Novel Role For Caspase-Activated Dnase In The Regulation Of Pathological Cardiac Hypertrophy

Cardiac hypertrophy is a response of the myocardium to an increased workload and is characterized by an increase in myocardial mass and an accumulation of extracellular matrix. Although cardiac hypertrophy might initially represent an adaptive response of the myocardium, it frequently progresses to ventricular dilatation and heart failure. Heart failure is a debilitating disease with high morbidity and mortality rates, and its prevalence is increasing. Although hypertrophy was first described more than a century ago and several studies have identified the signal transduction pathways that promote hypertrophic responses, the mechanisms that regulate these pathways have not been clearly elucidated. A better understanding of these regulatory mechanisms in cardiac myocytes might lead to novel strategies for suppressing cardiac hypertrophy.Caspase-activated DNase (CAD), also known as DNA fragmentation factor40kDa (DFF40), is a double-strand-specific endonuclease responsible for the cleavage of nucleosomal spacer regions and subsequent chromatin condensation during apoptosis. CAD not only induces DNA fragmentation during apoptosis but also functions as a prerequisite for inducing cell differentiation. The functions of CAD are similar to those of other endonucleases, such as DNase Ⅰ, DNase Ⅱ and Endog, which are important modulators of apoptosis and cardiac hypertrophy. Investigations have revealed that CAD interacts with several transcriptional regulators of cell proliferation, including HSP70, HSP40and HMGB1, which play a critical role in cardiac hypertrophy and heart failure. These observations ensure that CAD is an attractive target for therapeutic intervention to treat or prevent cardiac hypertrophy and heart failure.In this study, part Ⅰ aims to explore the expression of CAD in heart samples collected from pathological cardiac hypertrophy and the role of CAD on cardiac hypertrophy at the cellular level; part Ⅱ observated the role of CAD in cardiac hypertrophy and fibrosis in response to AB; part Ⅲ aims to discovere the molecular mechanisms of CAD in modulating cardiac remodeling. Part I The Expression of CAD in Hypertrophic Hearts and the Role of CAD on Cardiac Hypertrophy at the Cellular LevelAims:This part aims to investigate the expression of CAD in dilated cardiomyopathy human hearts and murine hypertrophic hearts, to examine the protein levels of CAD in the cultured neonatal rat cardiomyocytes treated with angiotensin Ⅱ or phenylephrine, and the role of CAD on cardiac hypertrophy at the cellular level.Methods:Samples of failing human hearts were collected from the left ventricles of dilated cardiomyopathy patients who were under treatment following heart transplants. Control samples were obtained from the left ventricles of normal heart donors (n=15in each group). The male wild-type C57BL/6mice aged8to10weeks and body weight ranged from23.5-27.5g were included. Aortic banding was used to establish pressure overload-induced cardiac hypertrophy in mice. The mice were randomly divided into3groups, respectively, for the sham operation group, AB-4weeks and8weeks groups (n=10in each group). In the cultured neonatal rat cardiomyocytes treated with angiotensin II (AngⅡ,1μM) or phenylephrine (PE,100μM) for48h to induce hypertrophy. By using primary cultured NRCMs we determined the functional contribution of CAD on cardiomyocyte hypertrophy in vitro NRCMs were infected with AdshCAD to knockdown CAD or with AdCAD to overexpress CAD. Subsequently, these infected cardiomyocytes were exposed to Ang II or to PBS as a control for48hours, followed by immunostaining with a-actinin to measure the cell size and real-time quantitative PCR (RT-PCR) to examine the expression of hypertrophic hallmarks myosin heavy chain (β-MHC) and atrial natriuretic factor (ANP).Results:Our data revealed that in dilated cardiomyopathy hearts the protein levels of CAD were reduced, whereas the level of β-MHC and ANP were markedly increased compared with their levels in healthy donor hearts. In the experimental mouse model of AB-induced cardiac hypertrophy (evidenced by P-MHC and ANP levels), CAD expression was down-regulated in mouse hearts4weeks after AB and was maintained at a low level until8weeks after AB compared with its level in sham-operated hearts. In the cultured neonatal rat cardiomyocytes (NRCMs) treated with angiotensin II or phenylephrine for48h to induce hypertrophy, the CAD protein levels were both significantly reduced by angiotensin II and phenylephrine, respectively. Under basal conditions, neither AdshCAD nor AdCAD affected the size of the cultured NRCMs compared with the control AdshRNA and AdGFP cells. In response to Ang II-induced cell hypertrophy, AdshCAD significantly reduced the cross-sectional area compared with the AdshRNA-infected controls, whereas Ang II-induced cell hypertrophy was greatly enhanced in the AdCAD-treated NRCMs compared with the AdGFP cells. Accordingly, the Ang II-induced expression of hypertrophic hallmarks (ANP and β-MHC) was dramatically suppressed in the AdshCAD-infected cardiomyocytes, whereas it was remarkably enhanced in the AdCAD-infected NRCMs compared with the controls.Conclusions:These results indicate that the