Effects of Chemically Induced Ovarian Failure on Voluntary Wheel-Running Exercise and Cardiac Adaptation in Mice

Abstract

The role of exercise in decreasing the risk of cardiovascular disease in postmenopausal women has not been studied sufficiently. Accordingly, we investigated the effect of voluntary wheel-running and forced treadmill exercise on cardiac adaptation in mice treated with 4-vinylcyclohexine diepoxide (VCD), which selectively accelerates the loss of primary and primordial follicles and results in a state that closely mimics human menopause. Two-month-old female C57BL/6 mice injected with VCD (160 mg/kg) for 20 consecutive days underwent ovarian failure by 60 to 90 d after injection. Responses to voluntary wheel running and treadmill exercise did not differ between VCD- and vehicle-treated 7-mo-old C57BL/6 or outbred B6C3F1 mice. Moreover, adaptive cardiac hypertrophy, hypertrophic marker expression, and skeletal muscle characteristics after voluntary cage-wheel exercise did not differ between VCD- and vehicle-treated mice. Because 5′ AMP-activated protein kinase (AMPK) is a key component for the maintenance of cardiac energy balance during exercise, we determined the effect of exercise and VCD-induced ovarian failure on the AMPK signaling axis in the heart. According to Western blotting, VCD treatment followed by voluntary cage-wheel exercise differently affected the upstream AMPK regulatory components AMPKα1 and AMPKα2. In addition, net downstream AMPK signaling was reduced after VCD treatment and exercise. Our data suggest that VCD did not affect exercise-induced cardiac hypertrophy but did alter cellular cardiac adaptation in a mouse model of menopause.

In response to a decrease in ovarian function, several physiologic and psychologic consequences may occur including cessation of menstrual cycles, increased adiposity, and mood or sleep disturbances. Compared with their premenopausal counterparts, postmenopausal women are more susceptible to cancer,⁴ metabolic syndrome, and associated comorbidities including cardiovascular disease.¹⁴ Therefore, elucidating the cellular and molecular mechanisms of cardiac adaptation to physiologic and pathophysiologic stimuli in women and how the transition to menopause affects disease susceptibility becomes tantamount to the discovery of clinical treatment strategies.

The most commonly used inducible model for mimicking human menopause is the use of ovariectomized rodents. However, ovariectomized models are not an accurate representation of the natural progression of menopause in humans because: 1) ovariectomy results in an abrupt withdrawal of estrogen and other ovarian hormones, and as a result, post-ovariectomy can only approximate the postmenopausal phenotype; 2) ovariectomy prevents the natural transitional period from a cycling (premenopause) to noncycling (postmenopause) condition called perimenopause; 3) ovariectomy excises all ovarian tissue, whereas fewer than 13% of postmenopausal women have undergone surgical removal of the ovaries;²⁴ 4) ovariectomy in rodents results in little to no voluntary exercise, unlike the condition in postmenopausal humans.^12,45 Instead of ovariectomy, the occupational chemical 4-vinylcyclohexene diepoxide (VCD) has been shown to specifically target and destroy small primary and primordial ovarian follicles by accelerating their natural process of atresia, ultimately resulting in ovarian failure and depletion of estrogen.^28,51 Consequently, noncycling female mice are achieved in the absence of obvious extraovarian toxicity.^30,34,46 Therefore, follicle-deplete, ovary-intact rodents more closely approximates the natural human progression through the events leading to perimenopause and into the postmenopausal stage of life than do ovariectomized animals.⁵¹

5′ AMP-activated protein kinase (AMPK) is a key component for the maintenance of skeletal and cardiac muscle energy balance during exercise.^7,47 AMPK is a heterotrimeric enzyme complex consisting of a catalytic α subunit and regulatory β and γ subunits; direct phosphorylation at Thr172 (α subunit) by upstream AMPK kinases is required for enzyme activation. Activation of AMPK in the heart leads to direct phosphorylation of acetyl CoA carboxylase (ACC).²⁵ In general, activation of AMPK turns off energy-consuming processes, such as protein synthesis, and switches on ATP-generating mechanisms, such as fatty acid oxidation and glycolysis.¹⁵

Considering the effect of menopause on cardiovascular and metabolic status, we hypothesized that treatment with VCD, as a model of menopause, will affect exercise performance and cardiac adaptation to exercise differently when compared with untreated, control female mice. We measured voluntary wheel-running performance, forced treadmill exercise, and cardiac adaptation in female mice treated with VCD or vehicle. Due to its important role in cardiac energy homeostasis, components of the AMPK signaling axis were measured, including upstream and downstream mediators. Unlike previous studies that found hormone-dependent differences in exercise performance and cardiac adaptation,^11,38,41 we found that noncycling, VCD-treated female mice exercised to the same extent as did untreated mice. Although cardiac adaptation was similar between treated and untreated mice, differences in AMPK signaling were detected.

Materials and Methods

Experimental animals.

Treatment with 4-vinylcyclohexene diepoxide.

