Olmesartan Attenuates Cardiac Remodeling And Improves Heart Failure In Mice Through Antagonizing Cardiac Ankyrin Repeat Protein

Background:Heart failure (HF) is the final result of a variety of cardiovascular disease and the most common cause of death for cardiovascular disease. It has the characteristic of high mortality and the5-year survival is similar to the malignant tumors and affects seriously the patient’s quality of life. The exact effects of heart failure drugs include (3blockers, angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blocker (ARB), vasodilators and diuretics and so on. Olmesartan, a novel angiotensin Ⅱ AT1receptor blocker, because of the overall efficacy of the reducing diastolic blood pressure was significantly superior to other similar products, the drug medication aspects, a small dose, compared with angiotensin-converting enzyme inhibitors do not produce cough side effects, and significantly antihypertensive effect to different degrees of hypertension, is regarded as the best choice for first-line antihypertensive drugs. However, in recent studies have shown that olmesartan also has roles in reducing cardiac hypertrophy and improve cardiac function, and may become the best choice for people with heart failure accompanying with high blood pressure, exploring its new mechanisms to provide effective molecular targets for the new drugs of heart failure treatment researching and development.However, a large number of clinical and animal studies indicated that cardiac ankyrin repeat protein (CARP) is upregulated significantly in myocardial hypertrophy and heart failure. It is a new marker of myocardial hypertrophy and heart failure. However, the role of ANKRD1/CARP in both cardiac hypertrophy and HF remains unclear. Although clinical evidence showed that ANKRD1mutations are involved in the pathogenesis of hypertrophic and dilated cardiomyopathy. Studies have shown that increased the CARP and titin and muscle palladium protein binding may lead to hypertrophic heart disease and CARP is closely related to the Pro-apoptotic proteins. In addition, CARP would be a mediator in calpains-involved signal transduction process which leading to myocardial hypertrophy and heart failure including calcineurin, p53and induced apoptosis factor and so on. Therefore, we hypothesized that CARP would damage the cardiomyocytes and inhibiting the the expression of CARP and its role may be a new treatment for the treatment of heart failure.As we all know, the renin-angiotensin system (RAS) plays an important role in the pathological process of cardiac hypertrophy and heart failure. However, studies have shown that the expression of the CARP was up-regulated Significantly by angiotensin-Ⅱ. These demonstrated that exists close links between the CARP with RAS and both participate myocardial hypertrophy and heart failure physiological and pathological processes together. However, olmesartan exerts its effect by blocking specifically AT1receptor of the RAS system. Is there any relation between olmesartan and CARP, still no literature reports about olmesartan and CARP. According to the data obtained, we hypothesize that Olmesartan improves cardiac hypertrophy and cardiac function in mice by antagonizing CARP. Objective:Using Ang II-stimulated cultured cardiomyocytes and mice with transverse aortic constriction (TAC), we carried out this study to examine the following issues:(1) To explore the relationship of olmesartan and CARP, whether ANKRD1/CARP is a pharmaceutical target of olmesartan.(2) To estimate the effects of ANKRD1/CARP on the cardiomyocyte hypertrophy and apoptosis.(3) Whether ANKRD1/CARP modulates calcineurin, p53and mitochondrial dysfunction.(4) Whether overexpression of CARP aggravates the TAC-induced cardiac remodeling and heart dysfunction.Methods:1. Cell culture and treatmentVentricular myocytes were isolated from neonatal rats at1-3days after birth and cultured. Cells were treated with Ang II0.1-1uM or Cyclosporin A(CsA, luM), RNH-6270luM (RNH, active metabolite of olmesartan or recombinant adenovirus containing the rat ANKRD1cDNA (Ad-ANKRD1). Real Time-PCR analysis gene expression levels of ANP, β-MHC and the expression levels of CARP, P53, P-P53, Bax, and Calcineurin protein were detected by Western Blot. Cell viability and cardiomyocyte apoptosis were determined by the methyl thiazolyltetrazolium (MTT) assay and Hochest33258, respectively, according to the corresponding manufacturer’s instructions. Surface area measurement of neonatal rat cardiomyocytes:cell stained with rhodamine phalloidin and diamidino-2-phenylindole dihydrochloride (DAPI) by standard immunocytochemical techniques. At least30random cells from each group were measured by the Image J software. 