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J Appl Physiol 100: 548-559, 2006. First published October 27, 2005; doi:10.1152/japplphysiol.01029.2005
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Removal of ovarian hormones from mature mice detrimentally affects muscle contractile function and myosin structural distribution

Amy L. Moran,1 Gordon L. Warren,3 and Dawn A. Lowe2

1School of Kinesiology and 2Department of Physical Medicine and Rehabilitation, University of Minnesota, Minneapolis, Minnesota; and 3Department of Physical Therapy, Georgia State University, Atlanta, Georgia

Submitted 24 August 2005 ; accepted in final form 20 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The purposes of this study were to determine the effects of ovarian hormone removal on force-generating capacities and contractile proteins in soleus and extensor digitorum longus (EDL) muscles of mature female mice. Six-month-old female C57BL/6 mice were randomly assigned to either an ovariectomized (OVX; n = 13) or a sham-operated (sham; n = 13) group. In vitro contractile function of soleus and EDL muscles were determined 60 days postsurgery. Total protein and contractile protein contents were quantified, and electron paramagnetic resonance (EPR) spectroscopy was used to determine myosin structural distribution during contraction. OVX mice weighed 15% more than sham mice 60 days postsurgery, and soleus and EDL muscle masses were 19 and 15% greater in OVX mice, respectively (P ≤ 0.032). Soleus and EDL muscles from OVX mice generated less maximal isometric force than did those from sham mice [soleus: 0.27 (SD 0.04) vs. 0.22 N·cm·mg–1 (SD 0.04); EDL: 0.33 (SD 0.04) vs. 0.27 N·cm·mg–1 (SD 0.04); P ≤ 0.006]. Total and contractile protein contents of soleus and EDL muscles were not different between OVX and sham mice (P ≥ 0.242), indicating that the quantity of contractile machinery was not affected by removing ovarian hormones. EPR spectroscopy showed that the fraction of strong-binding myosin during contraction was 15% lower in EDL muscles from OVX mice compared with shams [0.277 (SD 0.039) vs. 0.325 (SD 0.020); P = 0.004]. These results indicate that the loss of ovarian hormones has detrimental effects on skeletal muscle force-generating capacities that can be explained by altered actin-myosin interactions.

force; skeletal muscle; electron paramagnetic resonance spectroscopy; estrogen

THE EFFECTS OF ESTROGEN AND estrogen deprivation on bone, the cardiovascular system, and the female reproductive system are well known, whereas relatively little is known about the effects on skeletal muscle, especially on fully developed skeletal muscle. Skeletal muscle contains estrogen receptors, leading to the premise that it is a tissue responsive to estrogen (14–16, 37). In mature muscle, estrogen has been associated with modulating myosin heavy chain (MHC) isoform expression and with muscle injury prevention (5, 11, 26, 29, 35), but whether it influences the primary function of skeletal muscle, i.e., force generation, is uncertain.

Muscle force-generating capacity has been shown to increase (27), decrease (35), and not change (21) after the removal of ovarian hormones from growing, immature rodents. Furthermore, estradiol replacement reversed the changes in force generation in those studies, suggesting that estrogen was the ovarian hormone responsible for the initial changes after ovariectomy (27, 35). The ages of rodents used in all three of those studies are equivalent to adolescent or younger in humans, illustrating the complex, critical roles of ovarian hormones during muscle development. No studies have been conducted on the effects of ovarian hormones on intact skeletal muscle contractile function using mature rodents. Thus the first purpose of this study was to determine the effects of ovarian hormone removal on muscle contractile function of adult female mice. We hypothesized that removing ovarian hormones via ovariectomy would detrimentally affect force- and power-generating capacities of soleus and extensor digitorum longus (EDL) muscles.