At 2 mo of age, C57BL/6 or B6C3F1 mice were randomized to either treatment or control groups. Mice were weighed and given daily intraperitoneal injections of VCD (V3630; Sigma Aldrich, St Louis, MO) at a dose of 160 mg/kg for 20 consecutive days (Figure 1). Control mice were injected similarly with vehicle (sesame oil). After the 20-d injection period, estrous cycles were monitored daily by vaginal cytology to determine when cycling ceased, and therefore when ovarian failure occurred. Mice were considered acyclic after 15 consecutive days in persistent diestrus. Estrous cycles were monitored closely throughout the study to confirm lack of estrous cycles in VCD-treated mice. Estrogen depletion was confirmed by using an estradiol enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). At the time of tissue harvesting, ovaries were collected, trimmed of adipose tissue, and fixed in Bouin fixative. They then were transferred to 70% ethanol and paraffin-embedded, and serial sections (thickness, 4 to 5 μm) were prepared by the University of Arizona Histology Lab. Every 20th section was saved, to avoid double counting of small preantral follicles. Sections were mounted and stained with hemotoxylin and eosin. In each mouse, follicles were counted and classified by a blinded observer as primordial, primary, secondary, or antral, as previously described.³¹

Figure 1.

VCD administration and experimental protocol. Mice (C57BL/6 or B6C3F1) received VCD (160 mg/kg) or sesame oil (controls) starting at 2 mo of age for 20 consecutive days. After injections, estrous cycles were monitored. Loss of ovarian function occurred within 60 to 90 d and was confirmed by vaginal cytology and measurements of serum 17β-estradiol (E2). At 7 mo of age, mice were exposed to a cage wheel for 4 wk. At 7 mo, a subset of mice (excluding B6C3F1 mice) underwent the treadmill exercise protocol.

Voluntary wheel-running exercise.

At the age of 7 mo, VCD- or vehicle-treated female C57BL/6 mice were randomized to either the sedentary or voluntary wheel-running (4 wk) groups (n = 5 to 7 per group). For voluntary wheel running, mice were housed individually (cage dimensions, 47 × 26 × 14.5 cm) with free access to a cage wheel for 4 wk (28 d). The exercise wheels used have been described previously.² Briefly, this system consists of a wheel (diameter, 11.5 cm) with a 5.0-cm-wide running surface (model 6208, PetSmart, Phoenix, AZ) equipped with a digital magnetic counter (model BC 600, Sigma Sport, Olney, IL) that is activated by wheel rotation. Daily exercise values were recorded for time and distance for each exercised mouse throughout the 4-wk exercise period. All mice were given water and standard hard rodent chow ad libitum. A summary of the experimental protocol is included in Figure 1. To determine whether the effect of estrogen loss on exercise performance is mouse strain-specific, we exposed an outbred strain (B6C3F1) of VCD- and vehicle-treated female mice to the same voluntary wheel-running exercise paradigm. At the end of the exercise period, exercised and sedentary control mice were euthanized by cervical dislocation under inhaled anesthesia within 30 min of removal from cages. Body mass was weighed, and hearts and skeletal muscle (whole hindlimb, soleus, tibialis anterior, quadriceps) were excised rapidly and washed with ice-cold modified PBS (136.9 mmol/L NaCl, 3.35 mmol/L KCl, 12 mmol/L NaH₂PO₄, 1.84 mmol/L KH₂PO₄; pH 7.4). Hearts, skeletal muscles, and ovaries were harvested by using the snap–freeze method and were frozen in isopentane cooled in liquid nitrogen. All experiments were performed according to protocols that adhered to guidelines and approved by the Institutional Animal Care and Use Committee at the University of Arizona, and to the Guide for the Care and Use of Laboratory Animals.¹⁸

Treadmill exercise.

As shown in Figure 1, groups of C57BL/6 mice treated with either VCD or vehicle at 2 mo of age for 20 d underwent treadmill exercise at 7 mo of age. Mice were exercised on a 6-lane treadmill with adjustable belt speed, as previously described.²³ Over a 1-wk period, mice were acclimated to the treadmill through three 15-min running sessions at a 7° incline daily as follows: 1) no shock activation and 4 m/min belt speed; 2) shock stimulation and 5 m/min belt speed; and 3) shock stimulation and 15 m/min belt speed. After acclimation, the treadmill was set at a 7° incline, and speed was increased every 10 min from 15 m/min to 20, 25, and 30 m/min. Exercise was terminated when mice were unable to continue or showed signs of exhaustion or distress. In addition, mice were exercised at 20 m/min (about 80% of maximum) to assess endurance capacity. Each mouse was tested 3 times (2 to 3 d between each test), and the average values across all exercise sessions for each mouse were calculated.

Real-time PCR.

