Voluntary run training but not estradiol deficiency alters the tibial bone-soleus muscle functional...

Voluntary Run Training But Not Estradiol Deficiency Alters the Tibial Bone:Soleus

Muscle Functional Relationship in Mice

Gordon L. Warren1, Amy L. Moran2, Harry A. Hogan3, Angela S. Lin4, Robert E. Guldberg4, and

Dawn A. Lowe2

1Division of Physical Therapy, Georgia State University, Atlanta, GA

2Department of Physical Medicine and Rehabilitation, University of Minnesota, Minneapolis, MN

3Department of Mechanical Engineering, Texas A&M University, College Station, TX

4Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA

Running head: Voluntary Running Alters the Bone:Muscle Relationship

Contact information: Gordon L. Warren, PhD

Division of Physical Therapy

P.O. Box 4019

Georgia State University

Atlanta, GA 30302-4019

Phone: (404) 413-1255

Fax: (404) 413-1230

Email: [email protected]

of 46Articles in PresS. Am J Physiol Regul Integr Comp Physiol (September 19, 2007). doi:10.1152/ajpregu.00569.2007

Copyright © 2007 by the American Physiological Society.

mailto:[email protected]

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Abstract

The study’s objective was to investigate how estrogen deficiency and run training affect the tibial

bone:soleus muscle functional relationship in mice. Female mice were assigned into one of two

surgical conditions, ovariectomy (OVX) or sham ovariectomy (Sham), and one of two activity

conditions, voluntary wheel running (Run) or sedentary (Sed). To determine if differences

observed between OVX and Sham conditions could be attributed to estradiol (E2), additional

OVX mice were supplemented with E2. Tibial bones were analyzed for their functional

capacities, ultimate load and stiffness. Soleus muscles were analyzed for their functional

capacities, maximal isometric tetanic force (Po) and peak eccentric force. The ratios of bone

functional capacities to those of muscle were calculated. The bone functional capacities were

affected by both surgical condition and activity but more strongly by surgical condition. Ultimate

load and stiffness for the Sham condition were 7-12% greater than those for OVX (p=0.002)

while only stiffness was greater for Run compared to Sed (9%; p=0.015). The muscle functional

capacities were affected by both surgical condition and activity but in contrast to the bone, the

muscle was more affected by activity. Po and peak eccentric force were 10-21% greater for Run

compared to Sed (p≤0.016) while only Po was greater in Sham compared to OVX (7%; p=0.011).

The bone:muscle ratios of functional capacities were affected by activity but not by surgical

condition or E2 supplementation. Thus, a mismatch of bone:muscle function occurred in mice

that voluntarily ran on wheels, irrespective of estrogen status.

Keywords: biomechanics, functional capacity, exercise, estrogen

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Introduction

A stress fracture is defined as a skeletal defect caused by repetitive application of a stress

that is less than that required to fracture a bone in a single loading but more than that from which

a bone can fully recover. A stress fracture may however worsen until a frank fracture occurs. The

incidence of stress fractures is rare in the general population but the annual incidence may be as

high as 20% in young athletes and military recruits (8). There is a marked gender difference in

the risk of developing a stress fracture. In the U.S. Armed Forces, the risk is 2-10 times greater in

women (6, 17, 25). The gender difference may be related to low estrogen levels often observed

in active, young women, particularly those with female athlete triad. Stress fracture incidence is

3.3-fold greater in female college athletes without regular menses compared to those with (22).

Other evidence that estrogen may play a role comes from observations that the use of oral

contraceptives decreases stress fracture risk (2, 29). Another important risk factor for stress

fractures is the volume of exercise performed by an individual. There is a clear association

between increased volume of exercise, in particular running, and the increased risk of stress

fracture (5, 12, 16, 19, 23, 24, 35).

The physiological mechanism underlying the cause of stress fractures is uncertain. Most

believe that the cause of stress fractures is due to interaction of bone with its attached or adjacent

musculature. However, there is debate whether the forces produced by the muscles actually

induce the bone injury or protect the bone from injurious externally-applied forces. Devas (7)

hypothesized that stress fractures are due to the pull of muscle on the bone. He suggested that

stress fractures resulting from training do so because muscles can adapt more rapidly than can

bones. Muscular strength increases during training before the bones can adapt and the stronger

muscles apply forces that overstress the bone. This hypothesis is supported by observations that

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the rate of stress fractures is elevated by 2-3 weeks into a training program with the peak

incidence occurring at 5-8 weeks (11, 31), a time course similar to that for the rapid, early

adaptations seen in muscle strength during training (20). On the other hand, Feingold and Hame

(8) and others (3, 10) have hypothesized that muscle can act as a shock absorber during activities

when high external forces are applied upon the body, e.g., foot strike during running. Contraction

of muscle during these activities can decrease the cortical bending strain experienced by bone,

thus protecting the bone. In this theory, stress fractures result when the muscle’s functional

capacity is decreased, e.g., from fatigue, injury, or atrophy, and is thus less able to protect the

bone.

