Mitigation of Subsequent Ovariectomy Responses through Prior Exercise Training in Rats

Keywords

exercise training - menopause - cardiometabolic state - autonomic dysfunctions

Introduction

Cardiovascular diseases are a major cause of death worldwide and can be prevented by addressing behavioral risk factors, such as physical inactivity [1]. For the last few decades, it has been well known that the risk of coronary heart disease gradually increases after menopause; consequently, women in their sixth decade have the same incidence as men owing to estrogen level reduction [2]. Therefore, these data suggest cardioprotective effects of endogenous and exogenous estrogen in premenopausal women [3].

A reduction in skeletal muscle mass and strength, characterized by ovariectomy (OVX), is a well-documented outcome of menopause [4]. Several mechanisms have been proposed to explain the loss of skeletal muscle mass and strength induced by estrogen deficiency; however, physical inactivity is a major contributor to OVX-induced sarcopenia [5]. Moreover, experimental studies have demonstrated that rats subjected to ovarian hormone deprivation due to OVX surgery present with increased blood pressure (BP), sympathetic tonus, oxidative stress, and body weight, and reduced baroreceptor reflex sensitivity (BRS) compared to control female rats. On the other hand, these studies showed that the exercise training (ET) recommended after OVX improved the cardiometabolic dysfunction [6] [7].

Accordingly, the preventive strategies during the climacterium should begin with screening and careful assessment for risk factors, and include lifestyle management, a healthy diet, and moderate exercise [8]. However, the mechanism by which prior ET can prevent complications caused by menopause is not known. Thus, the purpose of the study was to compare the effects of aerobic ET initiated during the premenopausal period and followed after OVX to the effect of this non-pharmacological approach after OVX on metabolic, hemodynamic, and autonomic dysfunctions induced by menopause in rats.

Materials and Methods

All experimental procedures were conducted according to the Guidelines for the Use and Care of Animals Research issued by the National Institute of Health (NIH Publications No. 8023, revised 1978), and complied with the ARRIVE guidelines for reporting animal research [9]. The study protocol was approved by the Ethics Research Committee of the University Nove de Julho (process N° 0017/2013). Experiments were performed on female virgin Wistar rats (200–220 g) obtained from the animal facility at University Nove de Julho. The rats were fed standard laboratory chow and water ad libitum. They were housed in collective polycarbonate cages in a temperature-controlled room (22°C) with a 12-hour dark-light cycle. The rats were randomly assigned to four groups (n=8, per group): control (C), sedentary OVX (SO), trained OVX (TO), and previously trained OVX (PTO). The control group did not undergo sham surgeries and did not receive any treatment; the control group was used only for normal baseline physiological parameters. All experimental evaluations were performed in non-ovulatory phases of the estrous cycle of rats [10].

Ovariectomy

At 8 weeks of age, animals were anaesthetized (ketamine 80 mg/Kg+xylazine 12 mg/Kg, ip), and a small abdominal incision was made; the oviduct was sectioned, and the ovaries were removed, as described in detail elsewhere [6]. The estrogen concentration in the blood was measured by immunoassay to confirm OVX [7].

Exercise training

All animals were adapted to the treadmill (TK-01; Ibramed, Porto Alegre, Brazil, for 10 min/day; 0.3 km/h) for 1 week prior to beginning the ET protocol. A maximal treadmill test [11] was performed in all groups: at the beginning of the experiment, and in the fourth and eighth weeks of the training protocol to determine aerobic capacity and ET intensity. ET was a voluntary exercise protocol performed without electrical stimulus, water restriction, or food restriction performed on a treadmill (Ibramed TK-01, Brazil) at low-to-moderate intensity (~50–70% maximal running speed) for one hour a day, 5 days a week, with a gradual increase in speed from 0.3 to 1.2 km/h.

The PTO group trained for 4 weeks prior to OVX and 8 weeks after OVX. The TO group trained 8 weeks after OVX ([Fig. 1]).

Fig. 1 Follow-up of groups studied with exercise training (ET) and ovariectomy (OVX).