Total RNA was isolated from the left ventricles of vehicle- and VCD-treated female mouse hearts harvested at the end of each exercise regimen by using the High Pure miRNA Isolation kit (Roche, Indianapolis, IN) according to the manufacturer's protocol. Total cDNA was generated by using the NCode miRNA First-Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). Real-time PCR was done according to the Universal ProbeLibrary Assay (Roche) by using the LightCycler 480 system (Roche). Primers for Universal ProbeLibrary Assay were designed by using probeFinder software (Roche, http://qpcr.probefinder.com). The LightCycler 480 Probes Master (Roche) was used. GAPDH was used as an mRNA internal control.

Separation of cardiac myosin heavy-chain (MyHC) isoforms.

Frozen heart samples were prepared for SDS–PAGE as described to determine relative cardiac MyHC. Briefly, samples were diluted in a sample buffer that contained 8 M urea, 2 M thiourea, 3% SDS (w/v), 75 mM DTT, 0.03% bromophenol blue, and 0.05 M Tris HCl, pH 6.8. MyHC isoforms were separated on a 6% acrylamide resolving gel (37.5:1 crosslinked with DATD [Bio-Rad, Hercules, CA]) and a 2.95% stacking gel (5.6:1 crosslinked with DATD [Bio-Rad]) by using a SE600 Hoefer gel system (Hoefer, Holliston, MA) at 16 mA constant current. The gels were silver-stained and analyzed by using a flatbed scanner (PowerLook 1120, Umax, Dallas, TX) with 1200 × 2400 dpi and 3.7 Dmax. To determine relative MyHC content, 6 to 7 (cardiac) or 2 to 3 (skeletal) dilutions of each sample were analyzed so that readings stayed in the linear densitometric range. From the linear relationships determined for each MyHC isoform, the relative MyHC content of each isoform was extrapolated. Soleus (containing predominately types I [β] and IIa MyHC) and neonatal rat ventricular cardiomyocytes (containing both α and β MyHC) were used as standards.

Immunohistochemistry.

Standard immunohistochemistry staining for skeletal MyHC was performed as previously described.² Briefly, frozen sections (thickness, 7 μm on a rotary microtome) were cut and stained with antibodies specific for each MyHC isoform: antiMyHC I (Sigma Aldrich), antiMyHC IIa (prepared from hybridoma SC71 deposited in the American Type Culture Collection by S Schiaffino), antiMyHC IIb (prepared from hybridoma BF-F3 [ATCC – S. Schiaffino]), and antilaminin (Sigma). The secondary antibodies were fluorescence-labeled Dylight secondary antibodies (Jackson Laboratory, Bar Harbor, ME). Whole-muscle montages were assembled from 20 to 40 individual magnified images by using Photoshop software (Adobe, San Jose, CA). Histochemical staining for NADH-tetrazolium reductase (NADH-TR) was accomplished by incubation of samples for 30 min at 37 °C in NADH-TR reaction solution (0.2 M Tris, 1.5 mM NADH, and 1.5 mM nitrotetrazolium blue), dehydration through serial dilutions of acetone, and mounting in Aquamount (Lerner Laboratories, Pittsburgh, PA). The percentage of muscle fibers expressing a particular MyHC isoform was determined by an observer blinded to experimental conditions, who counted positively stained fibers and expressed the amount as a percentage of the total fibers in a given muscle. After NADH-TR staining, 3 types of fibers were characterized: unstained, moderately stained, and darkly stained. Fibers that were moderately or darkly stained were classified as positive for NADH-TR (oxidative fibers).

Staining with picrosirius red and determination of collagen content.

Hearts were processed, embedded in paraffin, sectioned, and stained with Picrosirius Red according to standard protocols. In brief, the paraformaldehyde fixed tissue was prepared using a tissue processor and embedded in paraffin. The embedded tissue was sectioned in 7μm thickness. The specimens were dewaxed, rehydrated, and stained with picosirius red to detect collagen fibers. Stained tissue specimens were photographed by using a camera connected to a polarized light microscope (Axio Imager M1, Zeiss, Oberkochen, Germany) to detect birefringence of collagen fibers. Three random fields from each heart sample were evaluated. The images were quantified by a semiautomated imaging analysis program (AxioVision, Zeiss). A color threshold was defined in such a way to detect mature collagen. The area of birefringence was normalized by the total area of interest.

Western analysis.