2. Construction of recombinant adenovirus carrying ANKRD1or empty vectorThe full-length cDNA of rat ANKRD1was inserted into the vector pUC57, and then subcloned into the shuttle vector pDC316-mCMV-EGFP. The Ad-MAX system was used for the generation of recombinant adenovirus carrying ANKRD1cDNA or empty vector (pEGFP).Briefly, pDC316-mCMV-EGFP-ANKRDland virus backbone plasmid pBHGloxdelIE13cre were co-transfected into cultured HEK293cells by using lipofectamine2000(Invitrogen), then the recombinant adenovirus were collected and amplified in HEK293. The overexpression of ANKRD1was achieved by transfecting cultured cardiomyocytes with the recombinant adenovirus (multiplicity of infection (MOI)=10) or by direct injecting into the myocardium through a left thoracotomy in mice.3. Immunofluorescence assay.Immunofluorescence staining was used to examine the cellular distribution of the CARP proteins and nuclear factor of activated T cells (NFAT). Location of CARP and NFAT in the cardiomyocytes was detected with a confocal microscopy. To analyze the mitochondrial depolarization, the Cardiomyocytes were labeled with tetramethylrhodamine ethyl ester (TMRE, Molecular Probes) and DAPI. The fluorescence intensity of TMRE was monitored at582nm by confocal microscopy and the fluorescence images were acquired.4. Pressure overload model.C57BL/6male mice (8-10weeks old, weighing22-25g) were anesthetized with were anesthetized with pentobarbital (50mg/kg, ip), intubated and ventilated with room air. Pressure overload model was created by transverse aortic constriction (TAC) as described elsewhere. Transthoracic echocardiography was performed at4weeks after TAC or sham operation and then the mice were sacrificed. Some of the TAC mice were randomized to treatment with olmesartan (10mg/kg/d added in the food) or vehicle alone for4weeks and then were sacrificed. Cardiac-restricted overexpression of adenoviral CARP was direct injecting into the myocardium through a left thoracotomy in mice when TAC2weeks. The mice were killed2weeks after adenovirus injection. Myocyte cross-sectional areas were measured by staining with rhodamine-conjugated wheat germ agglutinin.5. Statistical analysisData were expressed as the mean±SEM. Statistical significance was analyzed by using Student’s unpaired t-test or one-way ANOVA, followed by Bonferroni’s correction for post hoc multiple comparisons. In addition, the least squares method was used to analyze linear of the selected variables. In all analyses, p<0.05was considered to indicate statistical significance.Results:1. Olmesartan attenuated cardiac hypertrophy and improved heart failure in TAC mice through down-regulation of CARP.RT-PCR and Western Blot shown that RNH-6270(10-6mol/L)(active form of olmesartan) significantly abrogated the Ang II stimulated upregulation of CARP protein and ANP gene in cultured cardiomyocytes, and TAC-induced CARP expression was markedly reduced when treated with olmesartan. As expected, olmesartan treatment significantly reduced the TAC-induced HW/BW ratio (6.23±0.09in TAC group,5.02±0.33in olmesartan-treated TAC group, P<0.01) and the LW/BW ratio (7.62±0.30in TAC group,6.04±0.17in olmesartan-treated TAC group, P<0.01).These results indicate that Olmesartan attenuated cardiac hypertrophy and improved heart failure in TAC mice through down-regulation of CARP, and CARP is a pharmaceutical target of olmesartan.2. ANKRD1/CARP is a reliable marker of cardiac hypertrophy. Because BNP (B-type natriuretic peptide) is a recognized marker of cardiac hypertrophy and HF, we analyzed the correlations between the mRNA levels of ANKRD and BNP in mice with TAC or sham-operation for4-8weeks. Real-time PCR results showed that ANKRD1mRNA levels were positively correlated with BNP levels (r=0.914, P<0.01, n=15). Similarly, the HW/BW ratio was positively correlated with ANKRD1expression (r=0.934, P<0.01, n=15). In protein level, myocardial CARP was also upregulated remarkably in TAC mice confirmed by Western blot analysis. In neonatal rat cardiomyocytes (NRCs), Ang Ⅱ stimulation markedly dose-dependently upregulated ANKRD1mRNA and time-dependently increased CARP protein levels. Immunofluorescence assay shown that Ang induced the cytosolic translocation of CARP, which was abrogated by using a caplain inhibitor. These findings indicate that ANKRD1/CARP is a reliable marker of cardiac hypertrophy.3. Forced ANKRD1overexpression aggravates cardiomyocyte hypertrophy in response to Ang Ⅱ stimulation.Hypertrophy-associated fetal genes ANP and β-MHC were significantly upregulated in Ad-ANKRD1transfected cardiomyocytes in the absence/presence of Ang-Ⅱ stimulation. Furthermore, overexpression of ANKRD1significantly enhanced the Ang Ⅱ-stimulated increase of cell surface area. It has been well documented that calcineurin/NFAT pathway is believed to play a pivotal role in myocardial hypertrophy. Thus we examined protein levels of calcineurin and NFAT. Western blot analysis showed that overexpressed ANKRD1significantly increased the expression of calcineurin, and the nuclear translocation of NFAT in the absence/presence of Ang-Ⅱ stimulation. The increase of cell surface area by Ad-ANKRD1and Ang Ⅱ was abrogated by calcineurin inhibitor Cyclosporin A. These results indicate that forced ANKRD1overexpression promotes cardiomyocyte hypertrophy through the calcineurin-NFAT pathway.4. ANKRDl overexpression increased cardiomyocyte apoptosisMTT and Hochest shown that forced ANKRD1overexpression significantly reduced cell viability and increased apoptosis of NRCs in the presence of Ang II stimulation. In order to clarify the underlying mechanism of CARP’s proapoptotic role on cardiomyocytes, we examined the levels of apoptosis-related molecules by western blotting, and found that ANKRD1overexpression significantly increased protein levels of p53, phosphorylated-p53(p-p53) and mitochondrial Bax. In addition, mitochondrial depolarization determined by TRME staining indicated that the fluorescence of TMRE was reduced when the cells were exposed to Ang Ⅱ, and the loss of fluorescence was increased in cells transfected with Ad-ANKRD1, suggesting that overexpression of ANKRD1increases mitochondrial permeability.5. Cardiac-restricted overexpression of ANKRD1exaggerates cardiac remodeling and cardiac dysfunctionANKRD1/CARP was achieved by myocardial injection of Ad-ANKRD1. Myocardial overexpression of ANKRD1for2weeks increased TAC-induced cardiac hypertrophy. Heart weight/body weight (HW/BW) ratio was significantly higher in Ad-ANKRD1-TAC group than in Ad-EGFP-TAC group (8.69±0.38mg/g in vs.6.04±0.15mg/g, P<0.01). Similarly, LW/B W ratio, an index of pulmonary congestion, was significantly larger in Ad-ANKRD1-TAC group than in Ad-EGFP-TAC group (11.0±1.25mg/g vs.6.97±0.38mg/g, P<0.05). TAC-induced increase of cardiomyocyte cross surface area and ANP mRNA expression levels were markedly enhanced by overexpression of ANKRD1(all P<0.01). Echocardiography examination showed that left ventricular end-diastolic dimension (LVEDd) and LV end-systolic dimension (LVESd) in Ad-ANKRD1-TAC group were larger than that in Ad-EGFP-TAC group. Furthermore, expression of myocardial calcineurin protein was significantly increased in TAC mice treated with Ad-ANKRD1than in untreated TAC mice. The expressions of p53and p-p53proteins measured by western blotting and cell apoptosis evaluated by TUNEL assay were significantly increased in Ad-ANKRD1-TAC mice than in Ad-EGFP-TAC mice. These data indicate that myocardial overexpression of ANKRD1exacerbates cardiac hypertrophy and cardiac dysfunction.Conclusions:1. Olmesartan can inhibit significantly the expression of CARP and its detrimental effects and was showed to attenuate cardiac remodeling and heart dysfunction, suggesting that CARP inhibition might be a novel therapeutic target for heart failure.2. ANKRD1/CARP is a marker of myocardial hypertrophy. Cytosolic CARP promotes cardiomyocyte hypertrophy mainly through calcineurin/NFAT signaling pathway.3. ANKRD1/CARP overexpression increased cardiomyocyte apoptosis by activating P53and exacerbating mitochondrial dysfunction. It promoted the development of cardiac hypertrophy to heart failure.