Study Of Effect Of3,3’-diindolylmethane On Cardiac Hypertrophy And The Mechanisms Involved In

BackgroundCardiac hypertrophy, largely caused by increased size and/or thickness of the ventricles in the heart, is an important compensatory mechanism for pathophysiological states. This may provide initial salutary compensation to the stress. However, sustained hypertrophic stimulation becomes a maladaptive response associated with untoward events, including cardiomyocyte fetal gene re-expression and cardiac interstitial fibrosis. Untreated, cardiac hypertrophy generally progresses to ventricular dilatation, systolic and diastolic contractile dysfunction, and heart failure. In fact, cardiac hypertrophy represents a well-established risk factor for cardiovascular mortality, and significant increased in the incidence of cardiovascular events, such as sudden death, ventricular arrhythmia, myocardial ischemia, heart failure, and may increase mortality. However, the mechanisms participate in the process of cardiac hypertrophy have not been clearly demonstrated. Up to now, there is no effective method to prevent and treat cardiac hypertrophy. Therapies for cardiac hypertrophy still focus on regulating hemodynamics. Thus, pharmacological interventions targeting the molecular changes involved in cardiac hypertrophy may provide promising approaches for protecting against cardiac hypertrophy and progression to heart failure.3,3’-Diindolylmethane (DIM) is the major in vivo product derived from the acid-catalyzed condensation of indole-3-carbinol (I3C) which is a Brassica food plant extract material. Increasing number of studies suggest that DIM has various properties, including eliminating free radicals, activating apoptotic signaling pathways, antioxidant and anti-angiogenic effects, and promoting the apoptosis of a variety of tumor cell. DIM can affect mitogen-activated protein kinases (MAPKs), phosphoinositide3-kinase (PI3K)/Akt and the nuclear factor-KB (NF-κB) signaling pathway to play anti-cancer, anti-angiogenic and anti-inflammatory roles. The molecular mechanisms of DIM inhibition of the hypertrophic response remain unknown. The purpose of this study were, therefore, to determine whether DIM attenuates cardiac hypertrophy induced by angiotensin Ⅱ (Ang II) in rat cardiac H9c2cells in vitro and pressure overload induced cardiac hypertrophy in C57BL/6wild type and AMPKa2knockout mice in vivo, as well as to identify the molecular mechanisms that may be responsible for its putative effects. MethodsPart one:To confirm the effect of DIM on cardiac hypertrophy, we used an in vitro model with Ang Ⅱ (1μM) in cultured rat cardiac H9c2cells.After stimulation with DIM (1,5, and10μM) with or without Ang Ⅱ (1μM) for24h, cells were characterized by immunofluorescence for cardiac a-actinin to assess cardiomyocyte hypertrophy and molecular markers of cardiac hypertrophy were determined by quantitative real-time PCR. After determine the optimal concentrationt, quantitative real-time PCR demonstrated the induction of ANP, BNP and β-MHC mRNA expression of the H9c2cells treated with DIM for6,12, and24h induced by Ang Ⅱ. The levels of phosphorylated and total protein of AMPKa and mTOR signaling in H9c2cells treated with DIM for0,15,30, and60min induced by Ang Ⅱ were determined by western blot analysis.Part two:The male wild-type C57BL/6mice aged8～10weeks and body weight ranged from23.5-27.5g were included. Surgery and subsequent analyses were performed in a blinded fashion for all groups. Mice received normal feed or feed containing0.05%DIM (dose:100mg/kg/day DIM). After1week we subjected the mice to either chronic pressure overload generated by AB or sham surgery as the control group. Mice were randomly assigned into four groups as DIM+Sham, DIM+AB, Vehicle+Sham, and Vehicle+AB. In a reverse experiment, normal feed containing0.05%DIM was administered to mice for7weeks beginning1week after aortic banding surgery to8weeks after surgery. Mice were randomly assigned into four groups:DIMR+Sham, DIMR+AB, Vehicle+Sham, and Vehicle+AB.8weeks after AB surgery, cardiac function of mice was examined by echocardiography and hemodynamics, and the heart weight/body weight (HW/BW), lung weight/body weight (LW/BW), and heartweight/tibia length (HW/TL) ratios were compared in mice of each group. H&E, WGA, and PSR staining were used in morphology detection. Molecular markers of cardiac hypertrophy and cardiac fibrosis were determined by quantitative real-time PCR. The levels of phosphorylated and total protein of AMPKa and mTOR signaling in myocardial tissue were determined by western blot analysis