Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Feb 1.
Published in final edited form as: J Endocrinol. 2021 Feb;248(2):181–191. doi: 10.1530/JOE-20-0391

Tissue selective effects of bazedoxifene on the musculoskeletal system in female mice

Christine A Cabelka 1,2, Cory W Baumann 1, Angus Lindsay 1,3, Andrew Norton 4, Nick C Blixt 5, Gengyun Le 1, Gordon L Warren 6, Kim C Mansky 4, Susan A Novotny 1,7, Dawn A Lowe 1
PMCID: PMC7933086  NIHMSID: NIHMS1658731  PMID: 33295882

Abstract

The actions of selective estrogen receptor modulators are tissue dependent. The primary objective of the current study was to determine the tissue selective effects of bazedoxifene (BZA) on the musculoskeletal system of ovariectomized (OVX) female mice, focusing on strengths of muscle-bone pairs in the lower hindlimb. Treatment with BZA after ovariectomy (OVX+BZA) did not prevent body or fat mass gains (p<0.05). In vivo plantarflexor muscle isometric torque was not affected by treatment with BZA (p=0.522). Soleus muscle peak isometric, concentric and eccentric tetanic force production were greater in OVX+BZA mice compared to OVX+E2 mice (p≤0.048) with no effect on maximal isometric specific force (p=0.228). Tibia from OVX+BZA mice had greater cortical cross-sectional area and moment of inertia than OVX mice treated with placebo (p<0.001), but there was no impact of BZA treatment on cortical bone mineral density, cortical thickness, tibial bone ultimate load or stiffness (p≥0.086). Overall, these results indicate that BZA may be an estrogen receptor agonist in skeletal muscle, as it has previously been shown in bone, providing minor benefits to the musculoskeletal system.

Keywords: Bone, Ovariectomy, Physical activity, SERM, Skeletal muscle

Introduction

As women age and progress through menopause, they experience a myriad of physiological and systemic changes including decreases in skeletal muscle function and bone health, increased incidence of hot flashes, and alterations in body composition, such as increased abdominal adiposity. There is evidence that menopausal hormone therapy is effective in mitigating these undesirable changes (Tchernof et al., 2000, Anderson et al., 2004, Clarkson et al., 2004, Greising et al., 2009, Ronkainen et al., 2009). However, since the Women’s Health Initiative raised concerns about increased risk for thromboembolic events, stroke, and breast cancer for women using hormone therapy, use of exogenous estrogens has declined substantially (Ettinger et al., 2012). This decline has prompted the development of a class of drugs known as selective estrogen receptor modulators (SERM). The ideal SERM will have an estrogenic effect on the target tissue(s), while at the same time have anti-estrogenic, or no effect, in non-targeted tissues. Bazedoxifene acetate (BZA) is a third generation SERM. It was approved in the European Union for prevention of postmenopausal osteoporosis in 2009, and then by the United States Food and Drug Administration in 2014 for use in combination with conjugated estrogens for treatment of moderate to severe hot flashes, as well as prevention of postmenopausal osteoporosis.

Results from the Selective Estrogen, Menopause, and Response to Therapy (SMART) trials indicate treatment with BZA and conjugated estrogen increases bone mineral density, improves sexual functioning in women with vulvar/vaginal atrophy, reduces number and severity of hot flashes, prevents endometrial hyperplasia, and does not affect mammographic breast density (Pickar et al., 2009, Pinkerton et al., 2009, Harvey et al., 2013, Pinkerton et al., 2014).

While such clinical investigations have shown several positive effects of BZA, additional pre-clinical studies are necessary to further determine effects on targeted and non-targeted tissues, as well as the mechanisms through which BZA elicits these effects independent of a simultaneous estrogen treatment. In terms of efficacy on the skeleton, both rodent and primate models show treatment with BZA following ovariectomy (OVX) increases total bone mineral density (BMD), reduces OVX-induced bone turnover, and increases bone volume and trabecular thickness, particularly of the vertebrae (Kharode et al., 2008, Komm et al., 2011, Saito et al., 2015, Smith et al., 2015). Work by Borjesson and coworkers implicates estrogen receptor alpha (ERα), specifically the ERα- activation function-1, as a mechanism through which BZA elicits its effects in bone (Borjesson et al., 2013, Börjesson et al., 2016). While it appears BZA functions through ERα to mediate positive effects on BMD and turnover, there has been minimal investigation into the effect of BZA on the mechanical properties of bone such as ultimate load and stiffness (Borjesson et al., 2013, Börjesson et al., 2016).

The effects of SERMs on skeletal muscle are largely unknown. This is important because skeletal muscle fibers, like bone cells, contain estrogen receptors (Lemoine et al., 2003, Wiik et al., 2003, Wiik et al., 2009). Some pre-clinical studies on mice have shown that OVX results in decreased skeletal muscle contractility, while treatment with estradiol (E2) mitigates decreases (Moran et al., 2006, Greising et al., 2011a, Greising et al., 2011b, Lai et al., 2016). Similarly, postmenopausal women who take estrogen-based hormone therapy have greater skeletal muscle strength than those who remain estrogen deficient (Phillips et al., 1993, Greising et al., 2009, Ronkainen et al., 2009). Studies on female mice have also demonstrated that deletion of ERα, specifically in skeletal muscle fibers, results in decreased skeletal muscle function (Ribas et al., 2016, Cabelka et al., 2018, Collins et al., 2018). While the SERM tamoxifen has been shown to be an estrogen receptor agonist in skeletal muscle as measured by positive effects on muscle strength (Warren et al., 2007), the extent to which BZA is an ER agonist in skeletal muscle is not known.