Cardiovascular assessments

Twenty-four hours after the last training session, two catheters filled with 0.06 ml of saline solution were implanted in anaesthetized rats (80 mg/Kg ketamine and 12 mg/Kg xylazine, i.p.), one into the right carotid artery to capture blood pressure (BP) signals, and a second into the jugular vein for drug administration. Twenty-four hours after surgical procedures, the arterial catheter was connected to a strain gauge transducer (Blood Pressure XDCR; Kent Scientific, Torrington, CT, USA), and BP signals were recorded over a 30-minute period in conscious animals using a microcomputer equipped with an analog-to-digital converter board (WinDaq, 2 kHz, DATAQ, Springfield, OH, USA). The recorded data were analyzed on a beat-to-beat basis to quantify changes in the systolic (SBP), diastolic (DBP) and mean BP (MBP) and heart rate (HR).

Autonomic assessments

Baroreceptor reflex sensitivity (BRS) was evaluated by a mean index relating changes in HR to changes in MBP, allowing a separate analysis of gain for reflex bradycardia and reflex tachycardia. Increasing doses of phenylephrine (0.25 to 32 μg/Kg) and sodium nitroprusside (0.05 to 1.6 μg/Kg) were given as sequential bolus injections (0.1 mL) to produce pressure responses ranging from 5 to 40 mmHg. A 3- to 5-minute interval between doses was necessary for blood pressure to return to baseline. Peak increases or decreases in MBP after phenylephrine or sodium nitroprusside injection and the corresponding peak reflex changes in HR were recorded for each dose of the drug. The mean index was expressed as beats per minute per millimeter of mercury, as described elsewhere [12].

The total power of heart rate variability (HRV) and systolic blood pressure variability (BPV) were evaluated using BP recordings obtained in conscious rats at rest (continuous 30 minutes, 2,000 Hz). Overall variability of HR and SBP were assessed in the time domain by means of variance. HR and SBP fluctuations were assessed in the frequency domain by using autoregressive spectral analysis, as described elsewhere [13]. Briefly, HR and SBP series were divided into segments of 350 beats and overlapped by 50%. A spectrum was obtained for each of the segments via the Levinson-Durbin recursion, with the model order chosen according to Akaikeʼs criterion, ranging between 10 and 14. The oscillatory components were quantified in low (LF: 0.2 to 0.75 Hz) and high (HF: 0.75 to 3.0 Hz) frequency ranges [14]. The power spectrum density was calculated for each recognizable component in the LF and HF bands by integrating the spectrum of the components. The power is expressed as LF and HF power, as described elsewhere [15].

Twenty-four hours after cardiovascular measurements, the animals were euthanized by decapitation after 4 hours of fasting, following the previous recommendation [16]. The white adipose tissue (WAT) and soleus muscle were removed and prepared for analysis immediately; the plasma and left ventricle (LV) were removed and frozen at −70°C for metabolic and oxidative stress analysis.

Metabolic evaluations

Body weight and visceral white adipose tissue

Animals were weighed once a week during all weeks of protocol. The WAT (parametrial, subcutaneous and retroperitoneal) were weighed at the end of the protocol.

Determination of blood glucose and triglycerides

Plasma glucose and triglycerides concentrations were determined by enzymatic colorimetric assay following the manufacturer’s protocol (Bioclin, Belo Horizonte, MG, Brazil).

Adipocyte isolation to measurement of lipolysis

Adipocyte isolation was performed as previously described [17], with some slight modifications. Briefly, parametrial fat pads were minced in a flask containing DMEM supplemented with HEPES (20 mM), sodium pyruvate (2 mM), bovine serum albumin (BSA, 1%), and collagenase type II (1 mg/ml), pH 7.4, and incubated for 40 min at 37°C in an orbital shaker. Isolated adipocytes were filtered through a plastic mesh (150 μm) and washed three times in the same buffer without collagenase. After washing, medium was thoroughly aspirated and adipocytes were harvested. A small number of adipocytes were photographed under an optic microscope (×100 magnification) using a microscope camera (Moticam 1000; Motic, Richmond, BC, Canada), and mean adipocyte diameter was determined by measuring 50 cells using Motic-Images Plus 2.0 software.

Lipolysis was estimated as the rate of glycerol release in the incubation medium. Primary parametrial adipocytes (1×10⁶ cells/ml) were incubated in Krebs-Ringer-phosphate buffer (pH 7.4) containing BSA (20 mM) and glucose (5 mM) for 30 min at 37°C in the presence or absence of isoproterenol (2×10⁻⁶ M). The reaction was stopped on ice, and medium was carefully collected for the measurement of glycerol release (Free Glycerol Determination Kit, Sigma). Results are expressed as nmol of glycerol released per 10⁶ adipocytes.

