Honokiol blocks and reverses cardiac hypertrophy in mice by activating mitochondrial Sirt3

Abstract

Honokiol (HKL) is a natural biphenolic compound derived from the bark of magnolia trees with anti-inflammatory, anti-oxidative, anti-tumour and neuroprotective properties. Here we show that HKL blocks agonist-induced and pressure overload-mediated, cardiac hypertrophic responses, and ameliorates pre-existing cardiac hypertrophy, in mice. Our data suggest that the anti-hypertrophic effects of HKL depend on activation of the deacetylase Sirt3. We demonstrate that HKL is present in mitochondria, enhances Sirt3 expression nearly twofold and suggest that HKL may bind to Sirt3 to further increase its activity. Increased Sirt3 activity is associated with reduced acetylation of mitochondrial Sirt3 substrates, MnSOD and oligomycin-sensitivity conferring protein (OSCP). HKL-treatment increases mitochondrial rate of oxygen consumption and reduces ROS synthesis in wild type, but not in Sirt3-KO cells. Moreover, HKL-treatment blocks cardiac fibroblast proliferation and differentiation to myofibroblasts in a Sirt3-dependent manner. These results suggest that HKL is a pharmacological activator of Sirt3 capable of blocking, and even reversing, the cardiac hypertrophic response.

Figure 1. HKL activates SIRT3 and deacetylates mitochondrial proteins

(A) Primary cultures of neonatal rat cardiomyocytes were treated with different doses of HKL as indicated. Mitochondrial lysate was prepared and analyzed for lysine-acetylation using anti-acetyl lysine antibody (Ac-K). Total MnSOD level served as a loading control. (B) Primary cultures of neonatal rat cardiomyocytes were treated with 10μM HKL at different time points as indicated. Mitochondrial lysate was prepared and analyzed for lysine-acetylation using anti-acetyl lysine antibody. (C) Primary cultures of cardiomyocytes were treated with 5 and 10μM HKL for 24 hrs. Cell lysate was analyzed by western blotting with indicated antibodies. (D, E, F) Quantification of relative SIRT3, acetylated (Ac) MnSOD and acetylated OSCP levels in cardiomyocytes treated with HKL. Values are average of four independent experiments, mean± SE. *P<0.05; Students t test.

Figure 2. HKL blocks induction of cardiac hypertrophic response in vitro

(A) Primary cultures of cardiomyocytes were treated with 20 μM phenylephrine (PE) in the presence or absence of 10 μM HKL. Forty eight hours after treatment, cells were harvested and mitochondrial lysate analyzed by western blotting with use of indicated antibodies. (B) Cultures of cardiomyocytes were labeled with [³H] leucine and then treated with PE (20 μM) in the presence or absence of 5 or 10μM HKL. Twenty hours after treatment cells were harvested and incorporation of [³H] leucine into total cellular proteins was measured. Mean ± SE, values are average of three independent experiments; Students t test. (C) 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 HKL for 8 hrs. HKL treatment was given 2hrs prior to PE treatment. The luciferase activity assay was performed using an activity assay kit from Promega, as per manufacturer’s protocol. Mean ± SE, values are average of three independent experiments; Students t test. (D) Cardiomyocytes were treated same as in panel ‘A’. Thereafter nuclear lysate was prepared and analyzed by western blotting with use of indicated antibodies. (E) Cardiomyocytes were treated with PE in the presence or absence of 10μM HKL. Cardiomyocytes were identified by α-actinin staining (green) and the release of ANF from nuclei was determined by staining cells with anti-ANF antibody (red). DAPI stain was used to mark the position of nuclei. scale bars, 25 μm (F) Cardiomyocyte size of α-actinin positive cells was quantified by use of Image J software. Values are expressed as fold change with respect to untreated control. Mean ± SE, values are average of three independent experiments; Students t test.