There is an inherent link between the function of the musculoskeletal system and physical activity. That is, as physical activity decreases so can skeletal muscle function and bone strength, whereas increasing physical activity can improve muscle function and bone strength. In mice, OVX causes significant decreases in levels of spontaneous physical activity and voluntary wheel running, while treatment with E2 can reverse or prevent the inactivity (Gorzek et al., 2007, Greising et al., 2011a, Bowen et al., 2012). Spontaneous physical activity has previously been defined as home-cage activity or other activity that is different from motivated behaviors such as wheel running (Garland Jr et al., 2011). If BZA were to function like E2 in terms of preventing OVX-induced declines in physical activity, that would be considered a beneficial effect. However, the extent to which BZA affects spontaneous physical activity in mice has been studied only minimally (Kim et al., 2014). Also, the extent to which BZA affects musculoskeletal function independent of any physical activity has not been thoroughly investigated. Thus, to more fully elucidate the tissue specific properties of BZA, this study was designed to test the hypotheses that in ovariectomized mice BZA will 1) improve spontaneous physical activity over that of placebo, 2) be an ER agonist and have beneficial effects on skeletal muscle contractility, and 3) enhance bone structure and strength compared to treatment with placebo.

Methods

Animals and experimental design

Thirty, 8-wk old, female C57BL/6 mice from Jackson Laboratories were obtained and group housed on a 14h:10h light:dark cycle, with free access to water and phytoestrogen free chow (Harland Teklad, 2919, Madison, WI, USA). After 3 wk of acclimation and prior to any intervention, body composition was measured on conscious, 11-wk old animals (Echo MRI 3-in-1, Echo Medical System; Houston, TX, USA). Body mass was measured weekly, from time of the first body composition measurement, for the duration of the study.

Mice were randomly assigned to one of three treatment groups (n=10 each): OVX+placebo, OVX+BZA, and OVX+E2. At 12–14 wk of age mice underwent surgery. Two hours prior to ovariectomy surgery (Moran et al., 2006) mice received subcutaneous injections of 2 mg/kg slow release buprenorphine (ZooPharm, Windsor, CO, USA). Immediately following surgery, treatment pellets (placebo, BZA, or E2) were implanted at the base of the neck via a 10-gauge trochar (Innovative Research of America, Sarasota, FL, USA). One mouse each in the OVX+E2 and OVX+BZA groups were euthanized secondary to non-healing incisions after surgery.

The estrogen pellet consisted of 0.18 mg 17β-E2, released over 60 d such that mice received approximately 3 μg/d (Innovative Research of America). This E2 dose was chosen to elicit physiological levels in mice (Nelson et al., 1981, Farr et al., 1995). The BZA pellet consisted of 1.44 mg BZA, released over 60 d such that the mice received approximately 24 μg/d. This dosage of BZA has been reported to be effective in protecting against bone loss (Andersson et al., 2016a, Andersson et al., 2016b), and was based on body surface calculations used to determine human doses (Reagan-Shaw et al., 2008, Silverman et al., 2008). BZA, a generous gift from Pfizer Inc., (New York City, NY, USA), was sent to Innovative Research of America to make BZA time-release pellets (Wardell et al., 2013).

Spontaneous physical activity was quantified using 24-h cage activity monitoring at 7 wk of treatment, body composition was remeasured at 8 wk of treatment, and then in vivo and in vitro skeletal muscle function was analyzed, immediately followed by sacrifice. At sacrifice, uteri as well as subcutaneous and visceral fat pads were dissected and weighed. Blood was then collected by cardiac bleed, and was allowed to clot at room temperature followed by centrifugation for 10 min at 10,000 g at room temperature. Serum, void of red cell hemolysis, was extracted, snap frozen in liquid nitrogen, and then stored at −80°C until analyzed for E2 or BZA. Lastly, tibial bones were removed, cleaned of all soft tissue, snap frozen in tubes containing 1 mL PBS, and stored at −80°C until bone density and structure measurements and mechanical testing were completed.

The University of Minnesota Institutional Animal Care and Use Committee approved all protocols and animal care procedures. Measurements of serum BZA and E2, physical activities, body mass and composition, in vivo and in vitro muscle physiology, and bone density and strength were conducted with investigators blinded to mouse treatment.

Serum E2 concentration

Serum was pooled when necessary to reach >300 μL per sample to be analyzed, precipitated, eluted, and measured using liquid chromatography – tandem mass spectrometry (LC-MS/MS) as previously described (Le et al., 2018). Briefly, a micro LC column (2.7 μm, C18 90 Å, Halo Fused core, 100 × 0.3 mm, Eksigen, SCIEX; Framingham, MA, USA) was used in series with an AB SCIEX QTRAP 5500 mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The E2 reporter was identified by a m/z of 271.2 Da and quantified by peak area of the chromatograph using MultiQuant™ (SCIEX). Mean serum concentrations of E2 were: OVX+placebo = 4.7 pg/ml (n=4, pooled samples), OVX+E2 = 44.9 ± 27.9 pg/ml (n=5, unpooled samples with SD) indicating successful ovariectomy and replacement of E2 in the physiological range for female mice, respectively.

Serum BZA Concentration

Serum samples were thawed on ice and an equal volume of acetonitrile was added to each. Samples were vortexed, place on ice for 10 min followed by centrifugation at 4°C and 20,817 g for 10 min, and the supernatant was used for analysis. Samples were spiked with 5 ng/ml BZA standard prior to analysis.

HPLC measurement of BZA was performed using a Shimadzu I-series 2030C with auto-sampler, RF-20A fluorescence detector and on-line degasser with 10μL of each sample injected onto a Luna 5 μm C-18 100 Å, 250 × 4.6 mm column (Phenomenex, CA, USA). The mobile phase was generated by mixing (A) 10 mM KH2PO4 (pH 3) with (B) 10:90 water:acetonitrile (v:v) by pre-pump mixing to give 90% A and 10% B at 1mL/min. BZA was eluted with a linear gradient from 10 – 90% B over 30 min and followed by 90% B for a further 10 min. The column was returned to the starting condition by 90% to 10% B gradient over two min and holding at 10% B for 3 min making a total run time of 45 min. BZA was detected at its natural fluorescence of 300 nm excitation and 380 nm emission (Chandrasekaran et al., 2010). The concentration and identity of BZA was compared to a standard, bazedoxifene acetate (Pfizer Inc., Groton, CT, USA). Concentration was quantified by peak area using Shimadzu Class VP software. Serum BZA concentrations were (mean ± SD): OVX+placebo = 0.19 ± 0.47 ng/ml (n=4), OVX+BZA = 3.62 ± 6.27 ng/ml (n=10), and OVX+E2 = −0.08 ± 0.37 ng/ml (n=4). These BZA concentrations are consistent with previous reports (Chandrasekaran et al., 2010, Smitha et al., 2015).

