3,3'-Diindolylmethane protects against cardiac hypertrophy via 5'-adenosine monophosphate-activated protein kinase-α2

Abstract

Purpose: 3,3'-Diindolylmethane (DIM) is a natural component of cruciferous plants. It has strong antioxidant and anti-angiogenic effects and promotes the apoptosis of a variety of tumor cells. However, little is known about the critical role of DIM on cardiac hypertrophy. In the present study, we investigated the effects of DIM on cardiac hypertrophy.

Methods: Multiple molecular techniques such as Western blot analysis, real-time PCR to determine RNA expression levels of hypertrophic, fibrotic and oxidative stress markers, and histological analysis including H&E for histopathology, PSR for collagen deposition, WGA for myocyte cross-sectional area, and immunohistochemical staining for protein expression were used.

Results: In pre-treatment and reverse experiments, C57/BL6 mouse chow containing 0.05% DIM (dose 100 mg/kg/d DIM) was administered one week prior to surgery or one week after surgery, respectively, and continued for 8 weeks after surgery. In both experiments, DIM reduced to cardiac hypertrophy and fibrosis induced by aortic banding through the activation of 5'-adenosine monophosphate-activated protein kinase-α2 (AMPKα2) and inhibition of mammalian target of the rapamycin (mTOR) signaling pathway. Furthermore, DIM protected against cardiac oxidative stress by regulating expression of estrogen-related receptor-alpha (ERRα) and NRF2 etc. The cardioprotective effects of DIM were ablated in mice lacking functional AMPKα2.

Conclusion: DIM significantly improves left ventricular function via the activation of AMPKα2 in a murine model of cardiac hypertrophy.

Figure 1. DIM attenuated cardiac hypertrophy induced by pressure overload.

(A and B) A, Echocardiography results and B, pressure volume results from four groups of mice at 8 weeks after AB or sham surgery. (C) Statistical results of HW/BW ratio, HW/TL ratio (n = 12) and myocyte cross-sectional areas (n = 100 cells per section) at 8 weeks after AB or sham surgery. (D) Histology: representative gross hearts (top), H&E staining (middle), and WGA–FITC staining (bottom) at 8 weeks after AB or sham surgery. (E) Real-time PCR analysis of hypertrophic markers including ANP, BNP, α-MHC and β-MHC from hearts of mice in the indicated groups (n = 6). (F and G) F, Echocardiography and G, pressure volume results from four groups of mice at 8 weeks after AB or sham surgery in reverse experiments. (H) Statistical results of HW/BW ratio, HW/TL ratio (n = 9) and myocyte cross-sectional areas (n = 100 cells per section) at 8 weeks after AB or sham surgery in reverse experiments. (I) Histology: representative gross hearts (top), H&E staining (middle), and WGA–FITC staining (bottom) at 8 weeks after AB or sham surgery in reverse experiments. (J) Real-time PCR analysis of hypertrophic markers including ANP, BNP, α-MHC and β-MHC, from hearts of mice in the indicated groups in reverse experiment (n = 6). Values are expressed as the mean±SEM. *P<0.05 compared with the corresponding sham group. ^# P<0.05 vs Vehicle+AB group.

Figure 2. The effect of DIM on AMPKα and mTOR/p-70S6K signaling.

(A and B) The protein levels of phosphorylated and total AMPKα, mTOR, p70S6K, S6, eIF4e and 4E-BP1 in mice from indicated groups. A, Representative Western blots. B, Quantitative results (n = 6). (C and D) The protein levels of phosphorylated and total AMPKα, mTOR, p70S6K, S6, eIF4e and 4E-BP1 in mice from indicated groups in reverse experiments (n = 6). C, Representative Western blots. D, Quantitative results. Values are expressed as the mean±SEM. *P<0.05 compared with the corresponding sham group. ^# P<0.05 vs Vehicle+AB group.

Figure 3. DIM blocked the fibrosis induced by pressure overload.

(A) Representative images of PSR staining form indicated groups. (B) Quantitative analysis of left ventricle interstitial collagen volume fraction in indicated groups (n = 6). (C) Real-time PCR analysis of the mRNA expression of TGF-β1, TGF-β2, collagen Iα, col1agen β and CTGF in the myocardium obtained from indicated groups (n = 6). (D) Representative images of PSR staining form indicated groups. (E) Quantitative analysis of left ventricle interstitial collagen volume fraction in indicated groups in reverse experiments (n = 6). (F) Real-time PCR analysis of the mRNA expression of TGF-β1, TGF-β2, collagen Iα, col1agen β and CTGF in the myocardium obtained from indicated groups in reverse experiments (n = 6). The results were reproducible in three separate experiments. Values are expressed as mean±SEM. *P<0.05 compared with the corresponding sham group. ^# P<0.05 vs Vehicle+AB group.

Figure 4. DIM inhibited cardiac hypertrophy by targeting AMPKα2.

(A and B) Echocardiography and pressure volume results from four groups of mice at 4 weeks after AB or sham surgery. (C) Statistical results of the HW/BW ratio, HW/TL ratio (n = 12) and myocyte cross-sectional areas (n = 100 cells per section) at 4 weeks after AB or sham surgery. (D) Histology: representative gross hearts (top), H&E staining (middle) and WGA–FITC staining (bottom). (E) Real-time PCR analysis of hypertrophic markers including ANP, BNP, α-MHC and β-MHC, from hearts of mice in the indicated groups (n = 6). Values are expressed as mean±SEM. *P<0.05 compared with the corresponding sham group. ^# P<0.05 vs KO+AB group.

Figure 5. AMPKα2 is necessary in the anti-fibrotic effect of DIM.

(A) Representative images of PSR staining form indicated groups. (B) Quantitative analysis of left ventricle interstitial collagen volume fraction in indicated groups (n = 6). (C) Real-time PCR analysis of the mRNA expression of TGF-β1, TGF-β2, collagen Iα, col1agen β and CTGF in the myocardium obtained from indicated groups (n = 6). The results were reproducible in three separated experiments. Data are expressed as means±SEM. *P<0.05 compared with the corresponding sham group. ^# P<0.05 vs KO+AB group.

Figure 6. DIM inhibited Cardiac Oxidative Stress through AMPKα2.

(A and B) Real-time PCR analysis of the mRNA expression of ERRα, Nrf2 and its downstream genes including GPx, HO-1, Txn-1, Txnrd-1, SOD1, SOD-2 and SOD-3 in the myocardium obtained from indicated groups (n = 6). A, in WT mice and B, in AMPKα2 KO mice. (C and D) Representative immunohistochemial staining of Nrf2 protein expression in WT mice and AMPKα2 KO mice after AB. (E and F) The protein levels of the cardiac expression of Nrf2 and ERRα in the myocardium obtained from indicated groups (n = 6). E, in WT mice and F, in AMPKα2 KO mice. Top, Representative Western blots; bottom, Quantitative results. *P<0.05 compared with the corresponding sham group. ^# P<0.05 vs Vehicle+AB/KO+AB group.