Muscle cross-sectional area

Muscle cross-sectional area (CSA)

Soleus muscle was chosen for the analysis because it has a predominance of oxidative fibers and a previous study has demonstrated the ability of this muscle to adjust to the exercise, mainly in terms of functionality [18]. Soleus muscle was carefully harvested, snap-frozen in isopentane and stored in liquid nitrogen. Afterwards, soleus muscles were cut into 10 μm-thick sections using a cryostat (Criostat Mícron HM505E; Germany). Muscle sections were then incubated for myofibrillar ATPase activity after alkali (mATPase, pH 10.3) or acid (mATPase, pH 4.6) pre-incubation, as previously described [19]. The myosin ATPase reaction was used to identify the muscle fiber type. Type I fibers reacted deeply after acid pre-incubation at pH 4.6, and lightly after formaldehyde pre-treatment and alkali pre-incubation at pH 10.3. The inverse occurred with type II muscle fibers. There is a limitation in this protocol, because ATPase staining cannot distinguish IIB fibers. In this sense, we assume that type II fibers will be darker, as Ph makes type I fibers lighter. Fiber cross-sectional area were evaluated in whole muscles at 200×magnification, and images were captured on a computer attached to a microscope (Leica Qwin, Leica Microsystems, Germany) and further analyzed on a digitizing unit connected to a computer (Image J software, USA). All analyses were conducted by a single observer (AVNB), blinded to the ratʼs identity.

Citrate synthase activity

Soleus muscle protein extracts were sonicated for 30 s, at 4°C, followed by centrifugation at 1,000 g, at 4°C, for 20 min. Total protein levels [20] and maximal enzyme activity were measured in the supernatants. To determine citrate synthase activity we used an extraction buffer containing 0.5 mM Tris-HCl and 1 mM EDTA, pH 7.4, as well as an assay buffer containing 100 mM), DTNB (0.2 mM), acetyl-CoA (0.1 mM), and Triton X-100 (0.1% v/v), pH 8.1. The reaction was initiated by the addition of 50 µL oxaloacetic acid (10 mM final concentration) and absorbance at 412 nm for 5 min [21].

Oxidative stress profile

For oxidative stress analyses, the LV was removed and homogenized. Homogenates were centrifuged and protein was determined as detailed in the supplementary material.

Membrane lipoperoxidation by chemiluminescence (CL) and thiobarbituric acid reactive substances (TBARS)

The CL assay was carried out with an LKB Rack Beta liquid scintillation spectrometer 1215 (LKB Producer) in the out-of-coincidence mode at room temperature. Supernatants were diluted in 140 mM KCl and 20 mM sodium phosphate buffer, pH 7.4, and added to glass tubes, which were placed in scintillation vials; 3 mM tert-butyl hydroperoxide was added, and CL was determined up to the maximal level of emission [22]. For the TBARS assay, trichloroacetic acid (10%, wt/vol) was added to the homogenate to precipitate proteins and acidify the samples [23]. This mixture was then centrifuged (3,000 g, 3 min), the protein-free sample was extracted, and thiobarbituric acid (0.67%, wt/vol) was added to the reaction medium. The tubes were placed in a water bath (100°C) for 15 min. The absorbances were measured at 535 nm using a spectrophotometer. Commercially available malondialdehyde was used as a standard, and the results are expressed as micromole per milligram of protein.

Determination of protein oxidation by carbonyl assay

This method uses the reaction of protein carbonyl groups with 2,4-dinitro phenylhydrazine (DNPH) to form a 2,4-dinitrophenylhydrazone. The product of the reaction was measured at 360 nm, as previously described [24]. The concentration of the carbonyl in LV homogenates was standardized on the protein unit (nmol carbonyl group/mg protein) in homogenates of LV. The amount of protein was calculated from the bovine serum albumin dissolved in guanidine hydrochloride and read at 280 nm. Results were expressed as nmDNPH/mg protein.