Our hypothesis that the removal of ovarian hormones would detrimentally affect force generation was based in part on the results of a study on single, permeabilized muscle fibers from ovariectomized mice (36). Those results were that specific force of soleus muscle fibers was 20% lower in mice that were ovariectomized for 10–14 wk compared with ovarian-intact mice, suggesting that contractile proteins may be directly affected by ovarian hormones (36). With our finding that muscles from ovariectomized mice generated less force, the second purpose of this study was to determine whether myosin and actin contents and actin-myosin interactions were altered by the removal of ovarian hormones. It has previously been hypothesized that ovarian hormones directly affect contractile protein function, however, this mechanism has never been tested (23, 36). Therefore, we used electron paramagnetic resonance (EPR) spectroscopy to measure the fraction of strong-binding, force-generating myosin heads during maximal isometric contraction. We hypothesized that myosin and actin contents would be unaffected by the removal of ovarian hormones but that the fraction of strong-binding myosin would be reduced.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   Female C57BL/6 mice aged 5 mo were purchased from the National Institute on Aging colony and used in this study. Mice were housed four per cage on a 12:12-h light-dark cycle and were given phytoestrogen-free commercial rodent chow (2019 Teklad Global 19% Protein Rodent Diet, Harland Teklad, Madison WI) and water ad libitum. At 6 mo of age, ovarian tissue was surgically removed from one group of mice (OVX; n = 13). For this procedure, mice were anesthetized via inhalation with 1.75% isoflurane mixed with oxygen at a flow rate of 200 ml/min. Body temperature was maintained by a 37°C recirculating-water heating pad. Under aseptic conditions, bilateral ovariectomy was performed through two small dorsal incisions between the iliac crest and the lower ribs. The abdominal muscle wall incisions were closed with a single simple interrupted stitch using 6-0 silk suture, and skin incisions were closed with 7-mm wound clips. Each mouse was administered 0.15 µg of buprenorphine subcutaneously ~5 min after the isoflurane anesthetic was withdrawn. The surgical procedure for control, sham-operated mice (sham; n = 13) was the same except that the ovaries were not excised. Mice were housed individually for 1 wk postsurgery and four per cage thereafter.

Approximately 60 days postsurgery (58–62 days), mice were weighed and anesthetized by an intraperitoneal injection of pentobarbital sodium (100 mg/kg body wt) with supplemental doses given as required. Mice were euthanized with an overdose of pentobarbital sodium (200 mg/kg body wt) after muscles were excised. All protocols and animal care procedures were approved by the institutional animal care and use committee and complied with guidelines set by the American Physiological Society.

In vitro muscle preparation.   The soleus muscle from one hindlimb and the EDL muscle from the contralateral hindlimb of each mouse were studied to determine muscle contractile function and how that function was altered by the loss of ovarian hormones. The isolated mouse muscle preparation used was similar to that described previously (33). After a muscle was excised, it was mounted in a 0.38-ml bath assembly filled with Krebs-Ringer-bicarbonate buffer maintained at 25°C by a recirculating-water bath. The proximal tendon was attached by 6-0 silk suture to a dual-mode muscle lever system (model 300B-LR, Aurora Scientific, Aurora, ON, Canada). Muscles were set to their anatomic Lo (i.e., a length halfway between the muscle's minimum and maximum in vivo lengths), which coincides with the length at which isometric twitch forces are maximized (32). All contractile measurements began at Lo.

Muscles remained quiescent for 10 min in the bath assembly, and then a protocol for testing contractile function began. First, passive stiffness of the inactive muscle was determined by passively stretching the muscle sinusoidally from 97.5% Lo to 102.5% Lo at 0.5 Hz while measuring the resulting force (33, 34). Thirty seconds later, peak twitch force was elicited by stimulating the muscle with a 0.5-ms pulse at 150 V (Grass S48 stimulator delivered through a SIU5D stimulus isolation unit, Grass Telefactor, Warwick, RI). A second twitch was elicited 30 s later. Maximal isometric tetanic contraction force (Po) was next elicited by stimulating muscles at 150 V and 400 ms at 120 Hz for soleus muscles and 200 ms at 180 Hz for EDL muscles. Two minutes later, a second maximal isometric tetanic contraction was elicited, and at peak force a sinusoidal oscillation of 0.01% Lo at 500 Hz was imposed to determine active muscle stiffness. Last, 12 shortening velocities were measured by quick releases from Po to given afterloads that corresponded to 5–50% of the previous Po. Maximal shortening velocity (Vmax) was determined by fitting a hyperbolic-linear curve to the force-velocity data Table Curve 2D (version 5.1, Systat Software, Richmond, CA) and extrapolating the curve to a zero afterload (32). Maximal power output by each muscle was calculated from force-velocity data using the nonlinear curve-fitting procedures in the application Math Cad (version 12, Cambridge, MA). The stimulator and lever system were controlled by computer (Pentium 4, 3.2 GHz) using a KPCI-3108 interface board (Keithley Instruments, Cleveland, OH) and TestPoint software (version 5, Capital Equipment, Billerica, MA).

