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

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.Abbreviations: ACC, acetyl CoA carboxylase; AMPK, 5′ AMP-activated protein kinase; LKB1, liver kinase B1; MO25, mouse protein 25 kinase complex; NADH-TR, NADH-tetrazolium reductase; p, phosphorylated form; VCD, 4-vinylcyclohexine diepoxideIn 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.⁵¹5′ 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.¹⁵Considering 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.