Cardioprotection and neurobehavioral impact of swimming training in ovariectomized rats

Abstract

Cardiovascular (CV) disease is the major cause of mortality. Estrogens (E) exert multiple CV and neuroprotective effects. During menopause, CV and cognitive pathologies increase dramatically. At present, it is known that E exert many of their beneficial effects through the G protein-coupled estrogen receptor (GPER). Exercise reduces the risk of developing CV diseases. Sodium/proton exchanger (NHE-1) is overexpressed in ovariectomized (OVX) rats, probably due to the increase in reactive oxidative species (ROS). Insulin-like growth factor 1 (IGF-1), the main humoral mediator of exercise, inhibits the NHE-1. We aim to explore the subcellular mechanisms involved in the heart and brain impact of physiological exercise in OVX rats. We speculate that physical training, via IGF-1, prevents the increase in ROS, improving heart and brain physiological functions during menopause. Exercise diminished cardiac ROS production and increased catalase (CAT) activity in OVX rats. In concordance, IGF-1 treatment reduces brain ROS, surely contributing to the improvement in brain behavior. Moreover, the aerobic routine was able to prevent, and IGF-1 therapy to revert, NHE-1 hyperactivity in OVX rats. Finally, our results confirm the proposed signaling pathway as IGF-1/PI3K-AKT/NO. Surprisingly, GPER inhibitor (G36) was able to abolish the IGF-1 effect, suggesting that directly or indirectly GPER is part of the IGF-1 pathway. We propose that IGF-1 is the main responsible for the protective effect of aerobic training both in the heart and brain in OVX rats. Moreover, we showed that not only it is possible to prevent but also to revert the menopause-induced NHE-1 hyperactivity by exercise/IGF-1 cascade.

Keywords: Cardioprotection; Exercise training; IGF-1; Neuroprotection; Ovariectomized rats.

Fig. 1

Experimental protocols. OVX-ex: ovariectomized and exercised rats; OVX-sed: ovariectomized and sedentary rats; RAd-IGF-1: adenovirus encoding Insulin-like growth factor-I

Fig. 2

Cardiac mass and fibrosis assessment: A Left ventricle mass assessed by echocardiography expressed as % of basal value before the treatment. B Heart weight/tibia length ratio. C Total collagen expressed as % of tissue assessed by Picro-Sirius red stain. D Ratio of type 1 collagen and type 3 collagen. Statistical differences were evaluated by one-way ANOVA with Tukey as post test. For D, the Welch ANOVA test was used as the SDs were unequal. *p < 0.05 vs Sham ** p < 0.01 vs Sham

Fig. 3

Oxidative stress evaluation in heart tissue: A Lipid peroxidation assessed by TBARs production in heart tissue expressed as nanomoles of malonaldehyde (MDA) normalized by mg of tissue. B Reactive oxygen species production expressed as fluorescence units, normalized by mg of heart tissue. C Catalase activity expressed as enzymatic units normalized by mg of protein. Statistical differences were evaluated by one-way ANOVA with Tukey as post test. *p < 0.05 vs Sham; **p < 0.01 vs Sham; ***p < 0.001; ****p < 0.0001

Fig. 4

NHE activity in isolated ventricular myocytes: Representative traces of ammonium pulse technique used to determine NHE activity in isolated cardiomyocytes. Results are expressed as proton flux at an intracellular pH of 6.8 (JH at pHi = 6.8). Statistical differences were evaluated by one-way ANOVA with Tukey as post test. *p < 0.05 vs Sham; ***p < 0.001 vs Sham

Fig. 5

ROS and NO production in cultured OVX myocytes: the effect of IGF1 and the intracellular pathway ROS (A) and NO (B) production expressed as % of control. Several drugs we used to evaluate the effect of IGF1 and depict the intracellular pathway involved. Statistical differences were evaluated by one-way ANOVA with Tukey as post test. ***p < 0.001 vs control; ****p < 0.0001 vs control; ###p < 0.001 vs IGF1; ####p < 0.0001 vs IGF1

Fig. 6

Oxidative stress evaluation in brain tissue: A Reactive oxygen species production expressed as fluorescence units, normalized by mg of brain tissue (hippocampus and cortex). B Lipid peroxidation assessed by TBAR production in the hippocampus and cortex expressed as nanomoles of malonaldehyde (MDA), normalized by mg of tissue. Statistical differences were evaluated by one-way ANOVA with Tukey as post test. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 7

Behavior experiments: Open field and new object recognition test (NOR): (A) and B Open field experiment. Number of crosses through the middle line (A) and time of freezing (B) measured at time = 5 min of the experiment. In B, data is expressed using a Box and whisker plot. C Exploration time spent with objects A and B of OVX control, OVXex, and OVXIGF1 rats. D Exploration time spent with object A (familiar) and object C (new) of OVX control, OVXex, and OVXIGF1 rats. Statistical differences were evaluated by: one-way ANOVA with Tukey as post test for A; Kruskal–Wallis test and Dunn’s as post test (residues did not pass Shapiro-Wilks test for normality) for B and two-way ANOVA with Sidak as post test for C and D. *p < 0.05; **p < 0.01 vs OVX control