Muscles were removed from the bath assembly at the end of the contractile protocol, trimmed, blotted, weighed, and then immediately frozen in liquid nitrogen and stored at –80°C for subsequent protein analyses. Muscle weight and length data, using a fiber length-to-muscle length ratio of 0.71 for soleus muscle and 0.44 for EDL muscle, were used to calculate physiological cross-sectional area (2, 33).

EPR spectroscopy.   EDL muscles that were not used for the in vitro contractile analyses from each mouse were glycerinated for 3 days as described previously (19, 20) and then stored in a 50% glycerol-rigor buffer for 1–5 mo. Permeabilized EDL muscles were dissected into fiber bundles that were ~0.5 mm in diameter and had fibers running in parallel from end to end. These fiber bundles were prepared for EPR spectroscopy by spin labeling with 0.5 mM 4-(2-idoacetamido)-2,2,6,6-tetramethyl-1-piperidinyloxy spin label (IASL; Sigma) specifically at the Cys707 in the catalytic domain of the myosin head (19, 20). IASL-labeled fibers were fixed in a glass capillary tube inside a TE102 cavity (model 4102ST/8838, Bruker Instruments, Billerica, MA) so that the longitudinal axis of the fibers was perpendicular to the magnetic field. Buffers were flowed over the fibers at a rate of 2 ml/min at 22°C. For each fiber bundle, EPR spectra were collected under conditions of rigor, relaxation, and contraction on an E500 EleXsys spectrometer (Bruker Instruments). The parameters used to collect the low-field portion of the EPR spectrum were 3,415-G central peak, 15-G sweep width, 5.0-G peak-to-peak modulation amplitude, and 15.9-mW microwave power. Spectra were analyzed to determine the fraction of myosin strongly bound to actin during contraction as previously described (19, 20). Two to three bundles from each EDL muscle were analyzed and their values averaged to represent the fraction of myosin bound to actin during contraction for that muscle.

During spectroscopy, one end of the permeabilized fiber bundle was attached to a force transducer (model 801 strain gauge, SensoNor Ackers, Aksjelskapet, Norway), and the other end stabilized to hold the fibers isometrically. Thus force was monitored throughout EPR spectra collection. Maximal Ca2+-activated force of each fiber bundle was normalized to contractile protein content divided by fiber bundle length. This was necessary as bundle cross-sectional area could not be accurately calculated because bundles were not uniformly shaped; i.e., the cross section of some bundles appeared circular, whereas other bundles were more striplike. The contractile protein content of each EPR sample was determined by gel electrophoresis as described below.

