Exogenous NAD blocks cardiac hypertrophic response via activation of the SIRT3-LKB1-AMP-activated kinase pathway

Abstract

Since the discovery of NAD-dependent deacetylases, sirtuins, it has been recognized that maintaining intracellular levels of NAD is crucial for the management of stress response of cells. Here we show that agonist-induced cardiac hypertrophy is associated with loss of intracellular levels of NAD, but not exercise-induced physiologic hypertrophy. Exogenous addition of NAD was capable of maintaining intracellular levels of NAD and blocking the agonist-induced cardiac hypertrophic response in vitro as well as in vivo. NAD treatment blocked the activation of pro-hypertrophic Akt1 signaling, and augmented the activity of anti-hypertrophic LKB1-AMPK signaling in the heart, which prevented subsequent induction of mTOR-mediated protein synthesis. By using gene knock-out and transgenic mouse models of SIRT3 and SIRT1, we showed that the anti-hypertrophic effects of exogenous NAD are mediated through activation of SIRT3, but not SIRT1. SIRT3 deacetylates and activates LKB1, thus augmenting the activity of the LKB1-AMPK pathway. These results reveal a novel role of NAD as an inhibitor of cardiac hypertrophic signaling, and suggest that prevention of NAD depletion may be critical in the treatment of cardiac hypertrophy and heart failure.

FIGURE 1.

Exogenous NAD blocks the PE-induced oxidative stress of cardiomyocytes. A, primary cultures of cardiomyocytes were treated with PE (20 μm) in the presence or absence of NAD (250 μm) for 15 min. Cells were stained with CM-H₂DCFDA. ROS production was measured by fluorescence-activated cell sorter. B, quantification of the mean fluorescence intensity in different groups of cells. Values are mean ± S.E. of five experiments.

FIGURE 2.

NAD treatment blocks cardiac hypertrophic response in vitro. A, cardiomyocytes were treated with PE either alone or together with NAD. After 48 h of treatment cells were harvested and NAD content measured. B, ATP content was measured in the same groups of cells. C, cardiomyocytes were treated with PE (20 μm) or Ang-II (2 μm) in the presence or absence of NAD and labeled with [³H]leucine. Forty-eight hours after treatment, cells were harvested, and [³H]leucine incorporation into total cellular protein was measured. D, cardiomyocytes were stimulated with PE and then treated with NAD, either alone or together with carbenoxolone (10 μm). Cardiomyocytes were identified by α-actinin staining using anti-α-actinin antibody (green). The release of ANF from nuclei was determined by staining cells with an anti-ANF antibody (red). 4′,6-Diamidino-2-phenylindole (DAPI) stain was used to mark the position of nuclei. E and F, cardiomyocytes were transfected with luciferase reporter plasmids responsive to ANF (E) or CARP (F). Luciferase activity was measured after 8 h of treatment with PE, in the presence or absence of NAD (G). Cardiomyocytes were infected with a NFAT responsive luciferase reporter adenovirus vector. Twelve hours after infection cells were treated with PE in the presence or absence of NAD, and luciferase activity was determined 8 h post-treatment. Cont., control. Values presented in histograms are mean ± S.E. of six experiments.

FIGURE 3.

NAD treatment blocks the agonist-induced cardiac hypertrophy in vivo. A, heart weight (HW)/body weight (BW) ratio of different groups of mice (vehicle, NAD, Ang-II, or Ang-II plus NAD-treated mice, as well as mice subjected to swim exercise). B, top panel, hematoxylin- and eosin-stained sections of whole hearts of different groups; middle panel, sections of hearts stained with Masson's trichrome to detect fibrosis (blue); bottom panel, heart sections stained with wheat germ agglutinin to demarcate cell boundaries. C and D, quantification of cardiac fibrosis and the myocyte cross-sectional area in different groups of mice. E and F, Anf, brain natiuretic peptide (BNP), β-myosin heavy chain (MHC), and Collagen-α mRNA levels in the heart samples of Ang-II alone or Ang-II plus NAD-treated mice. G and H, quantification of NAD and ATP contents in the heart lysate of different groups of mice as in panel A. Values are mean ± S.E. of five to eight experiments.

FIGURE 4.

Exogenous NAD blocks the agonist-induced signaling pathways involved in development of cardiac hypertrophy. A, cardiomyocytes were stimulated with PE in the presence or absence of NAD for 24 h. Cell lysate was prepared and analyzed by Western blotting with kinase-specific and phosphokinase-specific antibodies. Numbers in parentheses indicate the position of the phosphoamino acid recognized by the antibody. B, mice were treated with vehicle, NAD, Ang-II, or Ang-II plus NAD for 14 days. C, mice were subjected to swim exercise for 12 weeks. Heart lysate was analyzed by Western blotting with antibodies as indicated. In panel B and C, results are shown for two animals of each group.

FIGURE 5.

