Combination Treatment With Antihypertensive Agents Enhances the Effect of Qiliqiangxin on Chronic Pressure Overload-induced Cardiac Hypertrophy and Remodeling in Male Mice

Abstract

We previously showed that Qiliqiangxin (QL) capsules could ameliorate cardiac hypertrophy and remodeling in a mouse model of pressure overload. Here, we compared the effects of QL alone with those of QL combined with the following 3 types of antihypertensive drugs on cardiac remodeling and dysfunction induced by pressure overload for 4 weeks in mice: an angiotensin II type 1 receptor (AT1-R) blocker (ARB), an angiotensin-converting enzyme inhibitor (ACEI), and a β-adrenergic receptor (β-AR) blocker (BB). Adult male mice (C57B/L6) were subjected to either transverse aortic constriction or sham operation for 4 weeks, and the drugs (or saline) were orally administered through gastric tubes. Cardiac function and remodeling were evaluated through echocardiography, catheterization, histology, and analysis of hypertrophic gene expression. Cardiomyocyte apoptosis and autophagy, AT1-R and β1-AR expression, and cell proliferation-related molecules were also examined. Although pressure overload-induced cardiac remodeling and dysfunction, hypertrophic gene reprogramming, AT1-R and β1-AR expression, and ERK phosphorylation were significantly attenuated by QL alone, QL + ARB, QL + ACEI, and QL + BB, the attenuation was stronger in the combination treatment groups. Moreover, apoptosis was reduced to a larger extent by each combination treatment than by QL alone, whereas autophagy was more strongly attenuated by either QL + ARB or QL + ACEI. None of the treatments significantly upregulated ErbB2 or ErbB4 phosphorylation, and none significantly downregulated C/EBPβ expression. Therefore, the effects of QL on chronic pressure overload-induced cardiac remodeling may be significantly increased when QL is combined with an ARB, an ACEI, or a BB.

FIGURE 1

Effects of QL alone or QL in combination with olmesartan, captopril, or metoprolol on hemodynamic parameters. Mice were subjected to either a sham operation or TAC for 4 weeks and administered saline, QL (0.6 mg·kg⁻¹·d⁻¹), QL (0.6 mg·kg⁻¹·d⁻¹) plus olmesartan (5.4 mg·kg⁻¹·d⁻¹), QL (0.6 mg·kg⁻¹·d⁻¹) plus captopril (10 mg·kg⁻¹·d⁻¹), or QL (0.6 mg·kg⁻¹·d⁻¹) plus metoprolol (30 mg·kg⁻¹·d⁻¹). (A), Quantitative analyses of ABP, LVESP, and LVEDP are shown. (B), Quantitative analyses of +dP/dtmax and −dP/dtmax. Values are expressed as the mean ± standard errors of the mean from 7 mice. *P < 0.05 and **P < 0.01 versus the sham group; P < 0.05 versus the TAC group.

FIGURE 2

Effects of QL alone or QL in combination with olmesartan, captopril, or metoprolol on cardiac morphology, histology, echocardiography, and hypertrophic gene expression. (A), Representative images of the global heart, HE staining, Masson's trichrome staining (scale bar: 20 mm), and M-mode echocardiography. (B), The ratio of heart weight to body weight (HW/BW) and the cross-sectional and fibrotic areas of cardiomyocytes were analyzed. Values are expressed as the mean ± standard errors of the mean from 7 mice; *P < 0.05 versus the sham group; &P < 0.05 versus the TAC group; #P < 0.05 versus the QL group. (C), The expression of ANP, BNP, SAA, and SERCA2 mRNA was evaluated through real-time RT-PCR. GAPDH was used as an internal control. Values were calculated as fold changes compared with GAPDH and expressed as the mean ± standard errors of the mean from 7 mice. *P < 0.05 versus the sham group; &P < 0.05 versus the TAC group; #P < 0.05 versus the QL group.

FIGURE 3

Effects of QL alone or QL in combination with olmesartan, captopril, or metoprolol on myocardial apoptosis and autophagy. (A), Representative images of TUNEL staining (brown, scale bar: 50 mm) and immunohistological staining (scale bar: 20 mm) with antibodies against LC3b (green) and α-MHC (red); the nuclei were stained by DAPI (blue) in the LV tissues. The black arrows indicate TUNEL-positive cardiomyocytes. (B), Quantitative analysis of apoptosis and autophagy in the LV tissues. TUNEL-positive cardiomyocytes and LC3b-positive aggregates were analyzed in 20 fields that were randomly selected from each section of the LV wall. Five sections from each heart were measured, and the numbers of TUNEL-positive cardiomyocytes and LC3b-positive aggregates per 10⁵ cardiomyocytes were expressed. *P < 0.05 versus the sham group; &P < 0.05 versus the TAC group; #P < 0.05 versus the QL group. (C), Western blot analysis of LC3b-I and LC3b-II expression; GAPDH served as a loading control. The ratio of LC3b-I LC3b-II to GAPDH was calculated. All data are expressed as the mean ± standard errors of the mean from 7 mice (n = 7). *P < 0.05 versus the sham group; &P < 0.05 versus the TAC group; #P < 0.05 versus the QL group.

FIGURE 4

Changes in cardiac remodeling-associated protein expression levels induced by QL alone or QL in combination with olmesartan, captopril, or metoprolol. (A), Western blot analysis of phosphorylated ERK (p-ERK), ERK, AT₁-R, and β₁-AR; GAPDH served as a loading control. (B), Quantitative analysis of the ratio of p-ERK to ERK and the expression of AT₁-R and β₁-AR (expressed as fold changes compared with GAPDH). The data are expressed as the mean ± standard errors of the mean from 7 mice (n = 7). *P < 0.05 versus the sham group; &P < 0.05 versus the TAC group; #P < 0.05 versus the QL group.

FIGURE 5

Effects of QL alone or in combination with olmesartan, captopril, or metoprolol on cardiomyocyte proliferation during 4 weeks of TAC. (A), Quantitative analysis and representative images of Western blots of C/EBPβ, pErbB2, and pErbB4. GAPDH served as a loading control. Values were calculated for the ratio of C/EBPβ, ErbB2, or ErbB4 to GAPDH. (B), All data are expressed as the mean ± standard errors of the mean from 7 mice (n = 7). *P < 0.05 versus the sham group; &P < 0.05 versus the TAC group. (B), Representative images of immunofluorescence staining for Ki67 (green) and α-MHC (red) in LV sections from heart tissue of the QL treatment group (the white arrow indicates 1 Ki67-positive cardiomyocyte; scale bar: 10 mm).