There is minimal direct evidence to support either of these two theories. Both of the

above theories hinge on a mismatch occurring between the functional capacities of a bone and its

adjacent musculature. The former theory depends on the muscle becoming relatively strong

compared to the bone whereas the latter theory depends on the muscle becoming relatively weak

compared to the bone. We are not aware of any data from human studies directly supporting

either of these two possibilities and the data from animal studies are sparse. We have previously

reported that the functional relationship between muscle and bone is not disturbed by one stress

fracture risk factor, i.e., estrogen deficiency, at least when studied in young, growing mice over a

short period (37) or when the deficiency is combined with a high-force resistance training

program (14). However, in this latter study, the training program had minimal effects on either

bone or muscle. Though not directly relevant to conditions associated with stress fractures, we

have found that the bone:muscle functional relationship can be altered. During the recovery of

rats from one month of greatly reduced muscle and bone loading, the muscle becomes relatively

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strong compared to the bone during the second week of recovery and remains so for 6-10 weeks

(1).

The objective of this study was to investigate the independent and combined effects of

two major risk factors for stress fractures, i.e., prolonged estrogen deficiency and run training, on

the tibial bone:soleus muscle functional relationship in young adult, female mice. We

hypothesized that because of the more rapid adaptability of muscle to exercise training, the

bone:muscle functional relationship would be altered in favor of the muscle, at least for the early

portion of the run training period. To our knowledge, this is the first study of the bone:muscle

functional relationship following run training, either by itself or in combination with estrogen

deficiency.

Methods

Animals. Three-month old, female C57BL/6 mice (n=99) were purchased from the

National Institute on Aging aging colony. Mice were initially housed 4-5 per cage on a 12:12-

hour light:dark cycle at 20-23 oC. Mice were given phytoestrogen-free rodent chow (2019 Teklad

Global 19% Rodent Diet; Harland Teklad, Madison, WI) and water ad libitum. After 2 weeks,

the mice were housed individually. Animal care and use procedures were approved by the

University of Minnesota institutional animal care and use committee and complied with

guidelines set by the American Physiological Society. Data for some of these mice have been

published previously. Some of the soleus muscle properties for 44 mice used in the present study

were published by Moran et al. (26). Likewise, the voluntary wheel running data for 23 mice

were published by Gorzek et al. (13).

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Experimental design. Mice were randomly assigned into one of two surgical conditions,

i.e., ovariectomy (OVX) or sham ovariectomy (Sham) and one of two activity conditions, i.e.,

voluntary wheel running (Run) or sedentary (Sed). Voluntary wheel running was used as

opposed to motorized treadmill running because of the greater distances generally covered with

the former as well as that training mode being considered less stressful on the animal. The Sed

condition meant that a mouse was housed in a standard mouse cage (30 x 18 x 13 cm) without

access to a running wheel. Mice from the four combinations of conditions were randomly

assigned into one of two time points for sacrifice, i.e., 30 days (30d) or 60 days (60d) after OVX

or Sham surgery. The 30d time point was chosen because it was thought to reflect a time when a

muscle adaptation but not a bone adaptation could have occurred whereas it was anticipated that

by the 60d time point both muscle and bone adaptations would have occurred. The eight groups

of mice and their numbers were as follows: OVX+30d Run (n=8), OVX+30d Sed (n=7),

OVX+60d Run (n=12), OVX+60d Sed (n=12), Sham+30d Run (n=6), Sham+30d Sed (n=8),

Sham+60d Run (n=8), and Sham+60d Sed (n=8).

To determine if any differences observed between the OVX and Sham conditions could

be attributed to the hormone, estradiol (E2), additional mice were ovariectomized and

supplemented with E2 beginning 30 days after ovariectomy. Half of these mice were randomly

assigned to the Run condition and the other half to the Sed condition. These mice were sacrificed

after 30 days of supplementation, i.e., 60 days after the OVX surgery. The two groups of mice

and their numbers were as follows: OVX+E2+60d Run (n=12) and OVX+E2+60d Sed (n=12).

An additional group of OVX mice with access to running wheels was supplemented with

tamoxifen, an E2 analog (OVX+Tamoxifen+60d Run; n=6). Unlike E2, which has antioxidant

and membrane-stabilizing properties (33), tamoxifen is thought to exert its effect only by binding

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to E2 receptors. A similar response between the OVX+Tamoxifen+60d Run and OVX+E2+60d

Run groups in muscle and bone functional capacities and in the bone:muscle ratios of those

capacities would indicate that an E2 effect is mediated via the E2 receptor.