24-hour physical activity monitoring

Using open-field activity chambers (Med Associates Inc., St. Albans, VT, USA) for a 24-h period, spontaneous physical activities of individual mice were measured as previously described (Landisch et al., 2008, Greising et al., 2011a). Briefly, each mouse underwent a familiarization period by being housed in a mock chamber for a full day prior to initiating the 24-hour of cage activity monitoring on the second day. Activity data was acquired using Activity Monitor version 5 software (Med Associates), and a box size of three. Disruption of photo beam arrays in the chamber due to mouse movements register an activity count and duration. To note, an “activity count” represents any single photo beam break and because multiple photo beams may break with one jump or vertical movement, those counts do not represent the actual number of times a mouse jumps or rears. One mouse from each of the OVX+placebo and OVX+E2 groups had data excluded in the analysis secondary to software malfunction.

In vivo plantar flexor muscle function

Each mouse was anesthetized via isoflurane inhalation (1.5%) with an O2 flow rate of 125 mL/min and in vivo contractility of the plantar flexor muscles (gastrocnemius, soleus, and plantaris) was assessed by stimulating the sciatic nerve. As previously described, the peroneal branch of the sciatic nerve was first severed to avoid recruitment of the dorsi flexor muscles (Baltgalvis et al., 2012). The ankle joint was positioned so that the foot was perpendicular to the tibia and the knee was stabilized with a clamp to inhibit lower limb movement. The footplate was attached to the shaft of a servomotor (300-LR; Aurora Scientific, Aurora, Ontario, Canada). Peak isometric torque was measured, followed by a series of concentric contractions at various angular velocities (1200–100°/s) of plantarflexion over a 40° excursion. Contraction of the plantar flexors was elicited via percutaneous stimulation of the sciatic nerve with platinum needle electrodes attached to a stimulator (E2–12 electrodes and S48 stimulator; Grass Telefactor, Warwick, RI, USA). While still anesthetized, mice received an intraperitoneal injection of sodium pentobarbital (100 mg/kg body mass) followed by in vitro contractility testing of the soleus muscle.

In vitro soleus muscle contractile function

Soleus muscle isolation and preparation have previously been described (Moran et al., 2005). Briefly, the soleus muscle was placed in a 1.2 mL bath filled with oxygenated Krebs-Ringer buffer maintained at 25°C. The proximal muscle tendon was attached by 6–0 suture to a dual-mode muscle lever system (300B-LR; Aurora Scientific Inc.). Measures of peak isometric, concentric, and eccentric forces during tetanic stimulation were obtained as previously described (Greising et al., 2011b).

Cortical bone structure of the tibia

Cortical bone of tibia at the mid-diaphysis was assessed using a micro-computed tomography system (XT H 225, Nikon Metrology Inc., Brighton, MI, USA). The scanner was set to a voltage of 120 kVp and a current of 61 μA, and bones were scanned using an isotropic 7.4 μm voxel size with a 708 ms integration time. A 0.5 mm region (approximately 68 slices) was scanned and the average relative bone density was determined at each slice using regression analyses derived from Image J (NIH) analysis based on standardized phantoms (Bruker micro-CT, Belgium). The average BMD across each of the 68 slices was calculated and reported for each tibia. CT Pro 3D (Nikon Metrology Inc.) was used to make 3D reconstruction volumes for each scan. VGStudio MAX 3.2 (Volume Graphics GmbH, Heidelberg, Germany) converted 3D reconstruction volumes to bitmap datasets for each scan. Morphometric analyses were completed with the SkyScan CT-Analyzer (Bruker micro-CT). At completion of scanning, bones were stored at −80°C until mechanical testing.

Bone mechanical testing

Mechanical properties of the tibia at the mid-diaphysis were determined using three-point bending. A Mecmesin MultiTest 1-D test machine and a Mecmesin AFG-25 load cell (Mecmesin, West, Sussex, UK) was used as previously described (Novotny et al., 2011). Briefly, each tibia was thawed, length measured, and the mid-point was marked. Each bone was placed on its lateral side on two supports with 1 cm separation and a quasi-static load was applied to the mid-diaphysis at a displacement rate of 2 mm/min until failure. Load-displacement curves were analyzed for each bone using a custom-written TestPoint program (TestPoint version 7; Measurement Computing Corp., Norton, MA, USA) as previously described (Novotny et al., 2011, Mader, 2014). Mechanical properties analyzed were ultimate load and stiffness.

Statistical analyses

To analyze the effects of treatment (OVX+placebo, OVX+BZA, OVX+E2) and time (baseline/pre-surgery, OVX, wk 1–8) on body mass and compositions, repeated measures two-way ANOVAs were performed. When main effects or interactions were significant (p<0.05), Holm-Sidak post hoc tests were performed to determine which combination of conditions were different from one another. To assess differences in treatment on spontaneous physical activity, skeletal muscle contractility, fat pad masses, uterine masses, tibial bone structural and mechanical properties, one-way ANOVAs were performed. When an effect of treatment was significant, Holm-Sidak post hoc measurements were used to determine differences among treatments. When assumptions of equal variance or normality were violated, as assessed by Shapiro-Wilk testing, Kruskal-Wallis one-way Analysis of Variance on Ranks was performed with Dunn’s post hoc tests. For all post hoc testing, p<0.05 is considered significant. Statistical analyses were carried out using SigmaStat version 3.5 (Systat Software, Inc., Point Richmond, CA, USA).