Figure 3. HKL blocks induction of cardiac hypertrophic response in vivo

(A) Heart weight body weight (HW/BW) ratio of control (Ct), TAC (transverse aortic constriction) and TAC mice treated with HKL, mean ± SE, n = 8-10 mice. (B) Expression levels of collagen-1, β-MHC and ANF mRNA in different groups of mice, mean ± SE, n = 8-10 mice, *P<0.01 compared to TAC alone. (C)Top panel, Sections of hearts stained with Masson’s trichrome to detect fibrosis (blue); scale bars, 20 μm; bottom panel, heart sections stained with wheat germ agglutinin (WGA) to demarcate cell boundaries, scale bars, 10 μm. (D and E) Quantification of cardiac fibrosis and myocyte cross-sectional area in different groups of mice. Mean ± SE, n = 5 mice. For the panels A, B, D and E, ANOVA was applied to calculate the P value. (F) Heart lysate of different groups of mice was subjected to immunoblotting using indicated antibodies. Results of two mice in each group are shown.

Figure 4. HKL attenuates pre-established cardiac hypertrophy in mice

(A) Mice were subjected to TAC for 4 months and then treated with HKL for 28 days. Bar diagram shows HW/BW ratio of control, TAC, TAC mice treated with HKL and HKL alone, mean ± SE, n = 5-8 mice; ANOVA. (B, C) Echocardiographic measurements of ejection fraction and fractional shortening in control, TAC, TAC treated with HKL and HKL alone mice. For panels A-C, mean ± SE, n = 5-8 mice; ANOVA was applied to calculate the P value. (D) top panel, whole heart of control, TAC and TAC treated with HKL and HKL alone mice; Scale bars, 1 mm; middle panel, H & E-stained sections of whole hearts of different groups of mice; scale bars, 1mm; bottom panel, sections of hearts stained with Masson’s trichrome to detect fibrosis (blue); scale bars, 20 μm. (E) Quantification of cardiac fibrosis in different groups of mice, mean ± SE, n = 5-8 mice; ANOVA was applied to calculate the P value. (F) β-myosin heavy chain (MHC), collagen-1 and ANF mRNA levels in the heart samples of control, TAC alone and TAC plus HKL and HKL alone treated mice. (G) Heart lysates of different groups of mice were subjected to immunoblotting with antibodies as indicated. Results are shown for two animals of each group.

Figure 5. HKL treatment blocks cardiac hypertrophy of wild-type, but not SIRT3-KO mice

(A) Heart weight body weight ratio of control (sham), isoproterenol (ISO) or ISO plus HKL treated wild-type (WT) and SIRT3-KO mice. Mean ± SE, n = 5-8 mice. *P<0.05, NS, not significant; ANOVA. (B) Heart sections stained with Masson’s trichrome to detect fibrosis (blue); Scale, 20 μm. (C) Quantification of cardiac fibrosis in different groups of mice. Mean ± SE, n = 5 mice. *P<0.001, NS, not significant; ANOVA. (D) Heart lysates analyzed by immunoblotting for the indicated antibodies.

Figure 6. HKL attenuates cardiac fibroblasts proliferation and differentiation into myofibroblasts

(A) Rat cardiac fibroblasts cultured in complete growth medium were treated with 5 or 10μM HKL. Sixteen hours after HKL treatment cells were treated with Brdu (10 μM) for 2 hrs. Cells were harvested, stained with anti-Brdu antibody (Y-axis) and 7AAD (X-axis) and subjected to FACS analysis. (B) Quantification of S-phase cells. Mean ± SE, values are average of three independent experiments, *P<0.05 compared to control; Students t test. (C) Cardiac fibroblasts were treated with 100 nM angiotensin-II (Ang) in the presence of 500 nM HKL for 72 hrs. Cells were immunostained for α-SMA and fibronectin; Scale 10 μm. (D) Primary cultures of cardiac fibroblasts were treated with 100 nM Ang in the presence or absence of HKL for 72 hrs. Cell lysates were prepared and analyzed by western blotting with indicated antibodies. Results are shown for two samples in each group.

