Natl. Although high levels of NAD up-regulate the levels of sirtuins, high levels of NADH and nicotinamide do the opposite. FGF10 A recent report has indicated that exogenous NAD can enter the neurons and protect them from degeneration and ischemia-induced cell death (15). It is therefore likely that by maintaining an adequate intracellular level of NAD, cardiomyocytes could be also protected from hypertrophy and cell death by increasing the activity of one or more sirtuin analogues. ELX-02 disulfate In this study we report that pathologic cardiac hypertrophy is associated with depletion of cellular NAD levels. Exogenous supplementation of NAD restores the intracellular levels of NAD and blocks the cardiac hypertrophic response. We also demonstrate that anti-hypertrophic effects of NAD are ELX-02 disulfate mediated via activation of SIRT3, but not SIRT1. The experiments performed to delineate the downsteam mechanism of NAD-mediated cardiac protection demonstrated that exogenous NAD restored the activity of endogenous anti-hypertrophic signaling via activation of the SIRT3-LKB1-AMPK pathway. To the best of our knowledge, this report represents the first application of NAD in the treatment of cardiac hypertrophy. MATERIALS AND METHODS Induction of Hypertrophy in Mice Angiotensin-II or isoproterenol were dissolved in 150 mm NaCl and 1 mm acetic acid. Angiotensin-II was delivered chronically at a rate of 3.0 mg/kg/day for 14 days. Isoproterenol was infused at a rate of 8.7 mg/kg/day for 7 days by implanting osmotic mini-pumps (ALZET model 2002) in the peritoneal cavity of mice. Control mice underwent the same procedure, except that the respective pumps were filled only with vehicle (150 mm NaCl, 1 mm acetic acid). Exogenous NAD was given at the rate of 1 1 mg/kg/day for the entire duration of treatment with the hypertrophy agonist. Physiologic hypertrophy was induced by subjecting the mice to swimming for 12 weeks at 1-h/day for 5 days a week. Primary Cultures of Cardiomyocytes, Transfection/Infection, and Luciferase Assay ELX-02 disulfate Neonatal rat cardiomyocytes were cultured and infected with adenoviral vectors as described earlier (16). and knock-out mice were generously provided by F. W. Alt and M. W. McBurney, respectively. All experiments with transgenic mice, except interaction of with LKB1, were performed using transgenic mice expressing the short form of SIRT3 (9). For studying interaction of SIRT3 with LKB1, transgenic mice expressing the long form of with the C-terminal hemagglutinin tag was utilized. These transgenic mice were generated and characterized similar to the method described earlier (9). In Vitro Acetylation Assay Immunoprecipitated LKB1 bound to beads were incubated with 200 ng of active PCAF (or P300) enzyme (Upstate Biotechnology), 0.5 mm acetyl-CoA (Sigma), 50 mm nicotinamide, and 10 m trichostatin in 1 HAT buffer (50 mm Tris, pH 8.0, 10% glycerol, 0.1 mm EDTA, 1 mm dithiothreitol) for 30 min at 30 C on a rotator. Unacetylated LKB1 contained all components except acetyl-CoA. Reactions were terminated by adding SDS sample buffer and resolved on a 10% SDS-polyacrylamide gel. Proteins were transferred to a Hybond-P membrane (GE Healthcare) and detected by Western analysis with anti-Ac-K antibody. In Vitro Deacetylation Assay Immunoprecipitated and acetylated LKB1 bound to beads was resuspended in 1 HDAC buffer (25 mm Tris/Cl, pH 8.0, 137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2, and 0.1 mg/ml of bovine serum albumin). For SIRT3-mediated deacetylation, 40 ng/l of SIRT3 (catalytic domain, Abcam, Inc.) was added to HDAC buffer in the presence or absence of 500 m NAD. The reaction mixture was incubated for 1 h at 30 C on a nutator. Proteins were resolved by SDS-PAGE and analyzed by either autoradiography or Western blotting with anti-Ac-K antibody. Echocardiography of Mice Chest hair of mice were removed with a topical depilatory agent and transthoracic echocardiography was performed under inhaled isoflurane (1%) for anesthesia, delivered via nose cone. Limb leads were attached for electrocardiogram gating, and the animals were imaged in the left lateral decubitus position with a VisualSonics Vevo 770 machine, using a 30 MHz high frequency transducer. Body temperature was maintained using a heated imaging platform and warming lamps. Two-dimensional images were recorded in parasternal long- and short-axis projections, with guided M-mode recordings at the midventricular level in both views. LV (left ventricle) cavity size and wall thickness were measured in at least 3 beats from each projection and averaged. LV.