Total antioxidant capacity (TRAP)

TRAP was measured using 2,2-azo-bis(2-amidinopropane) (ABAP, a source of alkyl peroxyl free radicals) and luminol. A mixture consisting of 20 mmol/l ABAP, 40 μmol/l luminol, and 50 mmol/l phosphate buffer (pH 7.4) was incubated to achieve a steady-state luminescence from the free radical-mediated luminol oxidation. A calibration curve was obtained by using different concentrations (between 0.2 and 1 μmol/l) of Trolox (hydrosoluble form of vitamin E). Luminescence was measured in a liquid scintillation counter using the out-of-coincidence mode, and the results were expressed in units of Trolox per milligram protein [23].

Antioxidant enzymes activities

Superoxide dismutase (SOD) activity was measured spectrophotometrically in LV homogenates by the rate inhibition of pyrogallol auto-oxidation at 420 nm [24]. Enzyme activity was reported as U/mg protein. Catalase (CAT) activity was determined in LV homogenates by measuring the decreased absorbance (240 nm) of hydrogen peroxide (H₂O₂). The results are expressed as nmol of reduced H₂O₂/min/mg protein [24]. Glutathione peroxidase (GPx) activity was assessed in LV homogenates by adding a mixture of 1 U/mL glutathione reductase and 2 mmol/L glutathione in 1 mL phosphate buffer to the assay. Mixtures were pre-incubated at 37°C for 30 minutes. Subsequently, NADPH and tert-butylhydroperoxide were added, and the change in absorbance at 340 nm was recorded to calculate GPx activity, as previously described [25] [26].

Statistical analysis

Data are reported as means±SEM. After confirming that all continuous variables were normally distributed using the Kolmogorov-Smirnov test, one-way or two-way ANOVA followed by the Student-Newman-Keuls post-hoc test was used to compare groups. Differences were considered significant at p≤0.05 for all tests.

Results

Indicators of the establishment of menopause, metabolic state, and the role of exercise training in improving the metabolism

At the beginning of the protocol (pre-OVX), body weight was not statistically different between the study groups (control [C]: 186.3±7; sedentary OVX [SO]: 197±4.8; trained OVX [TO]: 185.5±4.7; and previously trained OVX [PTO]: 195.3±4.6 g). At the end of the experimental protocol, after 8 weeks of OVX-induced estrogen deprivation, the OVX induced an increase in body weight (SO: 346±7.2 g and TO: 345.5±5 g) when compared to the C (305±10 g). Interestingly, the previous ET rats (PTO) had a reduced body weight (315±7 g) when compared to SO and similar to C values. Glycemia was similar between groups (C: 93±2.8; SO 93±2.7; 95±1.8; and PTO: 92±1.8 mg/dL). However, the triglyceride levels were reduced by previous ET (PTO: 88±2.6 mg/dL) when compared to the other groups (C: 97±1; SO: 100±4; TO: 101±3.5 mg/dL).

In addition, the weight of white adipose tissue (WAT) (parametrial and subcutaneous) was increased by OVX, and the previous ET rats (PTO group) showed no weight gain in all WAT (parametrial, subcutaneous, and retroperitoneal) ([Fig. 2a, b, and c]). Corroborating these results, the adipocyte diameter in trained groups was reduced (TO: 81±1; PTO: 74±4 μm) when compared with that in the SO group (97±3 μm), and was similar to that in the C group (82±2 μm), suggesting that OXV has a reduced cellular function and that ET maintains preserved cellular function. According to these observations, OVX resulted in reduced lipolysis after adrenergic stimulation by isoproterenol compared to the baseline state. ET stimulated an increase in lipolysis only in the previously trained group (PTO) compared to the baseline state, preventing the increase in body weight and WAT induced by OVX ([Fig. 2d]).

Fig. 2 Weight of white adipose tissue (WAT). a) Parametrial WAT; b) Subcutaneous WAT; c) Retroperitoneal WAT; d) Lipolysis of parametrial adipocytes, and e) Body weight initial (week 0) and final (week 13) in control (C), sedentary ovariectomized (SO), trained ovariectomized (TO), previously trained ovariectomized (PTO) rats. Data are presented as mean±standard deviation (n=7–8/group) and were analyzed using one-way ANOVA followed by Tukey as a post hoc test. *p≤0.05 vs. C; †p≤0.05 vs.SO; # p≤0.05 vs.TO; § p≤0.05 vs. baseline.

