Histone Acetyltransferase p300 Inhibitor Improves Coronary Flow Reserve in SIRT3 (Sirtuin 3) Knockout Mice

Figures

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  Figure 1. C646 treatment improved coronary microvascular dysfunction and cardiac dysfunction in SIRT3KO mice. A, Representative images of coronary flow reserve recording in mouse coronary artery. Coronary flow reserve was significantly impaired in the SIRT3KO mice. C646 treatment significantly improved coronary flow reserve in the SIRT3KO mice (n=5 mice). Mean±SEM, *P<0.05. B, Representative images of PWV recording in mouse aorta. PWV was significantly elevated in the SIRT3KO mice. Increased PWV was significantly attenuated by C646 treatment in the SIRT3KO mice (n=6 mice). Mean±SEM; *P<0.05, **P<0.01. C, Representative images of M‐mode in a mouse heart. Knockout of SIRT3 reduced levels of EF% and FS% compared with WT mice. Treatment with C646 significantly reversed these changes (n=6 mice), Mean±SEM; *P<0.05, ***P<0.001. EF indicates ejection fraction; FS, fractional shortening; PWV, pulse‐wave velocity; SIRT3, sirtuin 3; SIRT3KO, SIRT3 knockout; and WT, wild type.

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  Figure 2. C646 reversed pre‐existing cardiac remodeling in SIRT3KO mice. A, Interstitial and perivascular fibrosis were increased in the SIRT3KO mice (n=3 mice) compared with WT mice (n=3 mice). C646 treatment suppressed increased interstitial and perivascular fibrosis in the SIRT3KO mice significantly (n=3 mice). Mean±SEM; *P<0.05, **P<0.01. B, Knockout of SIRT3 resulted in increased cardiomyocyte size, heart weight:tibia length ratio, and β‐MHC expression in the heart (n=3 mice), whereas treatment with C646 reversed these alterations (n=3 mice). Mean±SEM; *P<0.05, **P<0.01. C, Capillary density was reduced in the SIRT3KO mice (n=3 mice) in comparison with WT mice (n=3 mice). This reduction in capillary density was reversed by treatment with C646 (n=3 mice). Mean±SEM; **P<0.01. βMHC indicates β‐myosin heavy chain; DAPI, 4′,6‐diamidino‐2‐phenylindole; IB4, isolectin B4; SIRT3, sirtuin 3; SIRT3KO, SIRT3 knockout; and WT, wild type.

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  Figure 3. C646 improved eNOS and oxidative stress in SIRT3KO mice. A, Treatment with C646 reversed reduction of eNOS levels in the SIRT3KO mice (n=4 mice). Mean±SEM; *P<0.05, **P<0.01. B, By DHE staining, ROS formation was enhanced in the SIRT3KO mice (n=3 mice) compared with the WT mice (n=3 mice). This enhancement was suppressed by treatment with C646 (n=3 mice). Mean±SEM; **P<0.01. DHE indicates dihydroethidium; eNOS, endothelial nitric oxide synthase; ROS, reactive oxygen species; SIRT3, sirtuin 3; SIRT3KO, SIRT3 knockout; and WT, wild type.

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  Figure 4. C646 treatment‐inhibited arginase II expression and arginase activity in SIRT3KO mice. A, Arginase activity was enhanced in the SIRT3KO mice (n=5 mice) in comparison with the WT mice (n=5 mice). Treatment with C646 inhibited arginase activity significantly (n=5 mice). Mean±SEM; **P<0.01. B, No difference in arginase I expression was found among the WT mice (n=3 mice), SIRT3KO mice (n=4 mice), or SIRT3KO mice+C646 (n=4 mice). C, Treatment with C646 reversed the increased arginase II expression in the SIRT3KO mice (n=4 mice). Mean±SEM; *P<0.05, **P<0.01. SIRT3 indicates sirtuin 3; SIRT3KO, SIRT3 knockout; and WT, wild type.

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  Figure 5. Treatment with C646 suppressed VCAM‐1 expression in SIRT3KO mice. A, Knockout of SIRT3 increased VCAM‐1 expression (n=4 mice), whereas C646 treatment inhibited VCAM‐1 expression significantly (n=4 mice). Mean±SEM; *P<0.05, **P<0.01. B, By costaining IB4 and VCAM‐1, we found that VCAM‐1 expression was colocalized with endothelial cells. Knockout of SIRT3 increased the number of IB4+/VCAM‐1+ cells (n=3 mice), and treatment with C646 reversed this increase (n=3 mice). Similarly, enhanced expression of VCAM‐1 in the endothelial cells of the coronary artery was found in SIRT3KO mice compared with WT mice. Treatment with C646 suppressed the up‐regulation of VCAM‐1 in the endothelial cells of the coronary artery. C, C646 increased eNOS and reduced arginase II and VCAM‐1 expression in the cultured endothelial cells. Exposure of cultured endothelial cells with C646 (3 μmol/L) significantly increased eNOS expression (n=3 mice). Mean±SEM; *P<0.05. Treatment with C646 resulted in a dose‐dependent reduction of arginase II expression in the cultured endothelial cells (n=3 mice). Mean±SEM; *P<0.05, **P<0.01, ***P<0.001. Treatment with C646 (3 μmol/L) significantly reduced VCAM‐1 levels in the…

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  Figure 6. Treatment with C646 reduced expression of NF‐κB and associated factors in SIRT3KO mice. A, Knockout of SIRT3 increased the expression of NF‐κB, TLR‐4, MyD88, and IRAK‐4 in the mouse hearts (n=4 mice). This increase was reversed by treatment with C646 (n=3–4 mice). Mean±SEM; *P<0.05, **P<0.01, ***P<0.001. B, Treatment with C646 (3 μmol/L) significantly reduced the basal levels of NF‐κB, TLR‐4, MyD88, and IRAK‐4 in the H9C2 cell lines (n=3 cell lines). Mean±SEM; **P<0.01. C, Immunofluorescence analyses of SIRT3 localization in the cultured ECs using an antibody specific to SIRT3. DAP1 was used to visualize the nuclei. The SIRT3 antibodies recognized a protein that resided exclusively in the nucleus that was colocalized with the blue DAPI regions (white arrow). D, Western blot analysis showed that knockout of SIRT3 resulted in a significant increase in p300 expression in the mouse hearts. C646 treatment significantly reduced the levels of p300 in the SIRT3KO mice (n=4 mice, **P<0.01). E, Western blot analysis showed that knockout of SIRT3 led to a significant increase in H3K56 acetylation. C646 treatment significantly attenuated the acetylated H3K56 levels in the hearts of SIRT3KO…