Tongxinluo Protects against Pressure Overload–Induced Heart Failure in Mice Involving VEGF/Akt/eNOS Pathway Activation

<div><p>Background</p><p>It has been demonstrated that Tongxinluo (TXL), a traditional Chinese medicine compound, improves ischemic heart disease in animal models via vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). The present study aimed to investigate whether TXL protects against pressure overload–induced heart failure in mice and explore the possible mechanism of action.</p><p>Methods and Results</p><p>Transverse aortic constriction (TAC) surgery was performed in mice to induce heart failure. Cardiac function was evaluated by echocardiography. Myocardial pathology was detected using hematoxylin and eosin or Masson trichrome staining. We investigated cardiomyocyte ultrastructure using transmission electron microscopy. Angiogenesis and oxidative stress levels were determined using CD31 and 8-hydroxydeoxyguanosine immunostaining and malondialdehyde assay, respectively. Fetal gene expression was measured using real-time PCR. Protein expression of VEGF, phosphorylated (p)-VEGF receptor 2 (VEGFR2), p–phosphatidylinositol 3-kinase (PI3K), p-Akt, p-eNOS, heme oxygenase-1 (HO-1), and NADPH oxidase 4 (Nox4) were measured with western blotting. Twelve-week low- and high-dose TXL treatment following TAC improved cardiac systolic and diastolic function and ameliorated left ventricular hypertrophy, fibrosis, and myocardial ultrastructure derangement. Importantly, TXL increased myocardial capillary density significantly and attenuated oxidative stress injury in failing hearts. Moreover, TXL upregulated cardiac nitrite content and the protein expression of VEGF, p-VEGFR2, p-PI3K, p-Akt, p-eNOS, and HO-1, but decreased Nox4 expression in mouse heart following TAC.</p><p>Conclusion</p><p>Our findings indicate that TXL protects against pressure overload–induced heart failure in mice. Activation of the VEGF/Akt/eNOS signaling pathway might be involved in TXL improvement of the failing heart.</p></div

TXL reduces cardiac fibrosis and ameliorates myocardial ultrastructure derangement after TAC.

<p>(<b>A</b>) Masson trichrome–stained sections of left ventricles. Scale bar, 50 µm. (<b>B</b>) Quantification of cardiac fibrosis area from Masson trichrome–stained sections (n = 5 per group). (<b>C</b>) Transmission electron micrographs of cardiomyocytes the respective treatment groups. Scale bar, 2 µm. Data are mean ± SEM. **<i>P</i><0.01, ***<i>P</i><0.001. NS, not significant.</p

TXL prevents pressure overload–induced cardiac hypertrophy.

<p>(<b>A</b>) Representative photographs of hearts and HE staining of the hearts at 12 weeks post-surgery. (<b>B</b>) Heart weight/tibial length (HW/TL) and lung weight/tibial length (LW/TL) ratios at 12 weeks post-surgery (n = 7–8 per group). Reverse transcription–PCR (RT-PCR) of relative mRNA levels of (<b>C</b>) ANP, (<b>D</b>) BNP, (<b>E</b>) β-MHC, and (<b>F</b>) SERCA2a. (<b>G</b>) HE-stained transverse sections of left ventricles. Scale bar, 50 µm. (<b>H</b>) Quantification of cross-sectional area of cardiomyocytes from HE-stained sections (n = 5 per group). Data are mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. NS, not significant.</p

TXL attenuates 8-OHdG expression and MDA content after TAC.

<p>(<b>A</b>) 8-OHdG–immunostained sections of LV myocardium. Scale bar, 50 µm. (<b>B</b>) Quantitative analysis of the proportion of 8-OHdG–positive nuclei at 12 weeks post-surgery. (<b>C</b>) Quantification of MDA in homogenized fresh heart tissues at 12 weeks post-surgery. Data are mean ± SEM, n = 5 per group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. NS, not significant.</p

TXL improves cardiac function and reduces mortality following TAC.

<p>(<b>A</b>) Transthoracic echocardiography at the end of 12 weeks. Evaluation of (<b>B</b>) FS%, (<b>C</b>) EF%, (<b>D</b>) E/A ratio, and (<b>E</b>) LVPWd (n = 7–8 per group). (<b>F</b>) Kaplan-Meier survival curves for different groups (n = 15 per group). Data are mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. Sham, sham group; TAC, mice that underwent TAC surgery without treatment; TAC+TL, mice that underwent TAC surgery treated with low-dose TXL; TAC+TH, mice that underwent TAC surgery treated with high-dose TXL; NS, not significant.</p

TXL activates the VEGF/Akt/eNOS pathway after TAC.

<p>Western blot analysis of (<b>A</b>) VEGF, (<b>B</b>), VEGFR2 and p-VEGFR2 (Tyr1175), (<b>C</b>), PI3K and p-PI3K (Tyr508), (<b>D</b>), Akt and p-Akt (Ser473), (<b>E</b>), eNOS and p-eNOS (Ser1177), (<b>F</b>), Nox4, and (<b>G</b>) and HO-1 expression at 12 weeks post-surgery. (<b>H</b>) Nitrite content of the respective treatment groups at 12 weeks post-surgery. Data are mean ± SEM, n = 5 per group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. NS, not significant.</p

TXL promotes myocardial capillarity after TAC.

<p>(<b>A</b>) Representative immunostaining of LV myocardial capillaries (CD31+) at 12 weeks post-surgery. (<b>B</b>) Quantification of LV myocardial capillary density at 12 weeks post-surgery. (<b>C</b>) Capillary number/cardiomyocyte ratios at 12 weeks post-surgery. Data are mean ± SEM, n = 5 per group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. NS, not significant.</p