Cardiac lysates were prepared by mechanical disruption in a protein extraction buffer (in 50 mmol/L Tris[hydroxymethyl]-aminomethane, 0.5 mmol/L EGTA; 1 mmol/L EDTA; 0.5 mmol/L DTT; pH 7.0). The buffer also contained leupeptin, pepstatin, and phenylmethylsulfonyl fluoride (0.1 mmol/L each) to prevent nonspecific proteolysis and sodium pyrophosphate and sodium vanadate (1 mmol/L each) to prevent nonspecific phosphorylation and dephosphorylation, respectively. SDS–PAGE was performed followed by transfer to a membrane (polyvinylidene difluoride) for Western analysis. All antibodies were obtained commercially from Cell Signaling Technology, except for that for GAPDH, which was obtained from Abcam. Membranes were saturated in enhanced chemiluminescence substrate (Perkin–Elmer, Waltham, MA) and used to expose double-emulsion autoradiography film (GeneMate Blue Basic, BioExpressions, Kaysville, UT), which was processed automatically (X-OMAT 2000A Kodak, Rochester, NY). The processed films were scanned (Perfection V750 Pro, Epson, Long Beach, CA) by using professional scanning software (SilverFast Ai, LaserSoft Imaging, Sarasota, FL). Protein optical densities were quantified by using LabImage 1D software (Kapelan Bioimaging Solutions, Leipzig, Germany) and normalized to protein optical densities for total GAPDH. Previous reports indicate that GAPDH does not change with exercise.^22,36,42 In addition, prior to immunoblotting, all membranes were stained with Ponceau S acid red and quantified for total protein. In addition, total protein measured by Coomassie blue or Ponceau S staining was compared with GAPDH expression for equal loading. All immunoblot analysis was generated from the semiquantitation of individual blots and not compared across blots. All immunoblot images of a given target were taken from the same blot. The data then were adjusted such that the sedentary–vehicle group had a mean value of 1 to highlight the effect of either exercise or VCD. Data are reported as mean ± SEM.

Data and statistical analyses.

Results are presented as mean ± SEM. We performed 2-way ANOVA followed by a Student–Newman–Keuls post hoc test or a Student t test to compare mean values. Because we were unable to perform paired analysis to determine the extent of cardiac growth, the percentage change in cardiac mass with exercise was determined by comparing the mass of the heart from each exercised mouse to the mean cardiac mass of the sedentary group, as previously described.^23,24 The difference in cardiac mass then was expressed as percentage change from that in sedentary mice. In addition, 2-way ANOVA followed by a Student–Newman–Keuls post hoc test or a Student t test was performed to compare differences between mean values in gene expression studies. P values less than 0.05 were considered statistically significant.

Results

VCD-induced ovarian failure in C57/BL6 mice.

After 20 consecutive days of VCD treatment, vaginal cytology was used to determine estrous cycles of VCD-injected mice. Vehicle-injected controls demonstrated regular estrous cycle patterns, with cycles lasting 5.2 ± 0.9 d (Figure 2 A). In contrast, the estrous cycle of VCD-treated mice became irregular and then ceased within 60 to 90 d after the start of VCD injections. At the end of the study, VCD-treated ovarian tissue showed significant atrophy, compared with controls (Figure 2 B, top panels). At high magnification (Figure 2 B, bottom panels), a complete depletion of primordial follicles is visible. Identical criteria were used to establish persistent diestrus in B6C3F1 mice (data not shown). Moreover, persistent diestrus was accompanied by serum 17β-estradiol amounts that were below detectable levels, as previously demonstrated^29,30 (data not shown).

Figure 2.

VCD-induced ovarian failure in mice (n = 5 or 6 per group). (A) Average length (days) of estrous cycle for vehicle- and VCD-injected sedentary (Sed) groups. One cycle is measured from day 1 in estrus until the next entry into estrus, with vaginal cytology beginning at about 6 wk after the start of injections. Compared with those of controls, estrous cycles of VCD-injected mice become irregular, and after 12 estrus cycles, cytology of all VCD-injected mice no longer indicated cycling. (B) Ovarian tissue at 8 mo of age; hematoxylin and eosin stain. Cross-sections (thickness, 5μm) from vehicle-treated (left panel) and VCD-treated (right panel) mice at magnifications of 20× (top panel) and 200× (bottom panel). Ovarian tissue at 200× magnification shows numerous follicles in vehicle-treated compared with few in VCD-treated mice.

Exercise performance in VCD-treated mice.

Voluntary cage-wheel running.

There were no significant differences between VCD- and vehicle-treated groups (Figure 3 A, top panel). VCD-induced ovarian failure mice ran an average of 5.7 ± 0.9 h/d compared with control groups, which ran 4.6 ± 0.8 h/d. Over a 24-h period, this amount of time resulted in average daily rates of 6.6 ± 1.2 and 5.4 ± 1.0 km/h, for VCD- and vehicle-treated mice, respectively. The calculated wheel-running speeds gradually increased over the 4-wk running period, as previously shown,²³ but were not significantly different between groups. These exercise performance values were similar to those previously reported.²³

Figure 3.

Voluntary wheel-running and treadmill performance (mean ± SEM) in vehicle- and VCD-treated mice (n = 5 or 6 per group). (A) Average running distance (km/d), time spent on the wheel (h/d), and running speed (km/h) for every 24 h over the 4-wk study period in C57BL/6 (top panels) and B6C3F1 (bottom panels) mice. (B) Treadmill running parameters for C57BL/6 mice. Maximal time (top panel) and speed (bottom panel) were determined as described in the Methods section.

We obtained similar results from an outbred strain of female mice (B6C3F1; Figure 3 A, bottom panel), with no significant differences in exercise performance measured by average daily time, distance, and speed on the exercise wheel. Therefore, these studies indicate no effect of VCD-induced ovarian failure on voluntary wheel-running exercise in 2 mouse strains.