Determination of total, MHC, and actin protein contents.   Each soleus and EDL muscle from the in vitro contractile analysis was homogenized in 10 mM phosphate buffer (pH 7.0) and assayed in triplicate for total protein content using BCA protein assay with albumin standards (Pierce Biotechnology, Rockford IL). Because myofibril solubility is lower in low-ionic-strength buffers (e.g., 10 mM phosphate buffer) compared with higher ionic strength ones, extra precautions were taken to 1) homogenize each sample uniformly, 2) remove all visible connective tissue from the homogenate, 3) prewet the pipette tip before taking the sample to be assayed, and 4) vortex the homogenate thoroughly and immediately before pipetting. Quantitative gel electrophoresis was also performed on those homogenates to determine contractile protein content of each muscle. In preparation for electrophoresis, homogenates were diluted with equal volume of Laemmli sample buffer containing beta-mercaptoethanol and heated to 100°C for 3 min. Ten micrograms of total protein from each muscle were separated on a 7.5% Criterion SDS-PAGE gel (Bio-Rad, Hercules, CA) by running at 150 V for 1.5 h. MHC (2, 4, 6, 8, 10 µg) and actin (1, 2, 3, 4, 5 µg) protein standards, a broad-range molecular weight marker, and 10 muscle samples, alternating between OVX and sham, were run on each gel (Fig. 1). Purified MHC was a kind gift from the laboratory of David D. Thomas (University of Minnesota, Minneapolis, MN), and actin was purchased from Sigma (A2522). Soleus and EDL muscle samples were run on separate gels.


Figure 1
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  		Fig. 1. Representative SDS-PAGE gel for determining myosin heavy chain (MHC) and actin protein contents of each muscle. Lane 1 is a molecular weight standard. Lanes 2–6 are MHC and actin standards. Lanes 7–16 are extensor digitorum longus (EDL) muscle samples, each containing 10 µg of total protein.

 
EDL fiber bundle samples from the EPR experiments were also assessed for myosin and actin contents. After EPR, fiber bundles were stored in Laemmli sample buffer containing beta-mercaptoethanol at –80°C. Immediately before electrophoresis, EPR samples were heated to 100°C for 5 min. Quantitative gel electrophoresis was performed on 20 µl of each EPR sample similar to the procedure for whole muscle homogenates.

All gels were stained for 1 h with 0.1% Coomassie blue R-250, 30% methanol, and 10% glacial acetic acid, and they were destained overnight in 20% methanol and 10% glacial acetic acid. Stained gels were scanned using a Bio-Rad GS-700 imaging densitometer and analyzed using Molecular Analyst software (version 21, Bio-Rad). Linear regressions of the optical density (OD), adjusted for local background, for the MHC and actin protein standards were used to determine the contents of MHC and actin proteins of each sample loaded onto the gel. Regression analyses on these standards from the gels yielded correlations of ≥0.95 for the OD-protein content relationships. The actin and MHC content of each muscle or fiber bundle was then calculated from the gel sample contents and the dilution factors used the during sample preparation (10). Contractile protein content is operationally defined as the sum of MHC and actin contents.

Statistical analyses.   Independent Student's t-tests were used to determine differences between OVX and sham-operated mice for contractile parameters and protein contents of muscles. Statistical analyses were performed using SigmaStat version 2.0 (Systat Software). An alpha level of 0.05 was used for all tests of significance. Values are reported as means (SD).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Body mass, muscle mass, and protein contents.   The removal of ovarian hormones for 60 days caused increases in body and muscle masses. Body mass for OVX mice was 13% greater than for sham mice [27.3 (SD 3.9) vs. 23.9 g (SD 2.4); P = 0.015]. Soleus and EDL muscle masses for OVX mice were 20 and 16% greater than for sham mice, respectively (Table 1).


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  		Table 1. Soleus and extensor digitorum longus muscle composition of sham-operated and ovariectomized mice

 
Total protein content was measured to determine whether the muscle mass gains reflected an anabolic response or simply an increase in nonprotein content, in response to ovariectomies. Total protein content was not different between OVX and sham muscles (Table 1). Furthermore, total protein concentration, i.e., total protein per muscle wet weight, was ~10% lower in muscles from OVX mice compared with sham mice [soleus muscle: 0.147 (SD 0.009) vs. 0.162 mg/mg (SD 0.009), EDL muscle: 0.146 (SD 0.012) vs. 0.163 mg/mg (SD 0.012); P ≤ 0.002].

