Long-Term Caloric Restriction Improves Cardiac Function, Remodeling, Adrenergic Responsiveness, and Sympathetic Innervation in a Model of Postischemic Heart Failure

Abstract

Background: Caloric restriction (CR) has been described to have cardioprotective effects and improve functional outcomes in animal models and humans. Chronic ischemic heart failure (HF) is associated with reduced cardiac sympathetic innervation, dysfunctional β-adrenergic receptor signaling, and decreased cardiac inotropic reserve. We tested the effects of a long-term CR diet, started late after myocardial infarction on cardiac function, sympathetic innervation, and β-adrenergic receptor responsiveness in a rat model of postischemic HF.

Methods and results: Adult male rats were randomly assigned to myocardial infarction or sham operation and 4 weeks later were further randomized to a 1-year CR or normal diet. One year of CR resulted in a significant reduction in body weight, heart weight, and heart weight/tibia length ratio when compared with normal diet in HF groups. At the end of the study period, echocardiography and histology revealed that HF animals under the CR diet had ameliorated left ventricular remodeling compared with HF rats fed with normal diet. Invasive hemodynamic showed a significant improvement of cardiac inotropic reserve in CR HF rats compared with HF-normal diet animals. Importantly, CR dietary regimen was associated with a significant increase of cardiac sympathetic innervation and with normalized cardiac β-adrenergic receptor levels in HF rats when compared with HF rats on the standard diet.

Conclusions: We demonstrate, for the first time, that chronic CR, when started after HF established, can ameliorate cardiac dysfunction and improve inotropic reserve. At the molecular level, we find that chronic CR diet significantly improves sympathetic cardiac innervation and β-adrenergic receptor levels in failing myocardium.

Keywords: caloric restriction; heart failure; myocardial infarction; receptors, adrenergic; sympathetic nervous system.

Figure 1. Long-term caloric restriction decreases body weight and accordingly heart weight in ischemic HF

A, Overall design of the 13-months study. B, Body weight (BW) changes during the study period for all groups at 0-, 6- and 12-months after the beginning of the dietary protocol. C, Representative picture of HF-ND (right side) and HF-CR rats (left side) at the end of the study period (12 months after the beginning of the dietary protocol). Measures of (D) Heart weight (HW) and (E) HW normalized to tibia length (TL). F, Graph showing the direct correlation between HW and BW in HF groups (HF-ND group: black spots; HF-CR group: red spots). n. at the end of the study: Sham=10; HF-ND=11; HF-CR=12. Data are presented as mean±SEM *p<0.0001 vs HF-ND, **p<0.01 vs HF-ND, ***p<0.05 vs HF-ND #p<0.01 vs Sham, ##p< 0.001 vs Sham, ^p<0.0001 vs Sham. Two- or one-way ANOVA and Bonferroni test were used between groups.

Figure 2. Long-term caloric restriction decreases left ventricular dilatation in ischemic HF

Ejection fraction (EF) (A), LV internal diameter at diastole (LVIDd) (B) and systole (LVIDs) (C), and heart rate (HR) (D) as measured by echocardiography before the dietary protocol (4 weeks after MI). EF (E), LVIDd (F), LVIDs (G) and HR (H) as measured by echocardiography at the end of the study period (12 months after the dietary protocol started). n = 6 to 11 per group. Data are presented as mean±SEM. * p<0.0001 vs Sham, **p<0.001 vs Sham, #p<0.05 vs HF-ND, ^p<0.05 vs Sham. One-way ANOVA and Bonferroni test were used between all groups.

Figure 3. Long-term caloric restriction enhances in vivo LV β-AR inotropic reserve in ischemic HF

Average LV +dP/dt (A) and LV −dP/dt values (B) in the experimental groups (Sham, n=5; HF-ND, n=7; HF-CR, n=6) evaluated under basal conditions and after maximal isoproterenol stimulation (ISO). Data are presented as mean±SEM. *p<0.05 vs Sham at basal, **p<0.001 vs Sham at basal, #p<0.0001 vs Sham after ISO, ^p<0.001 vs HF-ND after ISO, ^^p<0.05 vs Sham and HF-ND after ISO. Two-way ANOVA analysis and Bonferroni test were used among groups.

Figure 4. Long-term caloric restriction restores β₁-AR membrane level in ischemic HF

Representative western blot (A) and quantitative data (B) showing β₁-AR protein levels in membrane extracted from cardiac lysates in the 3 experimental groups (Sham, HF-ND and HF-CR). Ponceau used as loading control. n = 6 to 10 per group. Data are presented as mean±SEM. *p<0.05 vs Sham, #p<0.01 vs HF-ND. One-way ANOVA analysis and Bonferroni test were used among groups.

Figure 5. Long-term caloric restriction reduces left ventricular fibrosis in ischemic HF

Masson-Trichrome staining denoting cardiac fibrosis. (A) Representative images of Sham as well as HF-ND and HF-CR in border zone (BZ). Black scale bar corresponding to 100 µm (B) Quantification of the % fibrosis in Sham as well as remote area (RA) and BZ of HF groups. N= 3 to 5 per group. Data are presented as mean±SEM. *p<0.0001 vs Sham, **p<0.05 vs Sham, #p<0.0001 vs HF-ND (BZ). One-way ANOVA analysis and Bonferroni test were used among all groups.

Figure 6. Long-term caloric restriction improves left ventricular adrenergic fibers in ischemic HF

Representative images (A) and quantitative data (B, C). Shown in (A) anti-vesicular acetylcholine transporter (VaChT), Dopamine β-hydroxylase (Dβh) stainings and merged color image (VaChT in red and Dβh in green) of the 3 experimental groups: Sham (top panels), HF-ND (middle panels) and HF-CR (bottom panels); images at full definition (300 dpi) and 40x magnification; yellow scale bar corresponding to 100 µm. Quantification of number of fibers/mm² of VaCHT staining (B) and Dβh staining (C) in 3 groups. N=5 per each group. Data are presented as mean±SEM. *p<0.5 vs Sham, #p<0.0001 vs Sham, ^p<0.01 vs HF-ND. One-way ANOVA analysis and Bonferroni test were used among all groups.