Treadmill running.

We used forced (involuntary) running on a treadmill and determined exercise capacity. On average, the maximal speed of vehicle-treated mice was 26.7 ± 0.95 m/min, and the maximal speed of VCD-treated mice was 28 ± 1.4 m/min before exhaustion. There was no significant difference between vehicle- and VCD-treated group (Figure 3 B), with vehicle-treated mice running 28.3 ± 1.3 min and VCD-treated mice running 29.7 ± 1.4 min before exhaustion. These findings indicate that VCD-induced ovarian failure had minimal effect on mouse running performance.

Cardiac adaptation in voluntary wheel-running mice.

After voluntary wheel-running exercise, body morphometrics were recorded, and the hearts were excised rapidly and weighed. Because of significant (P < 0.05) differences in body adiposity between 8-mo sedentary and exercised groups (26.2 ± 5.7% and 33.5 ± 1.8%, vehicle- and VCD-treated respectively), comparisons of cardiac adaptation were limited to absolute heart weight and heart weight normalized to tibial length. Cardiac hypertrophy in response to cage-wheel exercise was evident in both exercised groups (Table 1). Absolute heart mass was significantly (P < 0.05) greater in vehicle- and VCD-treated exercised mice compared with sedentary counterparts by 11.7% ± 3.1% and 12.5% ± 2.8%, respectively. Similarly, the heart weight:tibial length ratio of exercised mice was greater than that of sedentary counterparts by 13.9% ± 2.9% (vehicle-treated) and 14.6% ± 2.6% (VCD-treated). However, there were no measurable differences in cardiac hypertrophy between control and VCD-induced ovarian failure mice after voluntary wheel-running exercise. Similarly, VCD treatment did not affect cardiac adaptation in exercised B6C3F1 mice (data not shown).

Table 1.

Morphometric data from sedentary (Sed) and exercised (Ex) C57Bl/6J female mice treated with vehicle (Veh) or VCD

	Body weight (g)	Heart weight (mg)	Tibial length (mm)	Heart weight:body weight (mg/g)	Heart weight:tibial length (mg/mm)
Sed-Veh (n = 5)	36.2 ± 1.9	113.5 ± 2.5	19.1 ± 0.1	3.18 ± 0.03	5.94 ± 0.11
Sed-VCD (n = 5)	38.6 ± 2.4	119.0 ± 3.8	19.2 ± 0.1	3.11 ± 0.11	6.20 ± 0.21
Ex-Veh (n = 6)	24.1 ± 0.7a	126.8 ± 3.6a	18.7 ± 0.2	5.28 ± 0.13a	6.77 ± 0.17a
Ex-VCD (n = 5)	26.7 ± 2.1a	133.9 ± 3.3a	18.8 ± 0.1	5.08 ± 0.24a	7.10 ± 0.16a

^aValue significantly (P < 0.05) different from that of sedentary counterparts.

Gene expression, collagen content, and MyHC isoforms in cardiac and skeletal muscle.

We performed a real-time PCR screen that included primers for known pathologic hypertrophy markers (including atrial natriuretic peptide [ANP] and brain natriuretic peptide [BNP]). As shown in Figure 4 A, these markers were not affected by VCD treatment or exercise. In addition, we examined the expression of βMyHC protein in sedentary and exercised control mice and those with VCD-induced ovarian failure. Cardiac MyHC isoform expression did not differ between all experimental groups (Figure 4 B).

Figure 4.

Expression (mean ± SEM) of hypertrophic markers in vehicle- and VCD-treated C57BL/6 mice after voluntary wheel-running exercise. (A) Relative levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were determined by using quantitative real-time PCR; n = 5 per group. (B) Expression of β myosin heavy chain (βMyHC) according to SDS–PAGE. A series of 6 or 7increasing concentrations from 1× to 80× were used to calculate the relative expression of each MyHC isoform (top panel). Clearly separated αMyHC and βMyHC were sliver-stained and quantified as detailed in the Methods. Soleus expressing type I (β) and type IIa MyHC and neonatal rat myocytes expressing αMyHC and βMyHC (not shown) were used as MyHC standards; n = 6 or 7 per group. Veh, vehicle-treated; VCD, VCD-treated; SED, sedentary; EX, exercise.

To determine whether VCD treatment increased collagen deposition in the heart, we performed picrosirius red staining in sedentary mice. There were no significant differences in myocardial collagen content between vehicle- and VCD-treated hearts (data not shown).