Results

Spontaneous physical activity

Following 7 wk of treatment, 24-hour spontaneous physical activities and time being active tended to be low in OVX+BZA mice with ambulation distance significantly less than OVX+placebo mice (Table 1). Although the ANOVA for total active time reached significance, post hoc tests did not identify any treatment group differing from another (Table 1).

Table 1.

Twenty-four hour cage activities of ovariectomized mice treated with placebo, bazedoxifene, or 17β-estradiol for 7 weeks.

OVX+Placebo
(n=9)		 OVX+BZA
(n=9)		 OVX+E2
(n=8)		 One-Way ANOVA
p-value
Ambulation Distance (m) 1,211 (918) 549 (370)* 1,155 (1129) 0.020
Ambulation Time (min) 148 (37.4) 92.7 (43.8) 148 (70.3) 0.062
Jump Count 1,611 (484) 1,423 (514) 2,109 (757) 0.065
Vertical Count 6,206 (3,315) 5,400 (1,558) 11,934 (13,109) 0.066
Active Time (min) 284 (43.3) 221 (43.4) 290 (80.5) 0.042
Open in a new tab

Data are means (SD).

*

Different from OVX+Placebo.

Body mass and composition and uterine mass

All mice gained weight throughout the study with the amount of weight gained in each group dependent upon time (interaction p<0.001; Figure 1A). OVX+placebo mice weighed more than OVX+E2 mice at wk 2 through 8. OVX+BZA mice weighed more than OVX+E2 mice at wk 3, and wks 5–8, indicating that BZA treatment did not blunt weight gain as did treatment with E2.

Figure 1:

Figure 1:

Open in a new tab

Body masses of mice over the duration of the study and end point uterine masses. A) Weekly body masses of ovariectomized mice differed among treatment groups depending on time (interaction p <0.001). Significant results from post-hoc testing between groups are denoted at wk of treatment. B) Uterine mass at sacrifice. Data are means ± SD. *Different from OVX+placebo. #Different from OVX+BZA.

Uterine masses of OVX+placebo and OVX+BZA mice were low compared to OVX+E2 mice (p<0.05; Figure 1B). This result indicates that BZA did not stimulate uterine hyperplasia as did E2 in ovariectomized mice.

The percent of body mass that was lean mass changed over the course of the study depending on treatment (interaction p<0.001; Figure 2A). Within groups, OVX+placebo and OVX+BZA mice had less lean mass at wk 8 compared to baseline, and at wk 8 had less lean mass compared to OVX+E2 mice (Figure 2A). The opposite held for fat mass with mice treated with placebo or BZA, but not E2, gaining fat over the 8 wk study (Figure 2B). The relative adiposity of OVX mice treated with placebo and BZA measured by MRI was corroborated by fat pad masses. Subcutaneous and visceral fat pads from OVX+placebo and OVX+BZA mice weighed 96–126% more than those from OVX+E2 mice (p<0.05; Figure 2C).

Figure 2:

Figure 2:

Open in a new tab

Body composition of ovariectomized mice differed according to treatment group and time (interaction, p <0.001) and mass of fat pads from ovariectomized mice was low in mice treated with estradiol. A) Lean mass as a percent of total body mass differed between baseline (pre-surgery) and 8 wk after ovariectomy within OVX+placebo and OVX+BZA groups as indicated by horizontal lines. At wk 8, lean mass differed by treatment. B) Fat mass as a percent of total body mass differed between baseline and 8 wk after ovariectomy within OVX+placebo and OVX+BZA groups as indicated by horizontal lines. At wk 8, fat mass differed by treatment. C) Subcutaneous and visceral fat pad masses at sacrifice were affected by treatment. *Different from OVX+placebo within time (or within fat pad in C); #Different from OVX+BZA within time (or within fat pad in C). Horizontal bars indicate differences between Baseline and Week 8 within group.

Skeletal muscle contractile function

There was no difference in peak isometric torque produced in vivo by the plantar flexor muscles among the treatment groups (p=0.522; Figure 3A). Over a range of angular velocities, torque production differed at 800 and 1000°/sec (p≤0.020; Figure 3B) between OVX+placebo and OVX+E2 mice (p≥0.140 for all other angular velocities; Figure 3B).

Figure 3:

Figure 3:

Open in a new tab

In vivo muscle function of ovariectomized mice minimally differed among treatments. A) Peak isometric torque of the plantarflexor muscles. B) Torque production of the plantarflexor muscles at increasing velocities. Data are mean ± SD. *OVX+E2 different from OVX+placebo.

Isolated soleus muscles from OVX+BZA mice weighed more and produced greater peak isometric, concentric and eccentric forces compared to OVX+E2 mice (Table 2). Once normalized for size, isometric force did not differ significantly among treatment groups (Specific Po, Table 2).

Table 2:

In vitro soleus muscle contractile properties of ovariectomized mice treated with placebo, bazedoxifene, or 17β-estradiol for 8 weeks.

OVX+Placebo (n=10) OVX+BZA
(n=9)		 OVX+E2
(n=9)		 One-Way ANOVA
p-value
Muscle mass (mg) 8.48 (0.47) 8.69 (0.61) 7.59 (0.74)# 0.009
Peak isometric tetanic force (mN) 212 (26.3) 234 (38.0) 191 (20.7)# 0.048
Specific Po (N/cm2) 25.4 (2.88) 27.6 (4.80) 24.0 (2.08) 0.228
Peak concentric force (mN) 108 (18.4) 117 (24.0) 86.7 (15.1)# 0.009
Peak eccentric force (mN) 381 (49.2) 414 (66.2) 339 (39.8)# 0.020
Open in a new tab

Data are means (SD). Specific force (Po) was calculated as peak isometric tetanic force normalized by muscle physiological cross-sectional area.

