Research On The Mechanism Of Dilated Cardiomyopathy And Heart Failure Induced By Dual Specific Phosphatase 7

ObjectiveHeart failure(HF)is one of the most common but complicated end-stage syndromes in clinical practice.Dilated cardiomyopathy(DCM)is a myocardial structural abnormality that is associated with heart failure,a condition in which the left ventricle stretches and thins and is unable to effectively pump blood.Dual-specificity phosphatases(DUSPs)are a group of protein phosphatases that regulate signaling pathways in numerous diseases,and type 7 DUSP(DUSP7)has been previously associated to ear development,tumor progression and chromosome alignment.However,its physiological and pathological impact in cardiovascular disease remains unknown.Methods1.Generation of the DUSP7 knockout mouse model and cardiac-specific DUSP7 overexpression mouse model: Mouse models developed by the Shanghai Model Organisms Center,Inc.These models were generated using the CRISPR/Cas9 system in a C57BL/6J mouse background.All protocols were approved by the Institutional Animal Care and Use Committee of Shanghai Tenth People’s Hospital,Tongji University School of Medicine.The two models used in this study were designed and developed by the Shanghai Model Organisms Center,Inc(Shanghai,China),an institute that is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International(AAALAC).2.Serial echocardiographic measurements were performed using a Vevo 2100System(Visual Sonics,Toronto,Canada)in KO and OE mice as well as their WT littermates ranging from 5 to 25 months of age.The parameters measured included:LVEF,left ventricular ejection fraction;FS,fractional shortening;LVVOL;d,left ventricular volume at end diastolic;IVS;d,inter-ventricular septum diastolic thickness;and LVPW;d,left ventricular posterior wall diastolic thickness.3.We investigated the MAPK pathway by Western Blot,including ERK1/2,p-ERK1/2,p38,p-p38,JNK,p-JNK,MEK1/2,p-MEK1/2,p-c-FOS,c-FOS,p-c-JUN,c-JUN,c-MYC,we also tested ANP,BNP,DUSP7,GAPDH et al.by Western Blot.4.For detection of DUSP7,ANP,BNP,et al.mRNA in mice heart tissue,total RNA was extracted using TRIzol reagent(Invitrogen)according to the manufacturer’s protocol.qPCR was performed using TB Green Premix Ex Taq II(Takara)on a Light Cycler 96 PCR System(Roche).5.To investigate the effect of DUSP7 overexpression or knockout on other genes,we performed a transcriptome sequencing analysis on the hearts of OE,KO and WT mice at 25 weeks of age.6.Immunofluorescence assay and wheat germ agglutinin staining analyses demonstrated significantly differences in the KO and OE groups at both the size of cells and protein levels.7.We utilized NHK enhances ERK phosphorylation to delineate its effect on DCM and HF caused by DUSP7 overexpression.We first investigated the effect of HNK on WT and DUSP7 OE mice.In another set of experiments,we investigated the effect of HNK in cultured cardiomyocytes isolated from rats that were transfected with DUSP7 using an adenovirus.8.We hypothesized that DUSP7 knockout protects mice from diseases.To test this,we first infused isoproterenol,a β-adrenergic receptor agonist,into WT,KO and OE mice.We measured heart function of the mice during this period by echocardiographic assessments.Moreover,both ANP and BNP levels were measured by western blot.Results1.To investigate the association between DUSP7 and the DCM and HF pathologies in vivo,we generated DUSP7-knockout(abbreviated as KO)and DUSP7cardiac-specific overexpression(abbreviated as OE)mouse models.2.Overexpression of DUSP7 causes deteriorating dilated cardiomyopathy,heart failure,and cardiac death in vivo.The gross heart sizes of the OE mice were larger than both KO and WT mice,most of the OE mice hearts displayed a globular cardiac silhouette.An increased interstitial collagen amount,a significant left ventricular cavity dilation and thinning wall in the OE mice compared to WT mice.3.The heart weight normalized to body weight(HW/BW),lung weight normalized to body weight(LW/BW),and heart weight normalized to tibia length(HW/TL)were significant increased in OE mice compared to WT mice.4.The echocardiography results at 5,10,15,20,and 25 weeks of age of KO,OE mice and their WT littermates revealed that OE mice developed DCM and HF phenotypes gradually,with reduced EF and FS functions that were accompanied by a remodeling in LVVOL;d,IVS;d,and LVPW;d.5.Compared the survival of OE mice to WT and KO mice over a 1-year period,the Kaplan-Meier survival analysis showed that,beginning at 6 months of age,there was a significant increase in the mortality rate of OE mice.Within 10 months,all OE mice were dead,compared to an observed 90% survival rate for both WT and KO mice at the end of 12 months.6.The cardiomyopathy and heart failure markers atrial natriuretic peptide(ANP)and brain natriuretic peptide(BNP)were significantly elevated with increased DUSP7 overexpression.7.Compared to ISO-treated WT mice,the ISO-treated KO mice displayed slight improvement in FS function,IVS;d and LVPW;d.At the same time,the ISO-treated KO mice showed HW/BW,LW/BW,and HW/TL ratios recovery compared to ISO-treated WT mice.Moreover,both ANP and BNP markers were significantly reduced in ISO-treated KO mice compared to ISO-treated WT mice.8.RNA sequencing reveals the significant regulatory role of MAPK pathway in cardiac-specific DUSP7 overexpression mice.9.As observed in the expression levels of three major MAPK pathways,ERK1/2,JNK and p38 using western blot,the phosphorylation level of ERK1/2 was dramatically reduced,while no changes were observed in the phosphorylation levels of JNK and p38.The transcriptome sequencing analysis also revealed no significant changes in RAS,RAF and MEK expression.10.Western blot data showed the total protein levels of c-FOS and its phosphorylation level were reduced in OE mice,while the total protein level of c-MYC dramatically increased in OE mice,and there was no difference in the total levels of the c-JUN between WT,KO and OE mice,but a significant increase in the phosphorylation level of c-JUN in OE mice.11.In vivo experiment has showed that Honokiol(HNK)promoted ERK1/2phosphorylation,and that the level of DCM markers ANP and BNP were also recovered.12.In vitro experiment has showed that the effect of HNK on primary neonatal rat ventricular myocytes(NRVMs),which were transfected with DUSP7 using an adenovirus,also enhanced ERK1/2 phosphorylation,and that the level of ANP and BNP were recovered as well.ConclusionsIn summary,we have demonstrated mouse models of cardiac-specific DUSP7-overexpression exacerbated DCM with similar clinical presentation to human DCM,including ventricular dilation,wall thinning,elevated ANP and BNP levels and fibrosis.In vivo studies indicated that mice with DCM developed heart failure and died within a year.In vitro studies revealed that high levels of DUSP7 inhibited ERK1/2 and influenced downstream c-MYC,c-FOS and c-JUN gene expression without affecting upstream activators.Taken together,our results revealed a novel molecular mechanism that confirmed the involvement of DUSP7 in DCM,HF and cardiac death,and provided a pharmaceutical target and new clinical pathway to alleviate DCM and improve cardiac function.