Sex hormones and exercise affect MyHC expression in skeletal muscle.^2,52 A representative gel with 2 dilutions of each muscle studied is shown in Figure 5 A. Neither VCD treatment nor exercise affected MyHC isoform content in soleus, tibialis anterior, and gastrocnemius muscles, as measured by SDS–PAGE (Table 2). However, quadriceps muscles displayed a significant (P < 0.05) increase of type IIa MyHC in both vehicle- and VCD-treated groups after exercise (Table 2). MyHC isoform content measured by immunohistochemistry (Figure 5 B) illustrated a similar pattern of expression. Exercise increased type IIa MyHC expression from 8.5% ± 1.2% to 14.3 ± 1.5% and from 8.3% ± 0.9% to 11.2% ± 1.7% in the rectus femoris of vehicle- and VCD-treated mice, respectively. Oxidative capacity as determined by the number of NADH-TR–positive fibers demonstrated a similar pattern of expression (Figure 5 C), such that rectus femoris showed increased numbers of darkly stained fibers, indicative of an increase in oxidative capacity. MyHC content by immunochemistry or NADH-TR staining of the soleus, tibialis anterior, and gastrocnemius muscles was not different between exercised and sedentary mice (data not shown).

Figure 5.

Fiber-type distribution in vehicle- and VCD-treated C57BL/6 mice after voluntary wheel-running exercise. (A) Representative image of types IIa/IIx, IIb, and I myosin heavy chain (MyHC) from quadriceps (Quad), gastrocnemius (G), tibialis anterior (TA), and soleus muscles separated by using SDS–PAGE and then silver-stained and quantified. (B) Representative image of immunostaining for type IIa (green) and IIb (red) MyHC of rectus femoris muscle in vehicle- or VCD-treated sedentary (SED) or exercised (EX) groups. (C) Representative image of rectus femoris stained for NADH-tetrazolium reductase. Veh, vehicle-treated; VCD, VCD-treated; SED, sedentary; EX, exercise; n = 6 or 7 per group.

Table 2.

Expression of isoforms of myosin heavy chain in skeletal muscle relative to total myosin heavy chain from sedentary (Sed) and exercised (Ex) C57Bl/6J female mice treated with vehicle (Veh) or VCD

	Sed-Veh (n = 5)	Sed-VCD (n = 5)	Ex-Veh (n = 5)	Ex-VCD (n = 5)
Soleus
Type I	0.58 ± 0.02	0.52 ± 0.01	0.49 ± 0.01	0.49 ± 0.02
Type IIa	0.41 ± 0.02	0.47 ± 0.01	0.47 ± 0.02	0.50 ± 0.01
Tibialis anterior
Type IIa/IIx	0.35 ± 0.02	0.39 ± 0.01	0.39 ± 0.02	0.36 ± 0.02
Type IIb	0.65 ± 0.02	0.61 ± 0.01	0.61 ± 0.02	0.64 ± 0.02
Gastrocnemius
Type IIa/IIx	0.22 ± 0.02	0.21 ± 0.02	0.22 ± 0.02	0.25 ± 0.02
Type IIb	0.78 ± 0.02	0.79 ± 0.02	0.78 ± 0.02	0.75 ± 0.02
Quadriceps
Type IIa/IIx	0.25 ± 0.02	0.26 ± 0.01	0.32 ± 0.02a	0.37 ± 0.03a
Type IIb	0.75 ± 0.02	0.74 ± 0.02	0.68 ± 0.02a	0.63 ± 0.03a

^aValue significantly (P < 0.05) different from that of sedentary counterparts.

Exercise, VCD treatment, and the AMPK signaling axis.

We performed Western blotting of whole-heart extracts for AMPKα2, AMPKα1, and components of the upstream liver kinase B1 (LKB1)–STE20-related kinase adaptor protein mouse protein 25 kinase complex (MO25; Figure 6 A).¹⁶ Significant (P < 0.05) differences in the basal levels of AMPKα2 and LKB1 but not AMPKα1 total protein were present between vehicle- and VCD-treated sedentary mice (Figure 6 B and C). However, sedentary VCD-treated mice showed no difference in MO25 expression compared with that of sedentary control counterparts (Figure 6 B).

Figure 6.

(A) Western blot analysis of AMPK pathway signaling components. Indicated are paired samples taken from the same blot. (B) Bar graph representations of relative protein expression of components of upstream AMPK kinase complex, LKB1 and MO25, (C) AMPK α1 and α2 isoforms, and (D) target phosphorylation sites pAMPK¹⁷² and pAMPK⁴⁸⁵. (E) Dot plot representation of pACC:ACC as a relative indicator of AMPK activity (n = 4 per group). Veh, vehicle-treated; VCD, VCD-treated; SED, sedentary; EX, exercise. Indicated value is significantly (P < 0.05) different from those for (1) SED-Veh, (2) SED-VCD, and (3) EX-Veh groups.

Phosphorylation of AMPKα at Thr172 (pAMPK¹⁷²) directly correlates with AMPK activity.⁴⁴ Therefore, to estimate the level of AMPK activity, we performed Western analysis that detected AMPKα only when phosphorylated at Thr172 (Figure 6 D). VCD-treated sedentary mice showed a decrease in the level of pAMPK¹⁷² compared with that of their vehicle-treated counterparts. AMPKα also is phosphorylated at Ser485 (Ser491 for AMPKα2). Similar to the change in pAMPK¹⁷², we noted decreased phosphorylation of Ser485 in AMPKα in VCD-treated mice.

