cardiomyocytes when compared to the control group,which were found to be statistically different.6)q RT-PCR results revealed that TEAD1 ic KO can upregulate expression of heart failure markers such as connective tissue growth factor(CTGF),atrial natriuretic peptide(ANP)as well as inflammatory markers such as MAC2,Cluster of differentiation 68(CD68),and interleukin-6(IL-6),while at the same time downregulated myosin heavy chain 6(MYH6)expression.7)Western Blot results revealed that TEAD1 ic KO also induced upregulation of heart failure markers such as CTGF,upregulation of inflammatory markers such as MAC2 and CD45,and necroptosis pathway components such as receptorinteracting serine/threonine-protein kinase 1(RIP1),receptor-interacting serine/threonine-protein kinase 3(RIP3),p-RIP3,mixed lineage kinase domain like pseudokinase(MLKL),and PGAM family member 5,mitochondrial serine/threonine protein phosphatase(PGAM5).Part III: TEAD1 ic KO in adult mice lead to mitochondria dysfunction,which then caused cardiomyocyte necroptosis.Specifically,molecular mechanistic studies shown that cardiomyocytes specific deletion of TEAD1 interfered with the assembly of mitochondrial complex I and supercomplex by repressing the transcription of mitochondria complex I accessory subunit Ndufa5 and Ndufab1,which leading to mitochondrial electron transport chain dysfunction.1)RNA-seq analysis revealed that a large number of mitochondria-related gene expressions were downregulated in cardiomyocyte from TEAD1 ic KO mice,including mitochondrial complex I accessory subunits Ndufa5 and Ndufab1.2)CHIP-assay results indicated that the transcription factor TEAD1 binds to the mitochondrial complex I accessory subunit Ndufa5 and Ndufab1.3)q RT-PCR and Western Blot results suggested that the deletion of TEAD1 in cardiomyocyte can result in significant decrease both in the m RNA and protein expression levels of Ndufa5 and Ndufab1.4)Dual-luciferase reporter assay indicated that the transcription factor TEAD1 can upregulate the transcriptional activity of Ndufa5 and Ndufab1.5)The defective assembly of mitochondrial complex I and supercomplex in TEAD1 ic KO adult mice cardiomyocytes were also found contributing to the mitochondrial respiratory and electron transport chain dysfunction as analyzed by Seahorse XF24.6)TEAD1ic KO group mice had significant reduction in mitochondrial complex I enzyme activity in heart tissue when compared to the control group.7)Results from isolated adult primary cardiomyocytes cultured in vitro indicated that TEAD1 deletion group had significantly higher PI positive and mito SOX positive cells compared to the control group.In parallel,the TMRM positive cardiomyocytes in TEAD1 deletion group were found significantly lower in number when compared to control group.Part IV: Nec-1 partly attenuated heart failure of TEAD1 ic KO mouse by inhibiting activation of necroptosis signaling pathway.1)L929 in vitro cell studies suggested that Nec-1 treatment can inhibited TNF-? induced cell death.2)Echocardiography analysis suggested that the degree of ventricular dilatation and ventricular wall thinning were significantly attenuated in TEAD1 ic KO adult mouse heart treated with Nec-1when compared to PBS treatment group.In parallel,cardiac function were also found significantly improved.3)MT staining analysis also revealed that the collagen fiber deposition in TEAD1 ic KO adult mice hearts treated with Nec-1 was significantly reduced when compared to PBS treatment group.4)IF staining analysis also showed that the number of EBD-positive and MAC2-positive cardiomyocytes in TEAD1 ic KO adult mice hearts treated with Nec-1 were found to be significantly decreased compared to PBS treatment group.5)Western Blot results indicated that the expression levels of p-RIP3,RIP3,RIP1,and MLKL,which participated in the activation of necroptosis signaling pathways,were significantly downregulated in TEAD1 ic KO adult mouse heart when treated with Nec-1.Conclusion: 1.TEAD1 ig KO in adult mice can lead to the activation of necroptosis in cardiomyocytes.The resulting loss of cardiomyocytes can lead to the development of acute dilated cardiomyopathy and severe heart failure in adult mice.2.TEAD1 ic KO in adult mice can leads to the activation of necroptosis in cardiomyocytes.The resulting loss of cardiomyocytes can lead to the development of acute dilated cardiomyopathy and severe heart failure in adult mice.3.Deletion of TEAD1 gene in adult mice cardiomyocytes leads to dysfunction of mitochondrial electron transport chain.Specifically,deletion of TEAD1 can repress the transcription of mitochondrial complex I accessory subunits Ndufa5 and Ndufab1.Consequently,this can leads to activation of necroptosis in cardiomyocytes.4.Nec-1 treatments can partly attenuate dilated cardiomyopathy and heart failure progression in TEAD1 ic KO adult mice by inhibiting the activation of necroptosis in cardiomyocytes. |
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