Myosin and actin contents were measured to determine whether the proportion of contractile to noncontractile protein was affected by the removal of ovarian hormones. Contractile protein content of soleus and EDL muscles was not different between OVX and sham mice (Table 1), and the ratio of myosin to actin was not different either (P ≥ 0.127). The mean (SD) myosin and actin contents for all soleus muscles were 0.33 mg (SD 0.05) and 0.16 mg (SD 0.03), respectively. The mean myosin and actin contents for all EDL muscles were 0.38 mg (SD 0.06) and 0.18 mg (SD 0.04), respectively. The mean ratio of myosin to actin in soleus muscle was 1.52 and was 1.51 for EDL muscle. Thus the removal of ovarian hormones for 60 days did not alter the quantity of the major contractile proteins in hindlimb muscles of mice.

Force generation.   Soleus and EDL muscles from mice lacking ovarian hormones displayed lower force-generating capacity. Po of electrically stimulated whole muscles from OVX mice was significantly lower than those from sham mice (Table 2). Specific Po, i.e., Po divided by muscle physiological cross-sectional area, was 21.1 N/cm2 (SD 2.3) and 16.4 N/cm2 (SD 2.4) for soleus muscles from sham and OVX mice, respectively. Specific Po was 27.4 N/cm2 (SD 1.9) and 20.5 N/cm2 (SD 2.6) for EDL muscles from sham and OVX mice, respectively. However, because of the ovariectomy-induced increase in muscle mass without a concomitant increase in contractile protein content, we believed that it was inappropriate to normalize strength to physiological cross-sectional area (28). Instead, Po was normalized by contractile protein content per muscle fiber length (10). Po per contractile protein content per fiber length was 19% less in soleus and EDL muscles from OVX mice compared with those from sham mice (P ≤ 0.006; Fig. 2). Because neither protein content nor muscle length was different between OVX and sham mice (P ≥ 0.201), these data indicate that a qualitative difference in force-generating capacity exists between muscles exposed to and deprived of ovarian hormones.


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  		Table 2. In vitro contractile properties of soleus and extensor digitorum longus muscles from sham-operated and ovariectomized mice

 

Figure 2
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  		Fig. 2. Maximal isometric force normalized to contractile protein content per fiber length for intact soleus and EDL muscles and for permeabilized EDL fibers from mice that were ovariectomized (OVX) and those that had sham operations. Bars represent means (SD). *Significantly different from sham, P < 0.05.

 
Maximal Ca2+-activated isometric force of permeabilized EDL fibers was measured during EPR experiments and was less in fibers from OVX mice than sham mice [3.71 (SD 1.12) vs. 4.36 mN (SD 0.77); P = 0.042]. Similarly, maximal Ca2+-activated force normalized to contractile protein content per fiber length was 25% less in fibers from OVX mice compared with those from sham mice (P = 0.043; Fig. 2). Because the lower force in permeabilized fibers from OVX mice was similar in magnitude to the reduction in whole muscle force, these data indicate a qualitative difference between OVX and sham muscle in a contraction step occurring downstream of Ca2+ release from the sarcoplasmic reticulum.

To assess the possibility that actin-myosin interactions were altered by the removal of ovarian hormones, the structural distribution of myosin during contraction in EDL muscle fibers was measured. EPR spectroscopy showed that the fraction of strong-binding myosin during contraction was 15% less in EDL muscles from OVX mice compared with sham mice [0.277 (SD 0.039) vs. 0.325 (SD 0.020); P = 0.004; Figs. 3 and 4]. Presently, EPR measurements of myosin strong-binding in soleus muscle fibers are not possible.


Figure 3
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  		Fig. 3. Representative electron paramagnetic resonance (EPR) spectra of spin-labeled muscle fibers from 8-mo-old sham and OVX mice obtained during conditions of rigor (black), maximal Ca2+-activated isometric contraction (blue), and relaxation (red). In these samples, the fraction of strong-binding myosin during contraction was 0.347 (0.039) for the sham and 0.204 (0.041) for the OVX fibers [means (SD)]. The larger fraction of myosin bound to actin during contraction in the sham sample can be visualized in these spectra as a greater area of "blue hashes" compared with that of the OVX sample.