Activation of AMPK in the heart leads to direct phosphorylation of ACC.²⁵ We therefore compared the amount of phosphorylated ACC normalized to the total ACC protein content (that is, pACC:ACC) between vehicle- and VCD-treated sedentary mice. We detected a significant (P < 0.05) reduction in the amount of pACC:ACC in VCD-treated compared with vehicle-treated hearts (Figure 6 E).

Voluntary wheel-running exercise induced decreases in total AMPKα2, LKB1, pAMPK¹⁷² and pAMPK⁴⁸⁵ levels in vehicle- and VCD-treated mice (Figure 6 B through D). There was no difference in the levels of AMPKα2, LKB1, pAMPK¹⁷², and pAMPK⁴⁸⁵ between vehicle- and VCD-treated mice that were exercised. In addition, MO25 expression after voluntary wheel-running exercise was significantly (P < 0.05) lower in VCD-treated mice compared with vehicle-treated mice (Figure 6 B). AMPK-dependent targeted phosphorylation of ACC (pACC:ACC) was reduced by exercise and further reduced by VCD treatment; both groups showed exercise-dependent decreases in pACC:ACC ratios (Figure 6 E).

Discussion

In the current study, we achieved estrogen depletion of mice by chemical destruction of primary and primordial follicles accelerating atresia. Similar to a previous study,¹³ we show that estrogen depletion by VCD treatment does not after voluntary wheel-running exercise, unlike in the case of mice rendered estrogen-deplete by ovariectomy. In fact, voluntary wheel-running performance measured according to time and distance is similar between cycling and noncycling mice, and our findings match those of previous work.²³ This lack of effect on exercise after chemical (VCD) destruction of ovarian function leading to estrogen depletion occurs in at least 2 strains of mice, further supporting the physiologic significance of these observations. In addition, no significant differences were measured in treadmill-exercise capacity between vehicle- and VCD-treated mice.

Several studies suggest an association between estrogen and wheel-running activity in both sexes of mice and rats.^11,38,41 Estrogen depletion by surgical ovariectomy leads to voluntary inactivity in mice¹² and rats.²⁰ Recently, chemical depletion of estrogen was confirmed by using Zoladex, an agonist of luteinizing hormone-releasing hormone, results in decreased voluntary wheel-running activity, but to a much lesser degree than for ovariectomized mice subjected to ovariectomy.¹⁷ Interestingly, diminished voluntary activity as observed in ovariectomized animals has not been noted to occur in postmenopausal women. Although postmenopausal women exhibit a slight (less than 10%) decrease in maximal exercise capacity, this decrease is more likely due to aging or morbidity associated with menopause than to menopause itself.³²

At the end of the 4-wk exercise period, mice in both groups demonstrated adaptive cardiac hypertrophy to voluntary wheel running, as previously reported.²³ In our study, estrogen depletion does not affect the extent of cardiac hypertrophy, but the current study is the first to examine cardiac adaptation to exercise after VCD treatment. Therefore, the hypertrophic response to voluntary wheel running likely remains intact in the VCD-induced menopausal model.

The finding that exercise-induced cardiac adaptation is not different between VCD-treated and control mice is not surprising considering that, according to ANP and BNP (mRNA) and βMyHC (protein) expression, hearts from VCD-treated mice are not overtly pathologic. Interestingly, unexercised rats treated with Zoladex demonstrated elevated βMyHC protein expression that could be reversed with voluntary exercise,¹⁷ suggesting that Zoladex treatment initiates a pathologic signaling cascade in the heart similar to that for other pathologic stimuli.³⁹ Furthermore, these data support our contention that estrogen depletion through Zoladex treatment or ovariectomy represents pathophysiology different from that of the accelerated loss of ovarian follicles by VCD treatment. Although we cannot implicate a specific cellular or molecular mechanism for these findings in Zoladex-treated or ovariectomized mice, they likely are due to primary or secondary effects of the surgery or chemical treatment. For example, Zoladex administration and ovariectomy result in an impairment of cardiac function,^17,43 potentially affecting wheel-running performance. One important limitation to the cited previous studies is their lack of a detailed morphometric analysis to determine the type of hypertrophy (concentric compared with eccentric) experienced by vehicle-treated compared with VCD-treated mice.

Similar to previous studies,^10,13,19 we noted muscle-specific changes in skeletal myosin isoform content (immunohistochemistry and SDS–PAGE) and oxidative capacity (NADH-TR staining) among the experimental groups studied after the 4 wk of voluntary exercise. Consistent with exercise performance, vehicle- and VCD-treated mice after exercise showed similar changes. It is possible that the intensity of exercise may be too weak or the duration of exercise too short to elicit responses that differ between experimental groups. Future studies will address whether alternate exercise regimens can induce changes in skeletal muscles.