The physical capacity ([Table 1]), evaluated by the duration of the maximal treadmill test, was reduced in the SO group when compared with that in the trained groups (TO and PTO) at the end of the protocol (C: 12.56±0.17; SO: 12.07±0.32; TO: 25±1.05; and PTO: 26.51±1.01 minutes), suggesting that ET improves the loss in physical capacity caused by OVX (4 and 8 weeks after OVX). Interestingly, previous ET resulted in additional physical capacity before OVX induction, which was maintained until the end of the protocol. This observation demonstrated the role of ET in the prevention of menopausal effects, corroborating the above results.

Table 1 A maximal treadmill test expressed in minutes.

Measurements	C	SO	TO	PTO
Week 0	1.4±0.1	15±0.2	14±0.2	14±0.1
Week 4	1.4±0.1	13±1.3	15±0.1	24±1.3*†#&
Week 5	1.5±0.1	15±0.1	15±0.1	21±0.1*†#&$
Week 9	1.4±0.1	15±0.1	18±0.2*†&$%	26±0.1*†#&%
Week 13	12±0.17	12±0.32	25±1.05*†&$%@	28±0.1*†#&%$@

The aerobic physical capacity in control (C), sedentary ovariectomized (SO), trained ovariectomized (TO), previously trained ovariectomized (PTO) rats, at the beginning of the experiment and in the fourth and eighth weeks of the training protocol. Data are presented as mean±standard deviation (n=8/group) and were analyzed using ANOVA for repeated measures with Bonferroni’s correction, followed by Tukey as a post hoc test. *p<0.05 vs. C; †p<0.05 vs. SO; #p<0.05 vs. TO; &p<0.05 vs. 0 week; $ p<0.05 vs. 4ª week; % p<0.05 vs. 5ª week; @ p<0.05 vs. 9ª week.

In order to verify whether ET could prevent menopause-associated muscle wasting, we subjected rats with OVX to moderate-intensity ET for 8 weeks (5 days/week), and additionally, one OVX group was trained for 4 weeks (5 days/week) prior to OVX surgery to prevent the effects of menopause. As expected, the SO rats showed decreased metabolic muscle activity in both type I and type II fibers of the soleus muscle compared to the C and PTO groups ([Fig. 3a and b] ).

Fig. 3 Cross-sectional area (CSA) of soleus in control (C), sedentary ovariectomized (SO), trained ovariectomized (TO), previously trained ovariectomized (PTO) rats: a) type I; b) type II, and c) representative histological images of all types of fibers analyzed. Scale bar=100μm. Data are presented as mean±standard deviation (n=7–8/group) and were analyzed using one-way ANOVA followed by Tukey as a post hoc test. *p≤0.05 vs. C; †p≤0.05 vs.SO.

The previously trained rats (PTO group) exhibited prevention of muscle metabolic injury caused by OVX-induced estrogen deprivation in all types of fibers analyzed compared to the SO group ([Fig. 3a, b and c]). Notably, ET (TO and PTO groups) attenuated soleus-type II fibers compared to the SO group ([Fig. 3b]). A representative histological image of all types of fibers analyzed showed that the PTO group was similar to the C group, and there was a remarkable decrease in both oxidative glycolytic fibers induced by OVX ([Fig. 3c]). In addition, the citrate synthase activity was higher in both trained groups (TO: 34±0.2 and PTO: 31±1 μmol/min/per mg of soleus fresh weight) compared with that in the sedentary groups (C: 26±1 and SO: 25±1.4 μmol/min/per mg of soleus fresh weight).

Establishment of cardiovascular and autonomic dysfunctions by menopause state and the role of exercise training in preventing these dysfunctions

OVX-induced estrogen deprivation resulted in increased BP in sedentary rats after 8 weeks, including both mean BP and systolic BP (120.5±2.2; 134.4±2.3 mmHg), when compared to that in C (110±2.5; 121±2 mmHg) and trained groups (TO: 109.3±1.5; 120.3±0.3 and PTO: 114.2±0.9; 127±0.7 mmHg). The diastolic BP remained unaltered between groups (C: 98±3; SO: 102.6±2.5; TO: 96±3; PTO: 98.4±1.2 mmHg). The heart rate (HR) was unchanged between sedentary groups (C: 380±9; SO: 411±16 bpm), but ET induced rest bradycardia in both trained groups (TO: 353±5; PTO: 331±8 bpm) when compared to SO.