protein levels of CAD are markedly decreased in human dilated cardiomyopathy hearts and pressure overload-induced hypertrophic mouse hearts as well as in vitro AngⅡ/PE-treated cardiomyocytes, suggesting that CAD might be involved in the development of cardiac hypertrophy. Our ex vivo data suggest that the downregulation of CAD mitigates pathological cardiac hypertrophy, whereas the upregulation of CAD promotes pathological cardiac hypertrophy. Part Ⅱ The Role for CAD in the Regulation of Pathological Cardiac HypertrophyAims:This study tested whether decreased CAD levels or overexpression of CAD in the heart would influence the development of cardiac hypertrophy and failure.Methods: The male CAD knockout mice (sham, n=15; AB, n=20), CAD cardiacspecific transgenic mice (sham, n=15; AB, n=20), and their respective wild-type littermates (sham, n=15; AB, n=20), aged8to10weeks and body weight ranged from23.5-27.5g were included. The mice were randomly divided into AB group and sham group.8weeks after AB in knockout mice and4weeks after AB in transgenic mice, cardiac function of mice was examined by echocardiography and hemodynamics, and the HW/BW, LW/BW, and HW/TL were compared in mice of each group. HE, WGA, and PSR staining were used for morphology analysis. Molecular markers of cardiac hypertrophy and fibrosis were determined by real-time quantitative PCR.Results: After8weeks of AB surgery, the HW/BW, LW/BW, HW/TL, cardiac myocyte cross-sectional area, and left ventricular collagen volume fraction of CAD knockout mice were significantly lower than those of wild-type mice. The echocardiographic and hemodynamics results showed that CAD knockout mic exhibited lower left ventricular wall thickness and ventricular diameter, and higher left ventricular systolic and diastolic function than wild-type mice. The mRNA levels of cardiac hypertrophic and fibrotic markers including ANP, BNP, β-MHC, collagen Ⅰα, collagen Ⅲα and CTGF in CAD knockout mice were decreased compared with those in wild-type mice. Conversely, CAD cardiac-specific transgenic mice exhibited the opposite phenotypes to CAD knockout mice after4weeks of AB surgery.Conclusions:The results indicate that the deletion of CAD suppresses pressure overload-induced cardiac remodeling, the overexpression of CAD exaggerates cardiac hypertrophy and fibrosis in response to chronic pressure overload. Part Ⅲ The Molecular Mechanisms of CAD in the Regulation of Pathological Cardiac HypertrophyAims:To investigate the molecular mechanisms of CAD in the regulation of pathological cardiac hypertrophy.Methods: The male CAD knockout mice (sham, n=15; AB, n=20), CAD cardiacspecific transgenic mice (sham, n=15; AB, n=20), and their respective wild-type littermates (sham, n=15; AB, n=20), aged8to10weeks and body weight ranged from23.5-27.5g were included. The mice were randomly divided into AB group and sham group. The levels of phosphorylated and total protein of MAPKs, Akt and Smads in hearts from each group were determined by Western blot analysis after AB. The cultures of cardiac myocytes were infected with AdCAD and AdshCAD for12h with MOIs of10, AdGFP and AdshRNA were used as the controls in each group. The culture medium was then replaced with serum-free medium for12h, followed by stimulation with1μM angiotensin II for48h{Jiang,2014#4;Jiang,2014#6;Li,2010#7}. The levels of phosphorylated and total protein of MAPKs and Akt in cells from each group were determined by Western blot analysis. Myocardial tissue sections were subjected to TUNEL staining to detect apoptotic cells. The Caspase3, Bax and Bc12signaling pathways related to AB-induced apoptosis were examined in the heart.Results:After8. weeks of AB, we detected that CAD deficiency was able to inhibit the activation of MEK1/2and ERK1/2induced by pressure overload using western blot analysis, while the cardiac-specific overexpression of CAD enhanced the phosphorylation of MEK1/2and ERK1/24weeks after AB. The western blotting results showed that the angiotensin Ⅱ-triggered activation of MEK1/2and ERK1/2was significantly attenuated by infection with AdshCAD; however, this activation was markedly increased by infection with AdCAD. Our results showed that AB-induced increased levels of Smad2/3phosphorylation and nuclear translocation were significantly attenuated in CAD knockout mice compared with the control hearts. Importantly, CAD transgene mice induced higher levels of Smad2/3phosphorylation and nuclear translocation compared with that of their control mice. TUNEL-positive nuclei were present in the control mice subjected to AB, whereas their number was significantly decreased in the CAD knockout mice and markedly increased in the transgene mice. The activation and cleavage of caspase-3induced by AB was modest in the control hearts and rarely observed in the CAD knockout hearts; however, the activity and cleavage of caspase-3was increased considerably in the transgene hearts. As predicted, the ratio of Bax to Bcl-2was remarkably lower in the CAD knockout mice and was significantly higher in the transgene mice after AB.Conclusions: These results demonstrate that the detrimental role of CAD in pathological cardiac hypertrophy is largely associated with the regulation of MEK-ERK1/2and Smads signaling, and CAD exacerbates AB-induced apoptosis.