Cytotoxic effects of cucurbitacin I in cultured neonatal rat cardiomyocytes.

<p>Cell viability was measured in cardiomyocytes treated with the DMSO (control) or cucurbitacin I at 0.1, 0.5, 1, 5, and 10 μM for 24 h (A), 48 h (B), and 72 h (C). (D) Cell viability at each concentration on a time-dependent manner. Data are expressed as the means ± S.D. from three independent experiments. Significance was determined via a two-way ANOVA. *<i>P</i> < 0.05 vs. control group. Cont, control; Cu I, cucurbitacin I.</p

The anti-hypertrophic impact of cucurbitacin I is attenuated in the CTGF-silenced, hypertrophic cardiomyocytes.

<p>(A) Expression of CTGF was measured by western blotting in cardiomyocytes transfected with control or CTGF siRNA. (B) Representative photograph of cardiomyocytes after transfection with a scrambled siRNA or CTGF siRNA and pretreatment with cucurbitacin I (1 μM), followed by exposure to PE (100 μM). Sarcomeric organization of the cardiomyocytes was visualized by staining with an anti-α-actinin antibody. (C) Cell surface areas were measured by using NIH Image J software (n = 100 cells). Scale bars, 50 μm. (D) Quantitative RT-PCR analysis of ANF and β-MHC mRNA expression levels in scrambled siRNA- or CTGF siRNA-transfected cardiomyocytes treated with cucurbitacin I and/or PE. All experiments were performed in triplicate with three independent samples. Data are expressed as fold changes ± S.D. vs. control group. Significance was measured via a two-way ANOVA. * <i>P</i> < 0.05. scr, scrambled siRNA; si-CTGF, si-RNA against CTGF; Cont, control; Cu I, Cucurbitacin I; NS, not significant.</p

Cucurbitacin I attenuates hypertrophic responses in PE-stimulated cardiomyocytes.

<p>(A) Representative photograph of cardiomyocytes treated with PE (100 μM) after cucurbitacin I (1 μM) pretreatment. Sarcomeric organization of the cardiomyocytes was visualized by staining with an anti-<b>α</b>-actinin antibody. The sarcomeric response was completely blocked by pretreatment with cucurbitacin I. (B) Cell surface areas were measured by using NIH Image J software (n = 100 cells). Scale bars, 50 μm. (C) Quantitative RT-PCR analysis for ANF and β-MHC mRNA expression in cucurbitacin I-pretreated/PE-treated rat cardiomyocytes. The RT-PCR analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± S.D. vs. the control group. Significance was measured via a two-way ANOVA. * <i>P</i> < 0.05. Cont, control; Cu I, Cucurbitacin I.</p

Cucurbitacin I inhibits CTGF expression and MAPK signaling in hypertrophic cardiomyocytes.

<p>(A) CTGF mRNA expression levels were measured by quantitative RT-PCR in hypertrophic cardiomyocytes stimulated with PE (100 μM) for 1, 6, 12, and 24 h. (B) Cardiomyocytes were pretreated with cucurbitacin I (1 μM), and then stimulated with PE (100 μM) for 6 h. Next, CTGF mRNA expression levels were measured by quantitative RT-PCR. (C) Cardiomyocyte extracts (50 μg) were subjected to Western blot analysis of CTGF and MAPK, (ERK1/2, JNK, and p38) protein expression levels. The expression levels of CTGF, the MAPKs, and the phosphorylated forms of the MAPKs (p-ERK1/2, p-JNK, and p-p38 kinase) were estimated by measuring band densities with NIH Image J software. GAPDH was used as a loading control, and Western blot analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± S.D. vs. control group. Significance was measured via a two-way ANOVA. * <i>P</i> < 0.05. Cont, control; Cu I, Cucurbitacin I.</p

Cucurbitacin I blocks the TGF-β/Smad signaling pathway in hypertrophic cardiomyocytes.

