Tetrandrine blocks cardiac hypertrophy by disrupting reactive oxygen species-dependent ERK1/2 signalling

Abstract

Background and purpose: Tetrandrine, a well-known naturally occurring calcium antagonist with anti-inflammatory, antioxidant and anti-fibrogenetic activities, has long been used clinically for treatment of cardiovascular diseases such as hypertension and arrhythmia. However, little is known about the effect of tetrandrine on cardiac hypertrophy. The aims of the present study were to determine whether tetrandrine could attenuate cardiac hypertrophy and to clarify the underlying molecular mechanisms.

Experimental approach: Tetrandrine (50 mg x kg(-1) x day(-1)) was administered by oral gavage three times a day for one week and then the mice were subjected to either chronic pressure overload generated by aortic banding (AB) or sham surgery (control group). Cardiac function was determined by echocardiography.

Key results: Tetrandrine attenuated the cardiac hypertrophy induced by AB, as assessed by heart weight/body weight and lung weight/body weight ratios, cardiac dilatation and the expression of genes of hypertrophic markers. Tetrandrine also inhibited fibrosis and attenuated the inflammatory response. The cardioprotective effects of tetrandrine were mediated by blocking the increased production of reactive oxygen species and the activation of ERK1/2-dependent nuclear factor-kappaB and nuclear factor of activated T cells that occur in response to hypertrophic stimuli.

Conclusions and implications: Taken together, our results suggest that tetrandrine can improve cardiac function and prevent the development of cardiac hypertrophy by suppressing the reactive oxygen species-dependent ERK1/2 signalling pathway.

Figure 1

Tetrandrine attenuated cardiac hypertrophy in vivo. (A) Gross hearts (top), representative whole hearts section (middle) and HE staining (bottom) at 8 weeks after aortic banding or sham operation(n= 6). (B–D) mRNA and protein expression levels of hypertrophic markers, ANP, BNP and Myh7, which were evaluated by real-time PCR (n= 3) and western blot (n= 4). *P < 0.01, compared with the vehicle-sham values. †P < 0.01, compared with the vehicle-AB values after AB. AB, aortic banding; ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; HE, haematoxylin and eosin; Myh7, myosin heavy chain 7.

Figure 2

Tetrandrine blocked fibrosis, but not apoptosis. (A,B) Representative images of PSR staining form indicated groups and quantitative analysis of left ventricle interstitial collagen volume fraction in indicated groups (n= 6). *P < 0.01, compared with the vehicle-sham values. †P < 0.01, compared with the vehicle-AB values after AB. (C) Protein expression of CTGF, collagen I and III were tested by western blot analysis. GAPDH was used as internal control (n= 4). (D) Real-time PCR analysis of TGF-β1, Col1a1, Col1a3 and CTGF mRNA level (n= 4). *P < 0.01, compared with the vehicle-sham values. †P < 0.01, compared with the vehicle-AB values after AB. (E) TUNEL positive cells from histological sections of indicated groups were quantified (n= 5). *P < 0.01, compared with the vehicle-sham values. (F) Western blot analysis of cleaved caspase-3, caspase-8 and caspase-9 as well as the expression of Bcl-2, Bad and Bax proteins in the indicated groups (n= 5). AB, aortic banding; BW, body weight; CTGF, connective tissue growth factor; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; PSR, picrosirius red; TUNEL, TdT-mediated dUTP nick end labelling.

Figure 3

Tetrandrine attenuated the excess production of reactive oxygen species (ROS) in vitro and in vivo. (A) The time courses for the effect of tetrandrine on the generation of ROS induced by angiogensin II (Ang II). Cardiac myocytes were pretreated with 10 µM tetrandrine for 30 min and subsequently incubated with 1 µM Ang II for 120 min. Four parallel experiments were indicated. *P < 0.05, compared with control (0 Ang II). (B) Tetrandrine inhibited the pressure overload-induced increase of signal decay rate (n= 5). (C) Representative blots for the oxidation of myofibrillar proteins tropomyosin and α-sarcomeric actin and their quantitative analysis for each indicated group of mice. *P < 0.01, compared with the vehicle-sham values. †P < 0.01, compared with the vehicle-aortic banding (AB) values after AB.

Figure 4

Tetrandrine inhibited the inflammatory response induced by pressure overload. (A) The DNA binding activity of NF-κB evaluated by EMSA in the hearts from the indicated groups (n= 6). (B) NF-κB activity was determined in cardiomyocytes treated with Ang II for the indicated times (n= 6). The concentration of tetrandrine was 10 µM. (C) Representative blots of IκBα degradation, and IκBα and IKKβ phosphorylation in the myocardium obtained from the indicated groups (n= 4). (D) Quantification of IL-6, TNF-α and MCP-1 mRNA levels by use of real-time PCR (n= 4). *P < 0.01, compared with the vehicle-sham values. †P < 0.01, compared with the vehicle-AB values after AB. AB, aortic banding; Ang II, angiogensin II; EMSA, electrophoretic mobility shift assays; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; IKK, IκB kinase; MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB; TNF-α, tumour necrosis factor α.

Figure 5

Tetrandrine inhibited the calcineurin/NFAT signalling in response to hypertrophic stimuli. (A) NFAT activity was determined in cardiomyocytes treated with Ang II for the indicated times (n= 6). (B,C) Expression of the nuclear protein NFATc4 in the myocytes induced by Ang II in vitro and AB in vivo (n= 4). The concentration of tetrandrine used for in vitro experiments was 10 µM. *P < 0.01, compared with the vehicle-sham values. †P < 0.01, compared with the vehicle-AB values after AB. AB, aortic banding; Ang II, angiogensin II; NFAT, nuclear factor of activated T cells.

Figure 6

The effects of tetrandrine on MAPKs signalling in response to hypertrophic stimuli. (A) Time course of phosphorylated and total ERK1/2, p38, JNK1/2, and the effects of tetrandrine (10 µM) on them, in cardiomyocytes treated with Ang II (n= 4). (B) Representative blots of ERK1/2, p38 and JNK1/2 phosphorylation and their total protein expression in the indicated groups of mice (n= 4). (C) The effect of NAC on ERK1/2 activation induced by Ang II in cultured myocytes. Cardiac myocytes were pretreated with 10 mM NAC for 30 min and incubated with Ang II for 120 min. (D) Luciferase assay of the effects of U0126 and NAC on the activities of NF-κB and NFAT (n= 6). Cells were incubated with 1 µM Ang II for up to 48 h. The luciferase assay was performed as described in the Methods. The results were reproducible in three separate experiments. *P < 0.05 versus corresponding control. Ang II, angiogensin II; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase; NAC, N-acetylcysteine; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-κB.