 

Figure 4
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  		Fig. 4. Fraction of strong-binding myosin in fibers from OVX mice relative to that fraction determined in fibers from sham mice. Left pair of bars represents the fraction of strong-binding myosin determined directly by EPR spectroscopy on permeabilized EDL fibers. Middle and right pairs of bars represent active stiffness data obtained on intact soleus and EDL muscles that were studied in vitro. The difference in the fraction of strong-binding myosin between muscles from sham and OVX mice was approximately the same, 10–15% as determined by EPR spectroscopy or active stiffness. *Significantly different from sham, P < 0.05.

 
Another way to indirectly estimate the degree of myosin strong binding during contraction is to measure active muscle stiffness. Active stiffness was determined on intact soleus and EDL muscles that were studied in vitro. Muscles from OVX mice had ~12% lower active stiffness than did muscles from sham mice (P ≤ 0.001; Table 2 and Fig. 4). These data reinforce the EPR results showing that ovarian hormones qualitatively affected contractile protein function.

Passive stiffness, which reflects an inactive muscle's resistance to lengthening, was also determined for all soleus and EDL muscles studied in vitro. We found that passive stiffness of muscles from OVX mice was 12–20% greater than that from sham mice (Table 2). The resistance to lengthening determined in passive stiffness measurements is due to elastic elements in parallel with force-generating elements, suggesting that other structural elements in addition to contractile proteins are altered by removing ovarian hormones.

Vmax and peak power.   Previous research on rodent skeletal muscle has shown shifts toward the expression of slower MHC isoforms after ovariectomy, leading to the hypothesis that contractile speed is affected by ovarian hormones (11). To test this hypothesis, Vmax of intact soleus and EDL muscles was determined in vitro. Vmax normalized to muscle fiber length was 9% faster in soleus muscle from OVX mice compared with sham mice, but Vmax of EDL muscle was not different between groups (Table 2). From the force-velocity experiment, peak muscle power generation was calculated. There were no differences in absolute peak power or peak power normalized to contractile protein content between muscles from OVX and sham mice (Table 2).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The key finding of this study was that the loss of ovarian hormones for 60 days resulted in a reduced force-generating capacity for soleus and EDL muscles from mature female mice. Contractile protein content of those muscles was not affected, but Ca2+-activated force and the proportion of strong-binding myosin during contraction were reduced in fiber bundles from ovariectomized mice. The reduction in the fraction of strong-binding myosin occurred to the same extent as the decline in Po, indicating that altered contractile protein function is a major mechanism underlying the force loss that is induced by removing ovarian hormones.

Mature, adult mice were used in this study, which is a key difference from previous muscle contractility studies on ovariectomized rodents (21, 27, 35). Our mice arrived at 5 mo of age and were fed a phytoestrogen-free diet to eliminate any potential influences of exogenous estrogen-like compounds, such as geinstein, that are common in many commercial rodent chows. Mice were ovariectomized at 6 mo of age, and muscle contractile function was assessed at ~8 mo of age. Previous muscle contractile studies were conducted on rodents that were ovariectomized between the ages of 6 and 10 wk (21, 35, 36), ages at which rodents are still growing and maturing and therefore better modeled amenorrheic adolescent girls than menopausal women. Because a long-term goal of our studies is to distinguish age-related from hormonally related contractile losses, we used mature mice. However, we also did not want to confound our surgical intervention of ovariectomy with age-induced ovarian failure, so we selected the age of 8 mo as the end point of our studies to avoid approaching the age at which ovaries begin to fail in mice, i.e., 11–16 mo of age (4).