NAD treatment blocks the agonist-mediated cardiac hypertrophy independent of SIRT1. A, endogenous LKB1 was immunoprecipitated (IP) from heart lysates of mice treated with Ang-II, Ang-II plus NAD, or NAD alone. It was analyzed by Western blotting with anti-Ac-K antibody. The stripped blot was probed with anti-LKB1 antibody for loading control. B, expression of phospho (P)-LKB1 and phospho-AMPK in the hearts of wild-type and Sirt1^−/− mice. Total LKB1 and AMPK were used as loading controls. Results are shown for two mice of each group. C, heart weight/body weight ratio of wild-type (WT) and Sirt1^+/− mice treated with vehicle (cont.) ISO or ISO plus NAD for 7 days. D and E, quantification of cardiac fibrosis and the myocyte cross-sectional area in wild-type and Sirt1^+/− mice subjected to different treatments. F, expression levels of SIRT1 in wild-type and Sirt1^+/− hearts. G, Collagen-α and Anf mRNA levels in heart samples of wild-type and Sirt1^+/− mice subjected different treatments. H, primary cultures of neonatal rat cardiomyocytes were pretreated with vehicle (control), 20 μm compound C (comp.c), or 50 μm splitomicin (splitom.) and then stimulated with PE in the absence or presence of NAD. Cells were subsequently labeled with [³H]leucine and incorporation of the isotope into total cellular proteins was measured 48 h after PE treatment. Bar diagrams represent values ± S.E. of five experiments.

FIGURE 6.

SIRT3 binds to and deacetylates LKB1, leading to increased activity of the kinase. A, SIRT3 and SIRT1 expression levels in the hearts of mice treated with Ang-II, or Ang-II plus NAD. B, expression of phospho (P)-LKB1 and phospho-AMPK in the hearts of wild-type (WT) and Sirt3^−/− mice. Total LKB1 and AMPK were used as loading controls. C and D, quantification of P-LKB1 and P-AMPK levels in the hearts of wild-type and Sirt3^−/− mice. E, expression of phospho-LKB1 and phospho-AMPK in the hearts of non-transgenic (N.Tg) and Sirt3 transgenic (SIRT3.Tg) mice. F and G, quantification of P-LKB1 and P-AMPK levels in the hearts of non-transgenic and Sirt3 transgenic mice. H, hemagglutinin-tagged SIRT3 was immunoprecipitated (IP) from heart lysates of Sirt3·HA-Tg mice, and the resulting complex was analyzed by Western blotting with anti-LKB1 antibody. I, endogenous LKB1 was immunoprecipitated from heart lysates of wild-type and Sirt3^−/− mice, and analyzed by Western blotting with anti-Ac-K antibody. The stripped blot was probed with anti-LKB1 antibody for loading control. J, LKB1 immunoprecipitated from the heart lysate of a control mouse was subjected to in vitro acetylation by recombinant PCAF or P300. Beads with LKB1 were separated, washed, and subjected to Western blotting with Ac-K antibody. The stripped blot was probed with anti-LKB1 antibody for loading control. K, LKB1 was immunoprecipitated from heart lysates and subjected to acetylation in vitro with PCAF. Beads containing acetylated LKB1 were then incubated with recombinant Sirt3, in the presence or absence of NAD. The acetylation status of LKB1 was determined by Western blotting with anti-Ac-K antibody. The stripped blots were probed with anti-LKB1 antibody for loading controls. L, rat cardiomyocytes were overexpressed with adenovirus vectors expressing Sirt3 wild-type or the mutant, and treated with vehicle (control) or PE for 24 h. Cells were lysed and LKB1 was immunoprecipitated and analyzed by Western blotting with anti-Ac-K antibody. Stripped blots were probed with anti-LKB1 antibody for loading control. In panels A, B, E, and I, results are shown for two animals of each group.

FIGURE 7.

NAD treatment is unable to block the cardiac hypertrophic response of Sirt3^−/− mice. A, heart weight/body weight ratio of control (Veh), ISO, or ISO plus NAD-treated wild-type (WT) and Sirt3^−/− mice. B and C, quantification of the cross-sectional area of myocytes and fibrosis in different groups of mice. D and E, Collagen-α and Anf mRNA levels in heart samples of WT and Sirt3^−/− mice subjected to ISO-mediated hypertrophy in the presence or absence of NAD. F and G, echocardiographic measurements of LV fractional shortening and wall thickness of control (Veh), ISO, or ISO plus NAD-treated wild-type and Sirt3^−/− mice. Values are mean ± S.E., n = 6. H, scheme illustrating signaling pathways modified by NAD to block the cardiac hypertrophic response. Stimulation of cardiomyocytes with an agonist causes reduction of cellular Nampt and NAD levels, which results in the reduced activity of SIRT3. Deceased activity of SIRT3 negatively affects the activity of the LKB1-AMPK-Raptor signaling pathway, which leads to increased activity of mTOR. Reduced activity of AMPK also regulates positively the activity of Akt1, resulting in a further activation of mTOR and suppression of the activity of GSK3β. The net result of these changes is increased protein synthesis and development of cardiac hypertrophy. Exogenous NAD enters into cardiomyocytes via connexin 43 channels, and elevates cellular NAD levels, thereby activating SIRT3. SIRT3 activation stabilizes the activity of the LKB1-AMPK signaling pathway, thus blocking the prohypertrophic activity of mTOR and of Akt1. As reported before (9), SIRT3 activation can also block cardiac hypertrophy by reducing cellular ROS levels and in so doing suppress the ROS-mediated activation of Akt1 signaling.