| Heart failure has become the most important cardiovascular health problem worldwide and will probably become a major concern as the aging population grows in size. Cardiomyocyte apoptosis is greatly increased in dilated cardiomyopathy which is characterized by the gradual development of heart failure.Several studies have subsequently suggested that cardiomyocyte apoptosis is a key event and a central mechanism in the development of dilated cardiomyopathy and heart failure.Both the intrinsic(mitochondrial) and the extrinsic(death receptor mediated) pathway have been shown to activate caspase-3.Activation of caspase-3 is followed by cardiomyocyte apoptosis,which is sufficient to induce dilated cardiomyopathy and heart failure.Recently, loss of a basal survival mechanism has been noted to undergo apoptosis in various animal models of heart failure.Chronically elevated but low levels of apoptosis may play a causal role in the pathogenesis of dilated cardiomyopathy and heart failure.SIRT1,the mammalian ortholog of Sir2,is a NAD-dependent deacetylase belonging to the classⅢhistone deacetylase(HDAC) family and functions in a wide array of cellular processes,including gene silencing,longevity,muscle differentiation,and DNA damage repair.Sir2αoverexpression was shown to inhibit p53 transcriptional activity and p53-dependent apoptosis in response to DNA damage and oxidative stresses.SIRT1 also inhibits apoptosis and promotes DNA repair by deacetylating FOXO transcription factors. However,a point mutation at the highly conserved histidine residue at the core domain (SIRT1-363Y) effectively abolished the deacetylase activity and overexpression of this catalytically inactive Sir2αprotein potentiated these cellular stress responses.Mice deficient in Sir2αexhibit developmental abnormality in the heart and only infrequently survive postnatally.Recent published data indicate that SIRT1 plays an essential role in mediating survival of cardiomyocytes under stress in vitro.Importantly,dominant negative Sir2αwhich express mouse Sir2H355A in neonatal rat ventricular myocytes has been showed to enhance basic cell death as well as slightly potentiating hydrogen peroxide-induced cell death,but in vivo the function of the deacetylase activity of SIRT1 in the heart is still largely unknown.We develop our hypothesis that cardiac overexpression of dominant Negative SIRT1 in cardiomyocytes can induce cardiac apoptosis and cause cardiac dysfunction.We carried out the following experiments to test the hypothesis.SIRT1 expression is up-regulated in the failing heart induced by 8 weeks of pressure overload.In an attempt to determine the requirement for the deacetylase activity of SIRT1 in the heart,we generated transgenic(TG) mice with cardiac-specific expression of a dominant negative form of SIRT1(SIRT1H363Y).Reverse transcription polymerase chain reaction(RT-PCR),Western blot and Immunohistochemistry show that SIRT1H363Y was properly expressed in cardiomyocytes.Unexpectedly,these mice exhibiting cardiac specific dominant negative SIRT1 overexpression had dilated atrial and ventricular chambers and lead to premature death and died from one week to four weeks after birth. Fortunately,three lines of TG founder mice survived to generate TG-positive offspring, and their offspring died mainly from postnatal days 8 to 12.The heart weight/body weight ratio of transgenic mice was significantly higher compared with nontransgenic mice. Pathology,echocardiography and molecular phenotype confirmed a dilated cardiomyopathy.Electronic microscopy revealed mitochondrial deterioration in hearts of transgenic mice and myofilaments degeneration in some limited regions of the hearts. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling analysis revealed greater abundance of apoptotic nuclei in TG mice hearts.To further investigate the mechanism of apoptosis in TG mice hearts,we found that the level of Bax protein was significantly increased.The level of Apaf-1 protein and the cleavage of Caspase-9 were significantly increased in transgenic mice hearts,whereas the protein abundance of c-FLIP and Caspase-8 were similar with WT controls.These data suggest that increased apoptosis in TG mice hearts is predominantly due to increased Bax-mediated mitochondria apoptosis pathway rather than FasL/TNF signaling pathway.To further examine potential connections between SIRT1H363Y and Bax-mediated apoptosis,we found that the level of p53 acetylation was upregulated upon dominant negative SIRT1 overexpression. Consistent with results in TG mice,adenoviral expression of SIRT1H363Y significantly upregulated Bax protein in neonatal rat ventricular myocytes,and the level of p53 acetylation was increased upon adenoviral SIRT1H363Y overexpression.In the present study,we generated TG mice exhibiting cardiac dominant negative SIRT1 overexpression using theα-myosin heavy chain(α-MHC) promoter.Cardiac overexpression of dominant negative SIRT1 is sufficient to cause cardiomyocytes apoptosis and dilated cardiomyopathy leading to a rapid deterioration in cardiac function and death in the early postnatal period. |
