A novel rat model of heart failure induced by high methionine diet showing evidence of association between hyperhomocysteinemia and activation of NF-kappaB

Abstract

Heart failure is one of the most serious diseases worldwide, and can be caused by many factors, among them hyperhomocysteinemia can increase the risk for development of heart failure. In this study, we treated rats with high methionine diet (HMD), which can be conversed to homocysteine in human body, to induce a novel model of heart failure. We proved the successful establishment of this model by echocardiography and pathological evaluation at the termination of treatment. Ejection fraction and fractional shortening were significantly deceased after HMD treatment, while left ventricular volume in systole was increased. HMD treatment caused hypertrophy of cardiomyocytes, disarrangement of myofibers, and infiltration of inflammatory cells, as well as abundant apoptotic cells appeared after HMD treatment. Plasmatic homocysteine level was elevated after HMD treatment. Furthermore, through electrophoretic mobility shift assay and chromatin immunoprecipitation, the activity of NF-κB in nuclear extract was also significantly elevated, showing evidence of positive relationship between hyperhomocysteinemia and activation of NF-κB in HMD-induced heart failure. The successful development and validation of this model have made it a new tool for translational medical research of metabolic disorders-related cardiovascular disease.

Keywords: Heart failure; NF-kappaB; high methionine diet; hyperhomocysteinemia; rat model.

Figure 1

Body weight and ventricle weight. Rats were administered with either high methionine diet which contains 1-3% methionine or control diet from age of 6 weeks for another 6 weeks. A. Body weight (BW) was recorded every week. &P < 0.05, 1% HMD vs naïve group; &&P < 0.01, 1% HMD vs naïve group; &&&P < 0.001, 1% HMD vs naïve group; ##P < 0.01, 2% HMD vs naïve group; ###P < 0.001, 2% HMD vs naïve group; ***P < 0.001, 3% HMD vs naïve group. B-E. Left ventricular weight (LVW) and right ventricular weight (RVW) were recorded at the termination. *P < 0.05 vs naïve group, ***P < 0.001 vs naïve group. N=8.

Figure 2

Cardiac function. At the termination, rats were anesthetized with pentobarbital sodium (30 mg/kg), and echocardiography was performed to dynamically evaluate cardiac function of the rats. Left ventricular internal dimension in systole (LVIDs), left ventricular anterior wall in systole (LVAWs), left ventricular posterior wall in systole (LVPWs), left ventricular volume in systole (LVs), ejection fraction (EF), fractional shortening (FS). *P < 0.05 vs naïve group, ***P < 0.001 vs naïve group. N=4.

Figure 3

H&E and TUNEL staining. After performed with echocardiography, rats were sacrificed by CO₂ euthanasia. The fixed heart sample was embedded in paraffin, and cut into sections with 6 μm thickness. The sections were stained for H&E and TUNEL analysis using commercially available kits. Magnification: 400.

Figure 4

Plasmatic homocysteine (HCY) level. At the termination, blood was collected by heart puncture and the EDTA-treated plasma samples were used to determine HCY level using commercially available kit. *P < 0.05 vs naïve group, ***P < 0.001 vs naïve group. N=8.

Figure 5

NF-κB activity. A, B. Nuclear extract was collected from heart tissue homogenate, and NF-κB/DNA binding activity was determined by electrophoretic mobility shift assay (EMSA) using commercially available kit. 1, Biotin-probe + nuclear extract (naive) + 200-fold molar excess of unlabeled probe; 2, biotin-probe + nuclear extract (3% HMD); 3, biotin-probe + nuclear extract (2% HMD); 4, biotin-probe + nuclear extract (1% HMD); 5, biotin-probe + nuclear extract (naive); 6, biotin-probe. ***P < 0.001 vs naïve group. C. Nuclear extract was collected from heart tissue homogenate, and NF-κB-regulated transcriptional activity was determined by Chromatin Immunoprecipitation (ChIP) using commercially available kit. ***P < 0.001 vs naïve group. N=8.