Silencing Of Ankyrin Repeat Domain 1 Attenuates Cardiomyocyte Apoptosis By Inhibiting P53 Activation And Mitochondrial Dysfunction In Rodents

Background and Objective:Heart failure (HF) is the final result of a variety of cardiovascular diseases, and is also the major cause of morbidity and death around the world. Although pharmacotherapy including angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blocker (ARBs), β blockers, diuretics and aldosterone receptor antagonists is effective in improving the outcomes of HF syndromes, the mortality of HF remains high. Therefore, we need to research new therapeutic target, and develop new drugs or strategies, which is of great importance to improve HF prognosis.Cardiac ankyrin repeat protein (CARP) is encoded by ankyrin repeat domain 1 (Ankrdl), while Ankrdl was identified as a fetal gene, the expression of which is augmented in both animals and humans with HF, but the role of Ankrdl/CARP in HF remains unclear. Although there is clinical evidence that Ankrdl mutations are involved in the pathogenesis of dilated or hypertrophic cardiomyopathy, there is no consensus on whether CARP is beneficial or detrimental to HF.Apoptosis has been demonstrated to play a critical role in the progression of HF. Coincidentally, there is recent evidence that CARP acts as a coactivator of p53 and modulates its transcriptional activity, and it is believed that the tumor-suppressor gene p53 contributes to cardiomyocyte apoptosis. Moreover, overexpression of CARP inhibits the phosphorylation of extracellular-signal-regulated kinase (ERK) and impairs the contractility of engineered heart tissues. These findings suggest that CARP might accelerate the progression of HF by enhancing cardiomyocyte apoptosis. Nowadays, mitochondria are considered to be the central executioners of apoptosis, but the influence of CARP on mitochondrial function is completely unknown.To date, there has been a paucity of literature on the topic of Ankrdl influencing apoptosis, among which three reports from a Korean laboratory have demonstrated that the ectopic expression of Ankrdl enhances apoptotic cell death in hepatoma cells, whereas it increases anoxia-induced apoptosis in rat embryonic heart-derived H9c2 cells; however, the down-regulation of Ankrdl is associated with the increase in cell apoptosis during myocardial ischemia/reperfusion. These findings suggest that the influence of Ankrdl on cell apoptosis might be context dependent.Angiotensin Ⅱ(AngⅡ) and left ventricular overload both can up-regulate Ankrdl and induce cardiomyocyte apoptosis, but it remains unclear whether the up-regulated Ankrdl inhibits or enhances cell apoptosis. In the present study, we used AngⅡ-stimulated cultured cardiomyocytes and mice that had undergone TAC (transverse aortic constriction) to examine the influence of Ankrdl silencing on apoptosis in cultured cardiomyocytes and left-ventricular-pressure overloaded mice, and whether Ankrdl/CARP modulates p53 and mitochondrial dysfunction of cardiomyocytes.Methods:1. Cell culture and treatmentThe neonatal rats at 1-3 days after birth were killed by 2% isoflurane inhalation, and the heart of each was removed. Isolation and culture of NRCs was performed as described previously. Cells were cultured for 4 days and then treated with 1μM AngⅡ or recombinant adeno-associated virus(AAV) carrying shRNA targeting Ankrdl(sh-Ankrdl) or negative control(AAV-ZsGreen). Cell viability and cardiomyocyte apoptosis were determined by the methyl thiazolyltetrazolium (MTT) assay and Hochest33258, respectively, according to the corresponding manufacturer’s instructions. Proteins were extracted from the cultured cardiomyocytes or their mitochondria, and the expression level of CARP, p53, p-p53 and Bax were detected by Western Blot.2. Pressure overload modelC57BL/6 male mice (8-10 weeks old, weighing 22-25 g) were anesthetized with pentobarbital (50 mg/kg, ip), intubated and ventilated. Pressure overload model was created by transverse aortic constriction (TAC) as described elsewhere.4 weeks before TAC or sham operation, mice were sacrificed and proteins were exacted from the whole heart. The expression level of p53, p-p53 and Bax were detected by Western Blot, and the expression of Bax was also detected by immunohistochemistry(IHC). Moreover, apoptosis in the myocardium was determined using TUNEL assay.3. Construction and infection by AAV-sh-AnkrdlpAAV2/9-CMV-ZsGreen vectors carrying sh-Ankrdlor negative control were generated by a professional company (Biowit, Shenzhen, China). For in vitro transfection, pAAV2/9-CMV-ZsGreen-sh-Ankrdlor negative control virus particles (5×105viral genomes/cell) were added to cultured NRCs. After transfection for 96 h (such a length of time is necessary to increase the efficiency of in vitro AAV infection), infection efficiency and silencing effect were evaluated using fluorescence microscopy and Western blotting respectively. For in vivo infection, pAAV2/9-CMV-ZsGreen-sh-Ankrdl or control virus particles (5×1012viral genomes/cell) were