#

Different from OVX+BZA.

Cortical structural and mechanical properties of the tibia

Tibia were imaged at the mid-diaphysis to determine the impact of BZA on cortical bone structure. There was no difference among OVX mice treated with placebo, BZA, and E2 in cortical BMD (p=0.528; Figure 4A). Cortical thickness of tibia also did not differ among groups (p=0.099). Cortical cross-sectional area of tibia differed among groups (p<0.001; Figure 4B) with OVX+BZA and OVX+E2 mice having greater areas than OVX+placebo, and tibia from OVX+BZA mice having less area than OVX+E2 mice. Cortical cross-sectional moment of inertia of the tibia differed among groups as well (p>0.001) being greater in OVX+BZA (0.078±0.005 mm4) than OVX+placebo (0.068±0.006 mm4) and OVX+E2 mice (0.072±0.006 mm4).

Figure 4:

Figure 4:

Open in a new tab

Tibial bone properties from ovariectomized mice were marginally different among the treatment groups. A) Cortical bone mineral density and B) cortical cross-sectional area at the mid-diaphysis. C) Mechanical properties, ultimate load and D) stiffness. *Different from OVX+placebo. #Different from OVX+BZA.

To determine if treatment with BZA improved bone strength, mechanical properties of the tibias were tested. Ultimate load differed among groups (p=0.033) with OVX+E2 being greater than OVX+placebo mice (Figure 4C). There was no difference among groups for tibial bone stiffness (p=0.086; Figure 4D).

Discussion

This study addressed three hypotheses related to effects of BZA on the musculoskeletal system. First, we did not detect a difference in spontaneous physical activity among the groups and thus were unable to support our hypothesis that BZA treatment would improve spontaneous physical activity more than treatment with placebo. Despite the absence of treatment effect on spontaneous physical activity, we did find effects on body and fat masses. While treatment with placebo and BZA resulted in ovariectomy-induced gains in body and fat masses, treatment with E2 maintained body mass after OVX. Second, BZA treatment preserved soleus muscle mass compared to treatment with E2 contributing to slightly greater isometric, concentric and eccentric force production and providing some support for the hypothesis that BZA is an ER agonist in skeletal muscle. Third, although tibial cortical cross-sectional area and cross-sectional moment of inertia were improved with BZA treatment, there was no significant difference between mice treated with placebo and BZA for tibia cortical BMD, ultimate load, or stiffness, thus the hypothesis that BZA would positively affect cortical bone structure and strength was not supported.

Because the function of the musculoskeletal system and physical activity are inherently linked, our experiment first sought to determine if BZA treatment affected spontaneous physical activity. Ovariectomy in rodents typically results in decrements in both spontaneous physical activities as well as voluntary wheel running (Moran et al., 2006, Gorzek et al., 2007, Greising et al., 2011a, Bowen et al., 2012). Two main parameters, ambulation time and total active time, were not different by treatment group though ambulation distance was significantly lower in OVX mice treated with BZA compared to placebo. Previous studies also found no differences in locomotor activity in BZA-treated OVX mice compared to untreated OVX or ovary-intact mice (Barrera et al., 2014, Kim et al., 2014). Because wheel running shows robust responses to manipulation of estrogenic compounds in rodents (Moran et al., 2006, Gorzek et al., 2007, Greising et al., 2011a, Bowen et al., 2012), future studies should consider measuring this type of physical activity. Our results combined with previous studies indicate that any effect of BZA on body composition, skeletal muscle, or bone in mice are not likely related to changes in physical activity.

Inconsistent with previous studies, our results show that BZA did not prevent gains in body and fat masses after OVX. Prior studies reported that BZA prevented OVX-induced weight and fat mass gains in mice (Barrera et al., 2014, Kim et al., 2014). A difference between those studies and ours was delivery of BZA; we used subcutaneous time-release pellets whereas other studies used osmotic mini-pumps (Barrera et al., 2014) or oral gavage (Kim et al., 2014). It is possible that the delivery system or dose of the BZA plays a role in the effectiveness of minimizing weight gain, and that the resulting BZA serum concentration we report may have been below therapeutic range. These results emphasize the importance of measuring and reporting concentrations of SERMs resulting from treatments. Consistent with previous studies (Barrera et al., 2014, Bernardi et al., 2014, Kim et al., 2014, Andersson et al., 2016a, Andersson et al., 2016b), our data clearly indicate that BZA treatment did not increase uterine mass.

A primary objective of our studies was to determine effects of BZA on the musculoskeletal system. We are aware of only one other study that investigated the effects of a SERM on both skeletal muscle and bone functions. Previously, Warren et al (Warren et al., 2007) treated mice 30 days following OVX with tamoxifen for 60 days. No significant differences between tamoxifen- and E2-treated groups were measured in rescuing maximal isometric tetanic force of the soleus muscle, micro-CT properties of the tibia, or mechanical properties of the tibia, from deleterious effects of OVX. The conclusion was that tamoxifen is an estrogen receptor agonist in skeletal muscle as well as bone. The current study also evaluated soleus muscle contractility and tibial bone cortical and mechanical properties, with expanded analyses to include measures of in vivo plantar flexor muscle contractility. Our results did not show differences among ovariectomized mice treated with placebo, E2, or BZA in plantar flexor torque production. Soleus muscles from OVX+BZA mice produced greater peak isometric, concentric and eccentric forces than did OVX+E2 mice; however once normalized for size, specific force did not differ among treatment groups.

Our imaging results of the mid diaphysis of the tibia showed that BZA increased cortical cross-sectional area and cross-sectional moment of inertia but did not increase cortical BMD. These results are consistent with the results from Anderson et al (Andersson et al., 2016a) which showed that BZA increased trabecular but not cortical BMD. In contrast, Komm et al (Komm et al., 2011) demonstrated that BZA treatment was effective in preventing OVX-induced reduction in cortical BMD, however this was after 52 wk of treatment. Our study provided only 8 wk of treatment. Additional studies have shown that BZA was effective in preventing OVX-induced decline in BMD, but these studies evaluated total BMD, that is, combining trabecular and cortical bone (Kharode et al., 2008, Bernardi et al., 2014).