after8weeks of AB surgery.Part three:The male AMPKa2knockout (KO) mice aged8-10weeks and body weight ranged from23.5-27.5g were included. KO mice were randomly assigned to four groups:KO+Sham, KO+AB, KO+DIM+Sham, and KO+DIM+AB. Mouse chow feed containing0.05%DIM (dose:100mg/kg/day DIM) was initiated one week prior to surgery and continued for4weeks after surgery. Cardiac function of mice was examined by echocardiography and hemodynamics, and the HW/BW, LW/BW, and HW/TL were compared in mice of each group. H&E, WGA, and PSR staining were used in morphology detection. Molecular markers of cardiac hypertrophy and cardiac fibrosis were determined by quantitative real-time PCR. The levels of phosphorylated and total protein of mTOR signaling in myocardial tissue were determined by western blot analysis after4weeks of AB surgery.ResultsAfter stimulation with Ang Ⅱ (1μM), H9c2cells showed enlarged cell surface area compared to those induced by DIM (1,5, and10μM) with Ang Ⅱ for24h (P<0.05). Quantitative real-time PCR demonstrated that DIM(1,5, and10μM) markedly decreased the induction of ANP, BNP, and β-MHC mRNA expression induced by Ang II (1μM), especially in DIM (5μM) group (P<0.05). There are no siginificant differences between DIM (5μM) group and DIM (10μM) group (P>0.05). In addition, quantitative real-time PCR demonstrated that cells treated with DIM (5μM) for6,12, and24h markedly decreased the induction of ANP, BNP and β-MHC mRNA expression induced by Ang II (P<0.05). The activation of AMPKa was increased and mTOR signaling was inhibited in H9c2cells treatment with DIM (5μM) compared to those in response to Angll(1μM)(P<0.05).After8weeks of AB, the echocardiography and the hemodynamics results showed that DIM-treated mice have lower left ventricular wall thickness and ventricular cavity size, and higher left ventricular systolic and diastolic function than vehicle-treated mice (P<0.05). DIM-treated mice showed attenuated cardiac hypertrophy with improvements in HW/BW, LW/BW, HW/TL, and cardiomyocyte cross sectional area compared with vehicle-treated mice in both former and reverse experiments (P<0.05). The mRNA levels of cardiac hypertrophy and cardiac fibrosis markers including ANP, BNP,β-MHC, TGF-β1, TGF-β2,collagen Iα, collagen Ⅲ and CTGF in DIM-treated mice were lower than those in vehicle-treated mice (P<0.05), and the mRNA levels of a-MHC were higher (P<0.05). We also detected that DIM-treated enhanced the phosphorylation of AMPKa, and inhibited the mTOR signaling pathway induced by pressure overload using western blot analysis both in former and reverse experiments (P<0.05).These findings indicated that DIM attenuated cell hypertrophy mainly through stimulating p-AMPKa activity and inhibiting the mTOR signaling pathway in vitro and in vivo.In AMPKa2konckout (KO) mice, after4weeks of AB surgery, the echocardiography and the hemodynamics results showed higher left ventricular wall thickness and ventricular diameter, and lower left ventricular systolic and diastolic function induced by pressure overload (P<0.05), cardiac function was not improved after DIM-treated (P>0.05). In KO mice, pressure overload induced increased HW/BW, HW/TL and myocyte cross-sectional, according with the morphology of the gross hearts, hematoxylin-eosin staining, and wheat germ agglutinin staining (P<0.05). However, these measures were not improved after DIM-treated (P>0.05). In addition, the mRNA levels of ANP, BNP, β-MHC, TGF-β1, TGF-(32, collagen la, collagen III and CTGF which were the induction of hypertrophic and fibrosis markers, were greatly escalated and a-MHC was decreased in both DIM-treated and vehicle-treated KO mice in response to AB (P>0.05). We detected that, in AMPKa2deficiency mice, DIM-treated couldn’t inhibit the mTOR signaling pathway induced by pressure overload using western blot analysis (P>0.05). DIM had no protective effect on cardiac hypertrophy induced by pressure overload in AMPKa2knockout mice.Conclusion1. DIM significantly inhibits rat cardiac H9c2cells hypertrophy induced by Ang Ⅱ in vitro through direct promotion the phosphorylation of AMPKa and inhibition of mTOR signaling pathway.2. DIM can not only prevent the development of cardiac hypertrophy and fibrosis but also reverse established cardiac hypertrophy and fibrosis induced by pressure overload in C57BL/6mice by promotion the phosphorylation of AMPKa and inhibition of mTOR signaling pathway.3. DIM has no protective property against cardiac hypertrophy and fibrosis induced by pressure overload in AMPKa2knockout mice; DIM can’t inhibit the mTOR signaling pathway in AMPKa2deficiency mice.