The greatest effect of removing ovarian hormones for 60 days was a decrease in force-generating capacity. We found ~18% lower Po values normalized to contractile protein content per muscle fiber length in intact soleus and EDL muscles from mature OVX mice. Our findings are in agreement with some (35, 36) but not all previous rodent work (21, 27). Warren and coworkers (35) showed that Po of EDL muscles from young OVX mice was 14% less compared with OVX mice treated with 17beta-estradiol. McCormick and coworkers (21, 35) showed that specific Po was unchanged by the removal of ovarian hormones from immature rats for 28 days, and another study on developing rats found that ovariectomy caused Po to increase (27). McCormick and coworkers normalized force generation to muscle cross-sectional area, but this cannot explain the discrepancy between our results and theirs because if nonprotein mass increases occurred in their study as in ours, then the specific Po difference between OVX and sham animals would be accentuated not blunted. It is more likely that the use of immature, growing rats in their study vs. the mature mice used in our study account for the different results. In addition, we found maximal Ca2+-activated force to be 25% lower in permeabilized EDL fibers from OVX mice compared with sham mice. These data are in agreement with those from Wattanapermpool and Reiser (36), who also showed that maximal Ca2+-activated force was reduced in single, permeabilized soleus fibers from immature ovariectomized rats.

The dynamic molecular interaction of myosin binding strongly to actin generates force. Thus, at the most elementary level, a loss of force results from 1) a decrease in the total number of myosin and actin molecules, 2) a decrease in the fraction of myosin molecules that are strongly bound to actin during the cross-bridge cycle, and/or 3) a decrease in the force per strongly bound myosin molecule. We measured myosin and actin protein contents of soleus and EDL muscles and found that contents were not affected by the removal of ovarian hormones, ruling out possibility 1. Possibility 2 was directly measured by EPR spectroscopy, which revealed that the fraction of myosin heads strongly bound to actin during maximal isometric contraction was 15% less in EDL muscles from OVX mice compared with sham mice. Possibility 3 can be indirectly determined because the ratio of Po to active stiffness gives an estimation of force per strong myosin-actin interaction (6). This ratio was not different between muscles from OVX and sham mice (P ≥ 0.392), indicating that ovarian hormones do not affect force per myosin-actin interaction. These results reveal that ovarian hormones directly affect contractile protein function, a mechanism previously hypothesized (23, 36) but until now never tested. Thus the loss of ovarian hormones did not affect contractile protein quantity but instead detrimentally affected myosin structural distribution during contraction, resulting in a reduced Po.

This result was substantiated by the active stiffness measurements made on intact muscles in vitro. According to Huxley's and Simmon’s cross-bridge model, stiffness of active muscle should be linearly related to the fraction of myosin heads strongly bound to actin (9). Following this model, early calculations predicted that up to 80% of myosin heads were strongly attached to actin during isometric contraction (7). Later, however, EPR spectroscopy, which directly measures the fraction of strong-binding myosin heads in functioning fibers during contraction, showed that the fraction was 20–30% for rabbit psoas fibers (3, 22). Recently, active stiffness measurements have been used in conjunction with new values of filament compliance, and these calculations predict that the fraction of myosin heads attached to actin during isometric contraction cannot be larger than 43% (18). Thus direct measurement by spectroscopy and calculations based on active stiffness agree that the fraction of myosin heads bound to actin during contraction is in the range of 30–40%, not 80% as initially predicted. In the present study, we found that that difference in active stiffness between muscles from sham and OVX mice was ~12%, agreeing closely with the 15% difference determined by EPR (i.e., 33 vs. 27% strong-binding myosin). Thus the removal of ovarian hormones for 60 days reduced the fraction of strong-binding myosin as determined both by EPR and active stiffness measurements on EDL muscles and by active stiffness measurements on soleus muscles. Again this ovariectomy-induced decrement in the proportion of strong-binding myosin can account for the entire reduction in Po for the OVX muscles.

Passive stiffness of soleus and EDL muscles was increased by 12–20% 60 days after ovarian hormone removal. Increased collagen content has been shown to increase passive stiffness (8) and could potentially have occurred in our study as a result of ovariectomy despite our finding that total protein content did not change. This is possible because our measure of total protein content does not reflect the contribution from collagen. Hence, if an increase in muscle collagen content occurred, it would be consistent with the observed increase in passive stiffness. However, further investigation is needed to directly determine whether ovarian hormones affect muscle collagen content.