Furthermore, in SO rats, OVX-induced estrogen deprivation impaired tachycardic (TR) and bradycardic (BR) responses demonstrated by baroreceptor activation during BP variations (2.48±0.39; −0.55±0.12 bpm/mmHg) when compared to C (4.22±0.41; −1.86±0.49 bpm/mmHg). On the other hand, both trained groups (TO and PTO) exhibited prevention of impaired baroreceptor activation induced by OVX in TR (TO: 4.5±0.8; PTO: 4.16±0.28 bpm/mmHg) and BR (TO: −376±0.41; PTO: −3.55±0.42 bpm/mmHg) responses ([Fig. 4]).

Fig. 4 Baroreflex sensitivity (BRS) evaluated by tachycardic (TR) and bradycardic (BR) responses to BP changes in control (C), sedentary ovariectomized (SO), trained ovariectomized (TO), previously trained ovariectomized (PTO) rats. Data are presented as mean±standard deviation (n=7–8/group) and were analyzed using one-way ANOVA followed by Tukey as a post hoc test. *p≤0.05 vs. C; †p≤0.05 vs.SO.

[Table 1] shows the impairment of HR and BP variability caused by OVX-induced estrogen deprivation. OVX (SO group) increased cardiac sympathetic modulation (the absolute and normalized power of the low frequency [LF]) when compared to the C group, indicating a sympathetic predominance. The sympathovagal balance (LF/HF) was higher in the SO group than in the C group. The ET (TO and PTO groups) reversed the autonomic dysfunction induced by OVX, increasing pulse interval variability and cardiac vagal modulation, and decreasing sympathovagal balance (LF/HF) in these animals. Moreover, only the previously trained group (PTO) prevented an increase in sympathetic modulation (absolute power of the LF).

In addition, systolic BP variance and vascular sympathetic modulation (LF component) were higher in the SO group than in the other groups ([Table 2]). ET (TO and PTO groups) reversed OVX-induced autonomic dysfunction.

Table 2 Cardiovascular autonomic modulation in groups studied.

Measurements	C	SO	TO	PTO
HRV
VAR-PI (ms 2 )	57±13	60±12	109±2*†	116±7.2*†
LF (ms 2 )	4.3±1.8	17±2.6*	11.6±3	9±0.8†
%LF (NU)	8±2	19.3±1.7*	11.4±2.6†	11.3±2†
HF (ms 2 )	26±4.8	25.3±3.4	47±5*†	43.±3*†
%HF (NU)	59±3	45.3±3.5	58±5.6	55±4
LF/HF	0.19±0.03	0.41±0.02*	0.24±0.05*†	0.28±0.05*†
BPV
Variance (mm Hg 2 )	15±2.8	40±3*	19±0.4†	21±1.2†
LF (mm Hg 2 )	3.3±0.8	8.6±1*	3.6±0.9†	4.3±0.4†

Systolic blood pressure (BPV) and heart rate (HRV) variabilities computed from 0.20 to 3 Hz (total power), pulse interval variability (VAR-PI); low-frequency (LF: 0.20–0.75 Hz) and high-frequency (HF: 0.75–3 Hz) bands of control (C), sedentary ovariectomized (SO), trained ovariectomized (TO), previously trained ovariectomized (PTO) rats. Data are presented as mean±standard deviation (n=7–8/group) and were analyzed using one-way ANOVA followed by Tukey as a post hoc test. *p≤0.05 vs. C; †p≤0.05 vs.SO.

Indicators of the establishment of the menopausal state in oxidative stress and the role of exercise training in preventing this injury

With regard to oxidative stress in the LV, membrane lipoperoxidation (CL and TBARS) and protein oxidation were higher in the SO group than in the C group. Furthermore, the antioxidant enzyme activities (CAT and GPx) and TRAP were decreased in the SO group compared to those in the C group, indicating impaired redox balance. ET initiated after OVX decreased membrane lipoperoxidation (only for TBARS) and protein oxidation and increased only CAT, accompanied by an increase in TRAP, when compared to the SO group. Interestingly, previous ET prevented oxidative stress in all parameters studied, decreasing membrane lipoperoxidation (CL and TBARS) and protein oxidation and increasing the activity of antioxidant enzymes (SOD, CAT, and GPx) and TRAP when compared to the SO, indicating an improved redox balance after OVX ([Table 3]).