There are only two studies that we are aware of which reported the effects of BZA on mechanical properties of bone. Borjesson et al (Borjesson et al., 2013), demonstrated that treatment with BZA increased maximal load to failure in femurs from orchidectomized male mice compared to femurs from control mice. In female mice, BZA treatment after OVX tended to increase cortical thickness and maximal load to failure but the increases did not reach statistical significance (Börjesson et al., 2016). Similarly, our analysis of bone mechanical properties measured no difference in ultimate load or stiffness between BZA and placebo treatment.

In summary, results of the current study show that BZA has minimal effects within the musculoskeletal system of female mice at the treatment dose used. The SERM caused modest improvements in soleus muscle mass and force production uniquely suggesting that BZA may be an estrogen receptor agonist in skeletal muscle. BZA had positive impacts on some but not all cortical bone structural properties, however there was no effect of BZA on mechanical properties of tibia. BZA treatment to ovariectomized mice did not prevent body or fat mass gains as did E2 treatment and thus overall did not demonstrate strong estrogenic properties in female mice. Further, results from this study did not appear to be confounded by treatment-induced changes in physical activities by the mice. Future studies are needed to recognize the tissue-selective nature of SERMs, including investigation into effects on multiple tissues and physiological systems.

Funding:

This work was supported by National Institutes of Health Grants R01-AG031743, T32-AR007612, T32-AR050938, T32-AG029796 and a Promotion of Doctoral Studies II Scholarship from the Foundation for Physical Therapy.

Footnotes

Declaration of interest: There is no conflict of interest to declare.

