Intense Caloric Restriction from Birth Protects the Heart Against Ischemia/Reperfusion Injury and Reduces Reactive Oxygen Species in Ovariectomized Rats

Abstract

This study investigates the cardioprotective effects of intense caloric restriction (ICR) from birth in ovariectomized rats, a model of estrogen deficiency mimicking menopause. Our findings demonstrate that ICR significantly improved both basal and post-ischemic cardiac function, even in the absence of estrogens. The restricted animals exhibited enhanced cardiac contractility and relaxation, particularly after ischemia/reperfusion (I/R) injury, with superior functional recovery compared to control groups. Notably, ICR reduced key cardiometabolic risk factors, including blood pressure, heart rate, and adiposity, while improving glucose tolerance and insulin sensitivity. Additionally, while mitochondrial biogenesis remained unaffected, ICR preserved mitochondrial integrity by reducing the number of damaged mitochondria. This was linked to a reduction in oxidative stress, as evidenced by lower reactive oxygen species (ROS) production in the hearts of restricted animals. These results suggest that ICR offers a protective effect against cardiovascular dysfunction induced by estrogen depletion, potentially through enhanced antioxidant defenses and mitochondrial protection.

Keywords: diet; menopause; mitochondria; oxidative stress.

Figure 1

Histological sections of retroperitoneal adipose tissue stained with hematoxylin and eosin. Scale bar, 40 µm; C-Sham: Control rats (ad libitum) without ovariectomy; C-OVX: Ovariectomized control rats (ad libitum); R-Sham: Rats with caloric restriction without ovariectomy; R-OVX: Ovariectomized rats with caloric restriction.

Figure 2

Basal and post-ischemic cardiac function in isolated hearts using the Langendorff technique. (A) Basal contractility index (+dP/dt). (B) Basal relaxation index (−dP/dt). (C) Basal heart rate basal. (D) Post-ischemic contractility index (+dP/dt). (E) Post-ischemic relaxation index (−dP/dt). (F) Post-ischemic heart rate basal index. Significant differences were determined by two-way ANOVA followed by Tukey’s post hoc test (p < 0.05, n = 10–11/group); * denotes significant difference between ovariectomized rats (C-OVX or R-OVX) and their respective Sham groups (C-Sham or R-Sham); # denotes significant difference between rats with caloric restriction (R-Sham or R-OVX) and their respective control groups (C-Sham or C-OVX). C-Sham: Control rats (ad libitum) without ovariectomy; C-OVX: Ovariectomized control rats (ad libitum); R-Sham: Rats with caloric restriction without ovariectomy; R-OVX: Ovariectomized rats with caloric restriction.

Figure 3

Mitochondrial content in cardiac tissue. (A) Volumetric density (Vv) of normal mitochondria. (B) Mitochondrial area. (C) Transmission electron micrographs of cross-sectional images of the post-ischemic left ventricle (ultrastructural view) from different experimental groups. (D) Pgc1-α expression. No difference by two-way ANOVA, n = 72 cells/group; Scale bar, 2.0 µm. C-Sham: Control rats (ad libitum) without ovariectomy; C-OVX: Ovariectomized control rats (ad libitum); R-Sham: Rats with caloric restriction without ovariectomy; R-OVX: Ovariectomized rats with caloric restriction.

Figure 4

Transmission electron microscopy analysis of the morphology of altered mitochondria in cardiac tissue. (A) Volumetric density (Vv) of altered mitochondria. (B) Transmission electron micrographs of cross-sectional images of the post-ischemic left ventricle (ultrastructural view), with representation of normal mitochondria (arrows) from R-Sham rat (left image) and altered mitochondria (arrowheads) from a C-OVX rat (right image), showing vacuolization, pleomorphism, and disordered and few cristae. Scale bar, 2.0 µm. Two-way ANOVA followed by Tukey’s post hoc test, n = 72 cells/group; C-Sham: Control rats (ad libitum) without ovariectomy; C-OVX: Ovariectomized control rats (ad libitum); R-Sham: Rats with caloric restriction without ovariectomy; R-OVX: Ovariectomized rats with caloric restriction.

Figure 5

Effects of intense caloric restriction on reactive oxygen species production by DCFH-DA in cardiac tissue. (A) ROS production evaluated by fluorescence microscopy images associated with DCFH-DA, Scale bar, 100 µm. (B,C) Fluorescence intensity at 0, 2, and 4 min. (D) Sod2 content; # denotes significant difference between C-OVX or R-OVX; in two-way ANOVA followed by Tukey’s post hoc test, n = 6–7/group; C-Sham: Control rats (ad libitum) without ovariectomy; C-OVX: Ovariectomized control rats (ad libitum); R-Sham: Rats with caloric restriction without ovariectomy; R-OVX: Ovariectomized rats with caloric restriction. Significant differences were determined by two-way ANOVA followed by Tukey’s post hoc test.