In general, muscle Vmax and peak power generation were minimally affected by ovariectomy. For EDL muscles, there were no differences in Vmax or peak power between OVX and sham muscles. Soleus muscles became ~10% faster as a result of removing ovarian hormones, in agreement with a previous study showing faster muscle twitch kinetics in soleus muscles from immature ovariectomized rats (21). It is possible that there was a shift in MHC isoform expression in the soleus muscle that contributed to the muscle becoming faster. However, this seems unlikely because previous studies have shown that the removal of ovarian hormones in rats had no effect on MHC isoform expression or caused a shift away from the faster IIa and IIb isoforms to the slower type I isoform (11, 21). Soleus muscle peak power, a product of force and velocity, was not affected by the removal of ovarian hormones because the decreased force-generating capacity was offset by an increased shortening velocity.

The levels of several hormones are altered when the ovaries are removed; therefore, it is unclear which ovarian hormone(s) contributed to the contractile changes we observed. We speculate that the lack of estradiol, the most biologically active form of estrogen, is the key hormone responsible for the force decrements. The effects of estradiol are mediated through estrogen receptors that are located in the nucleus and act as transcription factors (12). Gene expression is then regulated by the direct binding of estradiol-bound estrogen receptor to a specific sequence of DNA called the estrogen response element (13). We predict that some muscle genes have estrogen response elements in their promoter regions and are thus regulated, at least in part, by estradiol. Thus, in the absence of estradiol, it is possible that genes related to the regulation of myosin are perturbed, resulting in altered myosin structure and force-generating function. Estrogen receptors are also located on the cell membrane, but their function is not well defined (17). Alternatively, the mechanism by which the loss of ovarian hormones leads to altered myosin may be related to estradiol's antioxidant property (12, 30). For example, oxygen radical production after exercise-induced injury is low in mature female rats with normal estrogen levels, suggesting that estrogen may offer a line of defense against free radicals (31). Therefore, it is possible that a lowered antioxidant state occurs in the absence of estrogen, leading to some posttranslational oxidative modification at a critical site in myosin that in turn affects myosin's ability to bind actin and generate force. Both of these explanations attempting to connect the loss of ovarian hormones to myosin dysfunction are speculative and require further investigation.

Understanding how estradiol affects skeletal muscle contractility has health implications, particularly in light of the current debates over the benefits vs. the risks of hormone replacement therapy (HRT) for postmenopausal women. Clinical trials of HRT for menopausal women have shown mixed results on muscle strength. For example, a cross-sectional study by Phillips and coworkers (24) showed lower specific force of the adductor pollicis muscle in perimenopausal women; however, the reduction was prevented by HRT. Skelton and coworkers (25) conducted a randomized clinical trial of HRT and measured changes in adductor pollicis strength over a 12-mo period. They found that postmenopausal women on HRT increased strength by ~12%, whereas women not on HRT had a 3% loss of strength. On the other hand, Bemben and Langdon (1) found that upper body isotonic and handgrip strengths were similar in postmenopausal women regardless of HRT status.

In summary, the present study shows that the loss of ovarian hormones for 60 days has detrimental effects on the force-generating capacities of mouse soleus and EDL muscles. Furthermore, our data show that the decline in Po after the removal of ovarian hormones was not due to a reduction in contractile protein content but instead to a reduction in the fraction of strong-binding myosin during contraction. Additional research is needed to pinpoint which ovarian hormone(s) is (are) critical in regulating muscle contractile function and myosin structure and to determine whether those detrimental effects can be reversed.


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This research was supported by National Institutes of Health (NIH) Grant AG-20990 to D. A. Lowe, and A. L. Moran was supported by NIH Training Grant T32 AR-07612.


   ACKNOWLEDGMENTS
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We thank Igor Negrashov for assistance with Vmax and power data analyses.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. A. Lowe, Dept. of Physical Medicine and Rehabilitation, Univ. of Minnesota, MMC 388, 420 Delaware St. SE, Minneapolis, MN 55455 (e-mail: lowex017{at}umn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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