Mice long-term high-fat diet feeding recapitulates human cardiovascular alterations: an animal model to study the early phases of diabetic cardiomyopathy

Abstract

Background/aim: Hypercaloric diet ingestion and sedentary lifestyle result in obesity. Metabolic syndrome is a cluster of clinical features secondary to obesity, considered as a pre-diabetic condition and recognized as an independent risk factor for cardiovascular diseases. To better understand the relationship between obesity, metabolic syndrome and cardiovascular disease as well as for the development of novel therapeutic strategies, animal models that reproduce the etiology, course and outcomes of these pathologies are required. The aim of this work was to characterize the long-term effects of high-fat diet-induced obesity on the mice cardiovascular system, in order to make available a new animal model for diabetic cardiomyopathy.

Methods/results: Male C57BL/6 mice were fed with a standardized high-fat diet (obese) or regular diet (normal) for 16 months. Metabolic syndrome was evaluated testing plasma glucose, triglycerides, cholesterol, insulin, and glucose tolerance. Arterial pressure was measured using a sphygmomanometer (non invasive method) and by hemodynamic parameters (invasive method). Cardiac anatomy was described based on echocardiography and histological studies. Cardiac function was assessed by cardiac catheterization under a stress test. Cardiac remodelling and metabolic biomarkers were assessed by RT-qPCR and immunoblotting. As of month eight, the obese mice were overweight, hyperglycaemic, insulin resistant, hyperinsulinemic and hypercholesterolemic. At month 16, they also presented normal arterial pressure but altered vascular reactivity (vasoconstriction), and cardiac contractility reserve reduction, heart mass increase, cardiomyocyte hypertrophy, cardiac fibrosis, and heart metabolic compensations. By contrast, the normal mice remained healthy throughout the study.

Conclusions: Mice fed with a high-fat diet for prolonged time recapitulates the etiology, course and outcomes of the early phases of human diabetic cardiomyopathy.

Figure 1. Metabolic features of normal and obese mice.

(A) Representative glucose tolerance curves at eight and 16 months of normal and obese mice. (B) The area under the curve (AUC) of glucose tolerance test was calculated for each animal using the trapezoidal rule. n = 10. Mean ± SEM, p<0.0001 vs. normal mice (Student test).

Figure 2. Blood pressure parameters of normal and obese mice.

(A) Systolic blood pressure (SBP) was determined with sphygmomanometer (non-invasive determination) up to 12 months. (B) Mean arterial pressure was determined with cardiac catheterization (invasive determination) at eight and 16 months. n = 5–6. Mean ± SEM.

Figure 3. Vascular reactivity of normal and obese mice.

The aortic ring of normal and obese mice was exposed to different vasoactive agents at 16 months. (A) Vasoconstriction by norepinephrine. (B) Vasorelaxation by acetylcoline. (C) Vasorelaxation by sodium nitroprusside. n = 4. Mean ± SEM, p<0.05 vs. normal mice (Two-way ANOVA test).

Figure 4. Cardiac function under basal and stress conditions of normal and obese mice.

(A) Fractional shortening (FS%) was monitored by echocardiography under basal conditions at eight, 12 and 16 months. (B) Cardiac catheterization under basal and stress conditions was performed in order to obtained maximal positive pressure development (dP/dt_max), maximal negative pressure development (dP/dt_min). n = 5–7. Mean ± SEM, *: p<0.05, **: p<0.01 vs. normal mice (Two-way ANOVA test).

Figure 5. Light microscopy features of cardiac structure of normal and obese mice.

Transversal heart sections were stained with haematoxylin/eosin (H&E) in order to measure the mean cross-sectional area of cardiomyocyte. Capillary density was determined using IsoLectinB4 that specifically detects endothelial cells. Images are representative of six animals per group. Scale bars = 50 um.

Figure 6. Collagen type I and collagen type III content in myocardium of normal and obese mice.

(A and C) Representative immunoblots at eight and 16 months. Tubulin was detected as a loading control. (B and D) Densitometric measurements (arbitrary units). n = 7–8. p<0.05 vs. normal mice (Student test). COL I: collagen type I, COL III: collagen type III.