References

  1. ANDERSON GL, LIMACHER M, ASSAF AR, BASSFORD T, BERESFORD SAA, BLACK H, BONDS D, BRUNNER R, BRZYSKI R, CAAN B, et al. 2004. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial. JAMA : the journal of the American Medical Association, 291, 1701–12. [DOI] [PubMed] [Google Scholar]
  2. ANDERSSON A, BERNARDI A, NURKKALA-KARLSSON M, STUBELIUS A, GRAHNEMO L, OHLSSON C, CARLSTEN H & ISLANDER U 2016a. Suppression of Experimental Arthritis and Associated Bone Loss by a Tissue-Selective Estrogen Complex. Endocrinology, 157, 1013–1020. [DOI] [PubMed] [Google Scholar]
  3. ANDERSSON A, BERNARDI AI, STUBELIUS A, NURKKALA-KARLSSON M, OHLSSON C, CARLSTEN H & ISLANDER U 2016b. Selective oestrogen receptor modulators lasofoxifene and bazedoxifene inhibit joint inflammation and osteoporosis in ovariectomised mice with collagen-induced arthritis. Rheumatology (United Kingdom), 55, 553–563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. BALTGALVIS KA, CALL JA, COCHRANE GD, LAKER RC, YAN Z & LOWE DA 2012. Exercise training improves plantarflexor muscle function in mdx mice. Medicine & Science in Sports & Exercise, 44, 1671–1679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. BARRERA J, CHAMBLISS KL, AHMED M, TANIGAKI K, THOMPSON B, MCDONALD JG, MINEO C & SHAUL P 2014. Bazedoxifene and conjugated estrogen prevent diet-induced obesity, hepatic steatosis, and type 2 diabetes in mice without impacting the reproductive tract. American Journal Physiology-Endocrinology and Metabolism, 307, E345–E354. [DOI] [PubMed] [Google Scholar]
  6. BERNARDI A, ANDERSSON A, GRAHNEMO L, NURKKALA-KARLSSON M, OHLSSON C, CARLSTEN H & ISLANDER U 2014. Effects of lasofoxifene and bazedoxifene on B cell development and function. Immunity Inflammation and Disease, 2, 214–225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. BORJESSON AE, FARMAN HH, ENGDAHL C, KOSKELA A, SJOGREN K, KINDBLOM JM, STUBELIUS A, ISLANDER U, CARLSTEN H, ANTAL MC, et al. 2013. The role of activation functions 1 and 2 of estrogen receptor-alpha for the effects of estradiol and selective estrogen receptor modulators in male mice. Journal of Bone and Mineral Research, 28, 1117–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. BÖRJESSON AE, FARMAN HH, MOVÉRARE-SKRTIC S, ENGDAHL C, ANTAL MC, KOSKELA A, TUUKKANEN J, CARLSTEN H, KRUST A, CHAMBON P, et al. 2016. SERMs have substance-specific effects on bone, and these effects are mediated via ERαAF-1 in female mice. American Journal of Physiology - Endocrinology and Metabolism, 310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. BOWEN RS, KNAB AM, HAMILTON AT, MCCALL JR, MOORE-HARRISON TL & LIGHTFOOT JT 2012. Effects of Supraphysiological Doses of Sex Steroids on Wheel Running Activity in Mice. Journal of Steroids & Hormonal Science, 3, 110–110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. CABELKA CA, BAUMANN CW, COLLINS BC, NASH N, LE G, LINDSAY A, SPANGENBURG EE & LOWE DA 2018. Effects of ovarian hormones and estrogen receptor alpha on physical activity and skeletal muscle fatigue in female mice. Experimental Gerontology. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. CHANDRASEKARAN A, AHMAD S, SHEN L, DEMAIO W, HULTIN T & SCANTINA J 2010. Disposition of bazedoxifene in rats. Xenobiotica, 40, 578–585. [DOI] [PubMed] [Google Scholar]
  12. CLARKSON TB, FREEDMAN RR, FUGHBERMAN AJ, LOPRINZI CL & REAME NK 2004. Treatment of menopause-associated vasomotor symptoms: position statement of the North American Menopause Society. Menopause, 11, 11–33. [DOI] [PubMed] [Google Scholar]
  13. COLLINS BC, MADER TL, CABELKA CA, INIGO MR, SPANGENBURG EE & LOWE DA 2018. Deletion of estrogen receptor α in skeletal muscle results in impaired contractility in female mice. Journal of Applied Physiology, 124, 980–992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. ETTINGER B, WANG SM, LESLIE RS, PATEL BV, BOULWARE MJ, MANN ME & MCBRIDE M 2012. Evolution of postmenopausal hormone therapy between 2002 and 2009. Menopause, 19, 610–5. [DOI] [PubMed] [Google Scholar]
  15. FARR SA, FLOOD JF, SCHERRER JF, KAISER FE, TAYLOR GT & MORLEY JE 1995. Effect of ovarian steroids on footshock avoidance learning and retention in female mice. Physiology and Behavior, 58, 715–723. [DOI] [PubMed] [Google Scholar]
  16. GARLAND JR T, SCHUTZ H, CHAPPELL MA, KEENEY BK, MEEK TH, COPES LE, ACOSTA W, DRENOWATZ C, MACIEL RC, VAN DIJK G, et al. 2011. The biological control of voluntary exercise, spontaneous physical activity and daily energy expenditure in relation to obesity: human and rodent perspectives. J Exp Biol, 214, 206–229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. GORZEK JF, HENDRICKSON KC, FORSTNER JP, RIXEN JL, MORAN AL & LOWE DA 2007. Estradiol and tamoxifen reverse ovariectomy-induced physical inactivity in mice. Medicine & Science in Sports & Exercise, 39, 248–256. [DOI] [PubMed] [Google Scholar]
  18. GREISING SM, BALTGALVIS KA, KOSIR AM, MORAN AL, WARREN GL & LOWE DA 2011a. Estradiol’s beneficial effect on murine muscle function is independent of muscle activity. Journal of Applied Physiology, 110, 109–115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. GREISING SM, BALTGALVIS KA, LOWE DA & WARREN GL 2009. Hormone therapy and skeletal muscle strength: a meta-analysis. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 64, 1071–1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. GREISING SM, CAREY RS, BLACKFORD JE, DALTON LE, KOSIR AM & LOWE DA 2011b. Estradiol treatment, physical activity, and muscle function in ovarian-senescent mice. Experimental Gerontology, 46, 685–693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. HARVEY JA, PINKERTON JV, BARACAT EC, SHI H, CHINES AA & MIRKIN S 2013. Breast density changes in a randomized controlled trial evaluating bazedoxifene/conjugated estrogens. Menopause, 20, 138–145. [DOI] [PubMed] [Google Scholar]
  22. KHARODE Y, BODINE PVN, MILLER CP, LYTTLE CR & KOMM BS 2008. The pairing of a selective estrogen receptor modulator, bazedoxifene, with conjugated estrogens as a new paradigm for the treatment of menopausal symptoms and osteoporosis prevention. Endocrinology, 149, 6084–6091. [DOI] [PubMed] [Google Scholar]
  23. KIM JH, MEYERS MS, KHUDER SS, ABDALLAH SL, MUTURI HT, RUSSO L, TATE CR, HEVENER AL, NAJJAR SM, LELOUP C, et al. 2014. Tissue-selective estrogen complexes with bazedoxifene prevent metabolic dysfunction in female mice. Molecular Metabolism, 3, 177–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. KOMM BS, VLASSEROS F, SAMADFAM R, CHOUINARD L & SMITH SY 2011. Skeletal effects of bazedoxifene paired with conjugated estrogens in ovariectomized rats. Bone, 49, 376–386. [DOI] [PubMed] [Google Scholar]