Table 3 Cardiac oxidative stress profile in groups studied.

Measurements	C	SO	TO	PTO
Lipoperoxidation by CL (cps/mg protein)	1442±268	3017±206*	2280±543	1360±157†
Lipoperoxidation by TBARS (μmol/mg protein)	5.84±0.65	9.6±0.85*	5.3±0.87†	6.9±0.82†
Protein oxidation (nmol/mg protein)	5.19±0.6	8.42±0.8*	5.8±0.35†	6.6±0.29†
SOD (USOD/mg protein)	17±1	17.4±1.4	18.2±1.4	24.4±2.4*†#
CAT (nmol/mg protein)	1±0.1	0.72±0.09*	1.06±0.1†	0.94±0.08†
GPx (μmol/min/mg protein)	0.117±0.02	0.066±0.008*	0.031±0.002*†	0.1±0.009†#
TRAP (μM of trolox)	5.7±0.9	2.6±0.4*	7.6±1.2†	8±1.6†

Membrane lipoperoxidation by chemiluminescence (CL) and thiobarbituric acid reactive substances (TBARS), protein oxidation, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total antioxidant capacity (TRAP) of control (C), sedentary ovariectomized (SO), trained ovariectomized (TO), previously trained ovariectomized (PTO) rats. Data are presented as mean±standard deviation (n=7–8/group) and were analyzed using one-way ANOVA followed by Tukey as a post hoc test. *p≤0.05 vs. C; †p≤0.05 vs.SO; # p≤0.05 vs.TO.

Discussion

Previous studies have demonstrated the benefits of exercise in rats with OVX-induced estrogen deprivation with regard to metabolic, hemodynamic, and autonomic parameters [6] [27]. However, to the best of our knowledge, this is the first study to demonstrate that previous exercise training is a preventive tool against the deleterious effects of menopause. The hypothesis that sedentary conditions can worsen the damage caused by OVX-induced estrogen deprivation to metabolic, cardiovascular, and autonomic functions was tested using an experimental model of menopause (OVX). ET, used as a non-pharmacological treatment, could attenuate some parameters of metabolic and cardiovascular dysfunction triggered by the advent of menopause. Therefore, this study demonstrated that ET, when used as a preventive mechanism before OVX induction in the premenopausal period leading to physical conditioning, prevented metabolic, cardiovascular, and autonomic dysfunction.

This study showed that OVX in sedentary rats (SO group) increased the gain of body weight and WAT, probably due to decreased energy consumption due to the loss of estrogen. Indeed, lipolysis activation by a β-adrenergic agonist (isoproterenol) was reduced in these rats. The gain in body weight was previously observed in this model [28], suggesting that the damage to lipid metabolism corroborates central obesity and can be associated with lower circulating leptin in developing obesity by OVX [29]. Therefore, obesity impairs the autonomic nervous system by modulating the cardiovascular function, which is associated with increased BP [30]. Hence, in this study, SO rats presented an increase in mean BP, systolic BP, and autonomic dysfunction (increase in sympathetic modulation and BPV), as well as a reduction in baroreceptor reflex sensitivity in both TR and BR, corroborating previous studies [6] [7] [31].

Another mechanism that may be associated with increased BP after ovarian hormone deprivation is increased cardiac oxidative stress. Furthermore, some studies have shown that estrogen deprivation can induce deterioration of endothelial function [32] [33] [34]. Therefore, the increase in lipoperoxidation (CL and TBARS), protein oxidation, and reduction in antioxidant enzymes (CAT and GPx) and TRAP observed in this study in SO rats can reflect reduced nitric oxide bioavailability due to a worsening of the oxidative redox state, leading to endothelial dysfunction. These data corroborate those of previous studies [34] [35], demonstrating the role of oxidative stress in endothelial function in OVX rats, inducing alterations in BP.