Figure 7. HKL blocks differentiation of wild-type cardiac fibroblast to myofibroblasts, but not SIRT3-KO fibroblasts

(A, B) Primary cultures of mouse cardiac fibroblasts obtained from wild-type (WT) and SIRT3KO mice were treated with 100 nM Ang in the presence or absence of HKL for 72 hrs. Cells were immunostained for α-SMA and collagen-1; Scale 10μm. (C) Cell lysates were prepared from another set of plates and subjected to immunostaining for α-SMA. For loading control the blot was probed with an anti-GAPDH antibody.

Figure 8. HKL reduces ROS production and promotes cardiomyocyte survival under stress

(A) Primary cultures of cardiomyocytes obtained from wild type or SIRT3KO mice were treated with H₂O₂ (50 μM) in the presence or absence of HKL (10 μM) for 15 min. Cells were stained with CM-H₂DCFDA. ROS levels were measured by fluorescence-activated cell sorter. (B) Quantification of the mean fluorescence intensity in different groups of cells. Values are (mean ±SE) average of three independent experiments; Students t test. (C) Primary cultures of cardiomyocytes obtained from wild-type or SIRT3-KO mice were treated with H₂O₂ (500 μM) in the presence or absence of HKL (10 μM) for 2 hours. Extend of apoptosis was measured by estimating the percentage of Annexin V positive cells by FACS analysis. Data shows quantification of cell death, mean ± SE, Values are average of three independent experiments, Students t test. (D) Primary cultures of cardiomyocytes were treated with H₂O₂ (500 μM) in the presence or absence of HKL (10 μM) for 2 hours. Cell lysate was analyzed by western blotting with indicated antibodies. (E) Mitochondrial oxygen consumption rate (OCR) of WT and SIRT3-KO cardiac fibroblasts in response to HKL treatment, mean ± SE, Values are average of 4 independent experiments. *P<0.05 compared to WT untreated cells; Students t test.

Figure 9. HKL directly binds to and activates SIRT3

(A) Steady-state fluorescence anisotropy values are shown as a function of increased concentrations of HKL. The concentration of human SIRT3 was 3μM (a representative experiment). (B) In a deacetylase buffer 0.5ug of acetylated MnSOD was incubated with 0.5ug of SIRT3 in the presence or absence of HKL at the indicated concentrations of NAD. Samples were analyzed by immunoblotting with use of anti-MnSOD.AcK122 antibody. Blot was stripped and probed for MnSOD for equal loading. (C) Cardiomyocytes were treated with cycloheximide (10 μM) for 1hr and then with HKL (5 μM) for next 2 hrs. Mitochondrial lysate was prepared and analyzed by western blotting with use of indicated antibodies. (D) SIRT3 mRNA levels were measured after 6hrs of treatment of cardiomyocytes with 5 and 10 μM HKL, mean ± SE, values are average of three independent experiments; Students t test. (E) Cardiomyocytes were treated with 10μM HKL and PGC1α mRNA levels were measured 6 hrs after treatment. Values are average of three independent experiments (mean ± SE); Students t test (F) Wild-type or SIRT3KO fibroblasts were co-transfected with a PGC1α responsive promoter/ luciferase reporter plasmid. After 16 hours of transfection, cells were treated with 5 or 10μM of HKL for 8 hours. Cell lysates were prepared; luciferase activity was measured and normalized to protein content, mean ± SE, Values are average of four independent experiments; Students t test.

Figure 10. Model illustrating the mechanism of SIRT3 activation by HKL

In cardiomyocytes HKL can directly bind to and activate SIRT3. Increased activity of SIRT3 promotes deacetylation of mitochondrial targets, including MnSOD. These reactions lead to reduced synthesis of ROS and thereby reduced cellular oxidative stress. Activated SIRT3 can also cause activation of PGC1α, which activates SIRT3 gene promoter, leading to increased synthesis of SIRT3 mRNA transcripts. Increased activity of SIRT3 blocks cardiac hypertrophic response by suppressing ROS production and Akt activation, as reported by us before .