  25. LAI S, COLLINS BC, COLSON BA, KARARLGAS G & LOWE DA 2016. Estradiol modulates myosin regulatory light chain phosphorylation and contractility in skeletal muscle of female mice. American Journal of Physiology - Endocrinology And Metabolism, 310, E724–E733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. LANDISCH RM, KOSIR AM, NELSON SA, BALTGALVIS KA & LOWE DA 2008. Adaptive and nonadaptive responses to voluntary wheel running by mdx mice. Muscle Nerve, 38, 1290–1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. LE G, NOVOTNY SA, MADER TL, GREISING SM, CHAN SSK, KYBA M, LOWE DA & WARREN GL 2018. A moderate estradiol level enhances neutrophil number and activity in muscle after traumatic injury but strength recovery is accelerated. Journal of Physiology. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. LEMOINE S, GRANIER P, TIFFOCHE C, RANNOU-BEKONO F, THIEULANT M & DELAMARCHE P 2003. Estrogen receptor alpha mRNA in human skeletal muscles. Medicine & Science in Sports & Exercise, 35, 439–443. [DOI] [PubMed] [Google Scholar]
  29. MADER TL, NOVOTNY SA, LIN AL, GULDBERG RE, LOWE DA, WARREN GL 2014. CCR2 elimination in mice results in larger and stronger tibial bones but bone loss is not attenuated following ovariectomy or muscle denervation. Calcif Tissue Int, 95, 457–466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. MORAN AL, WARREN GL & LOWE DA 2005. Soleus and EDL muscle contractility across the lifespan of female C57BL/6 mice. Experimental Gerontology, 40, 966–975. [DOI] [PubMed] [Google Scholar]
  31. MORAN AL, WARREN GL & LOWE DA 2006. Removal of ovarian hormones from mature mice detrimentally affects muscle contractile function and myosin structural distribution. Journal of Applied Physiology, 100, 548–559. [DOI] [PubMed] [Google Scholar]
  32. NELSON JF, FELICIO LS, OSTERBURG HH & FINCH CE 1981. Altered profiles of estradiol and progesterone associated with prolonged estrous cycles and persistent vaginal cornification in aging C57BL/6J mice. Biology of Reproduction, 24, 784–794. [DOI] [PubMed] [Google Scholar]
  33. NOVOTNY SA, WARREN GL, LIN AS, GULDBERG RE, BALTGALVIS KA & LOWE DA 2011. Bone is functionally impaired in dystrophic mice but less so than skeletal muscle. Neuromuscular Disorders, 21, 183–193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. PHILLIPS SK, ROOK KM, SIDDLE NC, BRUCE SA & WOLEDGE RC 1993. Muscle weakness in women occurs at an earlier age than in men, but strength is preserved by hormone replacement therapy. Clinical Science, 84, 95–98. [DOI] [PubMed] [Google Scholar]
  35. PICKAR JH, YEH IT, BACHMANN G & SPEROFF L 2009. Endometrial effects of a tissue selective estrogen complex containing bazedoxifene/conjugated estrogens as a menopausal therapy. Fertility and Sterility, 92, 1018–1024. [DOI] [PubMed] [Google Scholar]
  36. PINKERTON JV, HARVEY JA, LINDSAY R, PAN K, CHINES AA, MIRKIN S & ARCHER DF 2014. Effects of bazedoxifene/conjugated estrogens on the endometrium and bone: a randomized trial. Journal of Clinical Endocrinology and Metabolism, 99, E 189–198. [DOI] [PubMed] [Google Scholar]
  37. PINKERTON JV, UTIAN WH, CONSTANTINE GD, OLIVIER S & PICKAR JH 2009. Relief of vasomotor symptoms with the tissue-selective estrogen complex containing bazedoxifene/conjugated estrogens: a randomized, controlled trial. Menopause, 16, 1116–1124. [DOI] [PubMed] [Google Scholar]
  38. REAGAN-SHAW S, NIHAL M & AHMAD N 2008. Dose translation from animal to human studies revisited. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 22, 659–661. [DOI] [PubMed] [Google Scholar]
  39. RIBAS V, DREW BG, ZHOU Z, PHUN J, KALAJIAN NY, SOLEYMANI T, DARAEI P, WIDJAJA K, WANAGAT J, DE AGUIAR VALLIM TQ, et al. 2016. Skeletal muscle action of estrogen receptor alpha is critical for the maintenance of mitochondrial function and metabolic homeostasis in females. Science Translational Medicine, 8, 334–354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. RONKAINEN P, KOVANEN V, ALEN M, POLLANEN E, PALONEN E, ANKARBERG-LINDGREN C, HAMALALNEN E, TURPELNEN U, KUJALA U, PUOLAKKA J, et al. 2009. Postmenopausal hormone replacement therapy modifies skeletal muscle composition and function: a study with monozygotic twin pairs. Journal of Applied Physiology, 107, 25–33. [DOI] [PubMed] [Google Scholar]
  41. SAITO M, KIDA Y, NISHIZAWA T, ARAKAWA S, OKABE H, SEKI A & MARUMO K 2015. Effects of 18-month treatment with bazedoxifene on enzymatic immature and mature cross-links and non-enzymatic advanced glycation end products, mineralization, and trabecular microarchitecture of vertebra in ovariectomized monkeys. Bone, 81, 573–80. [DOI] [PubMed] [Google Scholar]
  42. SILVERMAN SL, CHRISTIANSEN C, GENANT HK, VUKICEVIC S, ZANCHETTA JR, DE VILLIERS TJ, CONSTANTINE GD & CHINES AA 2008. Efficacy of bazedoxifene in reducing new vertebral fracture risk in postmenopausal women with osteoporosis: results from a 3-year, randomized, placebo-, and active-controlled clinical trial. Journal of Bone and Mineral Research, 23, 1923–34. [DOI] [PubMed] [Google Scholar]
  43. SMITH SY, JOLETTE J, CHOUINARD L & KOMM BS 2015. The effects of bazedoxifene in the ovariectomized aged cynomolgus monkey. Journal of bone and mineral metabolism, 33, 161–172. [DOI] [PubMed] [Google Scholar]
  44. SMITHA G, REDDY SS, RASAGA M, PRIYANKA R, BAKSHI V & JUKANT IR 2015. Bazedoxifene acetate quantification in rat serum with the aid of RP-HPLC: Method development and validation. World Journal of Pharmaceutical Sciences, 3, 2357–2363. [Google Scholar]
  45. TCHERNOF A, POEHLMAN ET & DESPRES JP 2000. Body fat distribution, the menopause transition, and hormone replacement therapy. Diabetes and Metabolism, 26, 12–20. [PubMed] [Google Scholar]
  46. WARDELL SE, NELSON ER, CHAO CA & MCDONNELL DP 2013. Bazedoxifene Exhibits Antiestrogenic Activity in Animal Models of Tamoxifen-Resistant Breast Cancer: Implications for Treatment of Advanced Disease. Clinical Cancer Research, 19, 2420–2431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. WARREN GL, MORAN AL, HOGAN HA, LIN AS, GULDBERG RE & LOWE DA 2007. Voluntary run training but not estradiol deficiency alters the tibial bone-soleus muscle functional relationship in mice. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 293, R2015–R2026. [DOI] [PubMed] [Google Scholar]
  48. WIIK A, EKMAN M, JOHANSSON O, JANSSON E & ESBJORNSSON M 2009. Expression of both oestrogen receptor alpha and beta in human skeletal muscle tissue. Histochemistry and Cell Biology, 131, 181–9. [DOI] [PubMed] [Google Scholar]
  49. WIIK A, GLENMARK B, EKMAN M, ESBJORNSSON-LIJEDAHL M, JOHANSSON O, BODIN K, ENMARK E & JANSSON E 2003. Oestrogen receptor beta is expressed in adult human skeletal muscle both at the mRNA and protein level. Acta Physiologica, 179, 381–387. [DOI] [PubMed] [Google Scholar]

RESOURCES