Considering that physical inactivity can aggravate the damage induced by ovarian hormone deprivation, our group has recently suggested ET as a treatment intervention to promote beneficial effects on metabolism and cardiovascular and autonomic functions and to reduce oxidative stress in OVX alone [31] or in association with other conditions such as hypertension [36] and metabolic syndrome [37]. However, in this study, ET was preconized during pre-menopause, demonstrating that a prior ET protocol was effective in markedly increasing aerobic physiological capacity by reducing body weight gain and WAT as well as promoting an increase in lipolysis by adrenergic stimuli in adipocytes ([Fig. 2]), thus reducing triglyceride levels. These findings prevented the increase in BP by allowing better mechanisms of BP control: autonomic modulation (reducing sympathetic modulation) and BRS (increasing tachycardic and bradycardic responses), as demonstrated in [Table 1] and [Fig. 4]. Moreover, previous ET reduced oxidative stress, increased antioxidant enzyme activity in the heart, maintained cellular function ([Table 2]), and promoted ET-induced cardiac responses such as an increase in the variance and vagal modulation of HR and rest bradycardia. Similarly, recent studies have shown that OVX aggravates cardiac and functional impairments in older female rats [31] and young adult rats [35], which is probably associated with exacerbated autonomic dysfunction, inflammation, oxidative stress [31], and endothelial dysfunction [35].

The reduction in skeletal muscle mass and strength characterized by OVX is a well-documented outcome of menopause [4], and several mechanisms have been proposed to explain the loss of skeletal muscle mass and strength induced by estrogen deficiency; however, physical inactivity is a major contributor to OVX-induced sarcopenia [5]. Furthermore, ovarian hormone deprivation could also have a negative impact on skeletal muscle energy metabolism, such as in the mitochondria, which are estrogen-sensitive organelles [38]. Although some studies addressed OVX-induced sarcopenia, none examined the impact of pre-estrogen deprivation exercise on preserving muscle mass and function.

In this study, a smaller proportion of oxidative, glycolytic, and intermediate fiber types was observed in the soleus muscle of the SO group; this reduction in the proportion of oxidative fibers (type 1) is a sign of oxidative metabolism, reflecting reduced physical performance. Interestingly, rats trained prior to OVX showed prevention of metabolic muscle damage caused by OVX-induced estrogen deprivation ([Fig. 3]). The preservation of muscle metabolism in the PTO group promoted better physical performance, as measured by the maximal exercise test and evidenced by the increase in citrate synthase activity. Some impairments in the skeletal muscles of OVX animals have been shown to be prevented by regular bouts of muscle exercise [39]. These observations raise the possibility that the impact of hormone deprivation on skeletal muscle is not only due to estrogen deficiency, but is also accompanied by physical inactivity.

Accordingly, estrogen promotes a cardioprotective effect by receptor β signaling in the female cardiovascular system through multiple mechanisms, demonstrating its vasodilator and anti-angiogenic properties that regulate the activity of nitric oxide, alter membrane ionic permeability in vascular smooth muscle cells, inhibit vascular smooth muscle cell migration and proliferation, and regulate the adrenergic control of the arteries [40]. Therefore, age and menopause-related endothelial injury, changes in vascular estrogen receptor expression, and intracellular signaling or genomics may alter the cardiovascular effects of this sex hormone [40]. Therefore, this should be considered when prescribing ET to women during individual interventions.

Notably, in this study, we sought to determine the isolated effect of OVX-induced estrogen deprivation on cardiometabolic status and autonomic dysfunction without other dysfunctions generated by advanced age. Therefore, we selected younger mice to verify the effects of physical training prior to estrogen deprivation. This may be a limitation of the study; however, we did not seek to identify dysfunctions related to aging, and only hormonal deprivation occurred during menopause. Furthermore, we reached conclusions based on a rat model; future studies in humans are required.

The key innovation of this study is the demonstration that engaging in exercise training prior to estrogen deprivation can effectively prevent or reduce the adverse effects of menopause on metabolic, cardiovascular, and autonomic functions. This preventative strategy, particularly when applied to young models, introduces a novel viewpoint and opens new avenues for future research and clinical applications. It suggests that early intervention may provide lasting benefits for womenʼs health during and after menopause.

Funding Information

Conselho Nacional de Desenvolvimento Científico e Tecnológico — http://dx.doi.org/10.13039/501100003593; 306768/2012–7 455326/2014–2

Fundação de Amparo à Pesquisa do Estado de São Paulo — http://dx.doi.org/10.13039/501100001807; 2012/21141–5 2015/04788–7

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