administered by direct injection in the left ventricular free wall (two sites, 10μl/site) in mice at 4 weeks of age using a syringe with a 30-gauge needle, and 4 weeks later, sham or TAC surgery was performed. Also 4 weeks after infection of AAV-sh-Ankrdl or AAV-ZsGreen, mice were sacrificed, the transduction efficiency of in vivo gene transfer by AAV and the experession of CARP were assessed in cryosectioned heart slices using a fluorescence microscopy and Western Blot respectively.4. Measurements of mitochondrial membrane potentialIt is believed that mitochondria play a central role in cell apoptosis. During apoptosis, mitochondrial membrane permeability transition pore (mPTP) opened and mitochondrial membrane potential(MMP) declined. To analyze the mitochondrial depolarization, the NRCs were labelled with TMRE (tetramethylrhodamine ethyl ester) and DAPL.The fluorescence intensity of TMRE was monitored at 582 nm.5. Statistical analysisData are 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<0.05 was considered to indicate statistical significance.Results:1. Ankrdl/CARP was up-regulated in response to HF and AngⅡ stimulationAccording to Western Blot analysis, myocardial CARP was markedly up-regulated in mice with HF induced by TAC, and in cultured cardiomyocytes, AngⅡ stimulation for 24h also markedly increased the expression of CARP. Because ANP is a recognized marker of HF, we analyzed the correlation between Ankrdl and ANP mRNA levels in mice with TAC or sham-operated mice over 4-8 weeks. Real-time PCR revealed that the level of Ankrdl expression was positively correlated with that of ANP, and CARP levels were also positively correlated with the lung weight/body weight ratio (LW/BW), a critical parameter of congestive HF in the pressure-overload model. These findings indicate that the expression pattern of myocardial Ankrdl/CARP is similar to that of natriuretic peptides in response to the pathological stimulation of HF.2. AAV-sh-Ankrdl exerted efficient infection in cardiomyocytes and myocardiumTo clarify the role of Ankrdl in AngⅡ stimulated cardiomyocytes, we infected NRCs with AAV-sh-Ankrd1. As a result, the infective efficiency was satisfactory, with the fluorescence microscopy showed that approximate 60%-70% cells were infected with sh-Ankrdl, and the level of CARP significantly decreased by-70% in sh-Ankrdl-infected cells. Similarly, intramyocardial injection of sh-Ankrdl 4 weeks before TAC or sham also produced satisfactory infection efficiency, with more than 50% myocardium was transfected with sh-Ankrdl, and the expression level of CARP reduced about 50%-60% in sh-Ankrdl infected hearts compared with Zs-Green-infected ones.3. Silencing of Ankrdl reduced cardiomyocyte apoptosisMTT and Hoechst33258 staining showed that AngⅡ stimulation for 24 hours reduced cell viability and increased apoptosis, which were partially antagonized by addition of AAV-sh-Ankrdl, while silencing of Ankrdl in the absence of AngⅡ had no effect on cell viability and apoptosis. These findings suggest that silencing of Ankrdl could partially reduce AngⅡ-induced cardiomyocyte apoptosis.4. Silencing of Ankrdl inhibited p53 activation and Bax translocationIn order to clarify the mechanism of the anti-apoptotic effect of silencing Ankrdl on cardiomyocytes, we examined the changes of apoptosis-related molecules by Western blotting, and found that AngⅡ stimulation markedly increased the level of p53, p-p53 and mitochondrial Bax, which were also partially abrogated by addition of AAV-sh-Ankrd1, indicating silencing of Ankrdl would inhibit AngⅡ-induced p53 activation and Bax translocation.5. Silencing of Ankrdl decreased mitochondrial permeabilityIn addition, mitochondrial depolarization was investigated using TMRE staining. We found that TMRE fluorescence was significantly reduces in the NRCs exposed to AngⅡ, while the decreased in fluorescence was attenuated when were infected with AAV-sh-Ankrdl. These findings suggest that silencing of Ankrdl would reduce AngⅡ-induced increase in mitochondrial permeability.6. Silencing of Ankrdl inhibited apoptosis in pressure-overload miceExpression of p53, p-p53 and Bax protein and apoptosis were evaluated. We noted that phosphorylation of p53 was increased in TAC and TAC+AAV-ZsGreen groups, but reduced in TAC+AAV-sh-Ankrdl mice detected by Western blotting. Immunostaining of Bax determined by immunohistochemistry and Western blotting was also reduced in TAC+AAV-sh-Ankrdl groups compared with TAC and TAC+AAV-ZsGreen mice. TUNEL assay showed that myocardial apoptosis was significantly enhanced in TAC and TAC+AAV-ZsGreen groups, and the percentage of cell apoptosis in TAC+AAV-sh-Ankrdl mice was markedly lower than in the TAC+AAV-ZsGreen group.Conclusion:Silencing of Ankrdl attenuates AngⅡ-and pressure overload-induced cardiomyocyte apoptosis, which is attributable to the inhibition of p53 activation and mitochondrial dysfunction. Regulating the expression of Ankrdl in cardiomyocytes might be a therapeutic potential in the progression of HF.