Nevertheless, despite the lack of different effects on cardiac hypertrophy after wheel-running exercise, we here provide evidence that the cellular adaptation to VCD treatment and the exercise stimulus differs between vehicle- and VCD-treated hearts. Studies indicate a crucial role for skeletal AMPK in exercise capacity, where either whole-body or skeletal-specific genetic deletion of AMPKβ (β1 and β2) causes marked impairment during treadmill running.^37,48 In addition, hormone status affects carbohydrate and lipid metabolism, especially when males and female rodents are compared.^50,53 Estrogen has been suggested to regulate adiposity, in part, through the AMPK signaling pathway.⁹

Because of the pivotal role of the AMPK pathway in orchestrating cellular metabolism and exercise and the ability of hormone status to influence metabolism, we focused our cellular investigation on the relationship between cardiac AMPK activity and voluntary exercise and on how VCD-induced ovarian failure may modify this pathway.^3,16,25 Therefore, we wished to determine 1) the effect of VCD-induced ovarian failure on the AMPK signaling axis and 2) the subsequent effect of exercise on this pathway in the heart. In this regard, we found that the loss of ovarian function suppresses basal (sedentary) levels of AMPKα2 and LKB1 total protein in VCD-treated compared with vehicle-treated mice, with no effect on AMPKα1 or MO25 expression. The pattern of pACC:ACC followed AMPKα2 and LKB1 expression and showed a reduction in AMPK-dependent target activation in hearts from VCD-treated mice. Previous work has shown that estradiol directly activates AMPK activity in vitro^8,9 and in vivo,⁴⁰ and postmenopausal women display increased adiposity and a predispostion to metabolic syndrome consistent with a decrease in AMPK signaling.^21,26 Therefore, we expected that the loss of ovarian estrogen after VCD treatment would result in a suppression of AMPK activity, a prediction that is consistent with our current findings.

Extending analysis of the AMPK signaling axis to the exercised groups shows a further suppression of this pathway. As an indicator of AMPK activity, the pACC:ACC ratio shows similar reduction after exercise in both vehicle- and VCD-treated groups. Notably, the level of pACC:ACC is lower in hearts from VCD-treated compared with vehicle-treated mice after exercise. This decreased pACC:ACC ratio can be explained by the suppression of AMPKα2, LKB1, and MO25 expression in VCD-treated mice after exercise. We suggest that the stoichiometric expression of each component of the AMPK signaling axis plays a crucial role in downstream target activation.

Several studies demonstrate that acute exercise can serve as a stimulus for AMPK activation in skeletal muscle^6,49,54 and heart.^7,33 However, the activation of AMPK may be dependent on exercise intensity or duration. For example, exercise below 60% of maximal aerobic capacity apparently leads to no significant change in AMPK activity.^6,54 Similarly, prolonged exercise training in humans attenuates the robust activation of AMPK activity induced by acute exercise.^35,54 We here illustrate that low-level, chronic exercise can serve as a regulatory feedback signal to suppress AMPK expression and suggest that prolonged exercise may improve the efficiency of AMPK signaling.

Several limitations in our study must be noted. First, because we did not include a VCD-treated ovariectomized control group, we cannot rule out the possibility that changes in AMPK activity may be due to an as-yet undiscovered effect of VCD in the absence of estrogen (that is, ovariectomy). Second, previous studies indicate that residual ovarian tissues in VCD-induced ovarian failure mice are still capable of producing androgen that can have a physiologic effect, just as do the ovaries of postmenopausal women.^27,29 Therefore, the observed inhibition of AMPK activity may be due to the androgen production, given that androgens inhibit AMPK activity in adipocytes that can be reversed by estrogen.³¹ Finally, in our current study, sedentary mice weighed more than did exercised mice. Perhaps the effect of VCD on AMPK activity differs depending on mouse body weight. Additional studies will address these limitations and clarify the relationship between estrogen loss, AMPK activity, and VCD administration.

Nonetheless, the relationship between exercise and AMPK is complicated and particularly depends on the type of activity performed (aerobic compared with anaerobic; acute compared with chronic) (refs. 7,34,53). Considering a potentially pathologic role of AMPK downregulation in menopause, we predict that the mechanisms by which VCD treatment and exercise suppress AMPK expression are mediated by distinct cellular pathways.

To summarize, our findings indicate that the ability of mice to perform voluntary wheel-running exercise is not impeded by the loss of estrogen after the administration of VCD. To date, no studies exist that directly compare daily, moderate activity levels in post- compared with premenopausal women.⁵ Our current data show different cardiac cellular phenotypes between vehicle- and VCD-treated mice. Although the depressed AMPK expression and activity in VCD- compared with vehicle-treated mice may be due to VCD exposure, the target specificity of VCD to the c-Kit survival pathway makes this hypothesis unlikely.^28,51 Furthermore, we here illustrate that VCD treatment suppresses AMPK signaling. Given that voluntary (chronic) wheel-running exercise further suppresses AMPK signaling, future studies will be aimed at delineating cellular signaling pathways in the AMPK suppression induced by VCD treatment and chronic exercise.