<p>Cell extracts (50 μg) were used for Western blot analysis of TGF-β and Smad (Smad2, 3, and 7) protein expression levels. The expression levels of TGF-β, Smad2, 3, and 7, and the phosphorylated forms of Smad2 and 3 (p-Smad2 and p-Smad3) were estimated by measuring band densities with NIH Image J software. GAPDH was used as a loading control. And Western blot analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± S.D. vs. control group. Significance was measured via a two-way ANOVA. * <i>P</i> < 0.05. Cont, control; Cu I, Cucurbitacin I.</p

β-Lapachone Ameliorates Lipotoxic Cardiomyopathy in Acyl CoA Synthase Transgenic Mice

<div><p>Lipotoxic cardiomyopathy is caused by myocardial lipid accumulation and often occurs in patients with diabetes and obesity. This study investigated the effects of β-lapachone (β-lap), a natural compound that activates Sirt1 through elevation of the intracellular NAD⁺ level, on acyl CoA synthase (ACS) transgenic (Tg) mice, which have lipotoxic cardiomyopathy. Oral administration of β-lap to ACS Tg mice significantly attenuated heart failure and inhibited myocardial accumulation of triacylglycerol. Electron microscopy and measurement of mitochondrial complex II protein and mitochondrial DNA revealed that administration of β-lap restored mitochondrial integrity and biogenesis in ACS Tg hearts. Accordingly, β-lap administration significantly increased the expression of genes associated with mitochondrial biogenesis and fatty acid metabolism that were down-regulated in ACS Tg hearts. β-lap also restored the activities of Sirt1 and AMP-activated protein kinase (AMPK), the two key regulators of metabolism, which were suppressed in ACS Tg hearts. In H9C2 cells, β-lap-mediated elevation of AMPK activity was retarded when the level of Sirt1 was reduced by transfection of siRNA against Sirt1. Taken together, these results indicate that β-lap exerts cardioprotective effects against cardiac lipotoxicity through the activation of Sirt1 and AMPK. β-lap may be a novel therapeutic agent for the treatment of lipotoxic cardiomyopathy.</p></div

β-Lap preserves mitochondrial structure and functions in ACS Tg mice.

<p><b>A</b>. Electron microscopy images of mitochondrial structure and quantification of the mitochondrial surface area in sections of hearts from Wt and ACS Tg mice administered either vehicle (Veh) or β-lap (βL). Scale bar is 1 µm. <b>B</b>. Western blot analysis of Complex II protein level. Mice heart lysates (50 µg) were subjected to western blot analysis, and band density was quantified using NIH Image J software. GAPDH was used as a loading control. <b>C</b>. qRT-PCR analysis of mt-Co1 and mt-Cyt b transcript levels in mouse hearts. <b>D</b>. qRT-PCR analysis of genes involved in mitochondrial biogenesis (NRF-1, PPARα, ERRαα and PGC-1β) and FA metabolism (MCAD, PDK4, GPAT, CPT1-β, UCP2, and ATP6i). n = 4−6 per group. Significance was measured via two-way ANOVA. *<i>p</i><0.05, **<i>p</i><0.001.</p

β-Lap prevents lipid accumulation in the heart of ACS Tg mice.

<p>Triglyceride contents in hearts from Wt and ACS Tg mice administered either vehicle (Veh) or β-lap (βL). n = 7−8 per group. Significance was measured via two-way ANOVA. *<i>p</i><0.05, **<i>p</i><0.001.</p

Echocardiographic parameters.

<p>Wt, wild-type mice; Tg, ACS overexpressing transgenic mice; Veh, vehicle; βL, β-lapachone; IVSTd, Interventricular septum in diastole; LVIDd, Left ventricular internal dimension in diastole; LVPWd, Left ventricular posterior wall thickness in diastole; IVSTs, Interventricular septum in systole; LVIDs, Left ventricular internal dimension in systole; LVPWs, Left ventricular posterior wall thickness in systole; EF, Ejection fraction; FS, Fractional shortening. Significance was measured via two-way ANOVA.</p><p>**<i>p</i><0.001 vs Wt,Veh.</p>≠<p><i>p</i><0.05 vs Tg,Veh.</p>≠ ≠<p><i>p</i><0.001 vs Tg,Veh.</p

β-Lap attenuates cardiac hypertrophy and fibrosis in ACS Tg mice.

<p><b>A</b>. Cross-sections of the hearts and assessment of heart weight/body weight (HW/BW) ratios in Wt and ACS Tg mice administered either vehicle (Veh) or β-lap (βL). Scale bar is 1 mm. <b>B</b>. Higher magnification images of the heart sections. Cell surface area (CSA) of individual cardiomyocytes was measured using the AnalySIS image analysis program. Scale bar is 50 µm. <b>C</b>. Trichrome staining of histological sections. Fibrotic areas were quantified using the AnalySIS image analyzer on histological sections. Scale bar is 100 µm. <b>D</b>. qRT-PCR analysis of collagen I and TGF- β1 transcript levels in mouse hearts. n = 4−6 per group. Significance was measured via two-way ANOVA. *<i>p</i><0.05, **<i>p</i><0.001.</p