Surgical procedures. OVX and Sham surgical procedures were conducted when the mice

were 3.75-4 months old. The procedures were done as previously described (13, 26, 27). In brief,

mice were anesthetized via inhalation of 1.75% isoflurane mixed with O2 at a flow rate of 200

ml/min. Under aseptic conditions, bilateral ovariectomy was performed through two dorsal

incisions between the iliac crest and lower ribs. The abdominal muscle wall incisions were

closed using 6-0 silk suture and skin incisions were closed with 7-mm wound clips. Following

recovery from the anesthetic, each mouse was administered 0.15 µg of buprenorphine

subcutaneously as an analgesic. Sham operations were done similarly to the OVX procedure

except the ovaries were not excised.

E2 and tamoxifen supplementation were administered using 60-day time-release pellets

(Innovative Research of America; Sarasota, FL) implanted subcutaneously. While a mouse was

under isoflurane anesthesia, a pellet was inserted through a ~3-mm incision on the dorsal aspect

of the neck. Each E2 pellet contained a total of 0.18 mg of 17β-estradiol so that the mice received

~3 µg/day. This dosage was based off our previous work and was designed to elicit a plasma E2

concentration in OVX mice similar to that observed in a young, intact female mouse, i.e., ~20

pg/ml (30). For OVX mice receiving tamoxifen supplementation, each pellet contained a total of

5 mg of tamoxifen that was released over 60 days. For OVX mice not receiving E2 or tamoxifen

supplementation, a placebo pellet was implanted that contained the same matrix as the E2 and

tamoxifen pellets.

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Voluntary wheel running. Exercise wheels (Prevue Mouse Wheel; Pets Warehouse,

Copiague, NY) were mounted to the tops of standard mouse cages. The wheels were 11 cm in

diameter with a 5-cm wide running surface. A digital magnetic counter was attached to each

wheel and interfaced to a microprocessor (PIC16F877A Development Board, Custom Computer

Systems, Inc.; Brookfield, WI), which was capable of storing the number of revolutions per 24-

hour period for 8 days. The Run mice were allowed to acclimate to the running wheels for one

week prior to the OVX or Sham surgery. Four to five days following the surgery, the mice were

re-introduced to the wheels.

To estimate the activity levels of sedentary mice, the average daily distance covered in a

cage was measured using activity monitors (Activity Monitors, Med Associates; St. Albans, VT),

which employ three sets of infrared beams that detect movement in the x, y, and z planes. Data

were recorded for 8 OVX+60d Sed and 8 OVX+E2+60d Sed mice over 24 hours during the last

1-2 days before being sacrificed. Prior to the measurements, mice were placed in mock activity

chambers for 24 hours to acclimate to the new environment. Mock chambers were exactly the

same as the activity monitor chambers except there were no infrared beams.

Tissue harvest. At the time of sacrifice, a mouse was anesthetized using sodium

pentobarbital (100 mg/kg). The left soleus muscle was then excised for measurement of in vitro

contractility as described below. Blood from the inferior vena cava was collected into a

heparinized 1-ml syringe and then centrifuged to collect plasma. The plasma sample was frozen

in liquid N2 and stored at -80 oC until the time the E2 concentration was assayed as described

below. Following blood collection, the mouse was killed by exsanguination and the left tibia was

dissected free. The tibia was placed in vial containing phosphate-buffered saline and stored at -

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80 oC until micro-computed tomography (micro-CT) of the tibial mid-diaphysis was done as

described below.

Determination of soleus muscle in vitro contractility and total protein content. Of the

lower-leg muscles, the soleus muscle was chosen for study for two reasons. For one, its strength

is known to be manipulated by both E2 (26) and run training (38). Second, it is the most

metabolically active muscle in the lower leg during rest and up to moderate-speed locomotion in

rodents (21) and thus one would presume that its functional capacity would normally be matched

to that of the adjacent tibial bone.

The isolated soleus muscle preparation used to assess contractile function has been

described in detail previously (26-28, 36). After being mounted in a glass chamber at optimal

muscle length (Lo), the muscle equilibrated in Krebs-Ringer buffer (pH 7.4) for 15 min at 25 oC.

Maximal isometric tetanic force (Po) was then determined by stimulating the muscle for 400 ms

with 0.5-ms pulses at 120 Hz and 150 V (Grass-Telefactor S48 stimulator with SIU-5 stimulus

isolation unit; Warwick, RI). Force production by the muscle was recorded using a servomotor

(Aurora Scientific 300B-LR; Aurora, ON, Canada) interfaced to a computer via an interface

board (Keithley KPCI-3108; Cleveland, OH) and software (Capital Equipment Corp. TestPoint

version 6.0; Billerica, MA). The stimulation was done twice with 2 min between trials. The

highest peak force for the two trials was considered the Po. Three min after the second isometric

stimulation, the muscle began a series of 10 eccentric contractions. For these contractions, the

muscle were passively shortened to 0.9 Lo over 3 s and then stimulated for 133 ms as the muscle

lengthened to 1.1 Lo at 1.5 Lo/s; the muscle was then passively shortened back to Lo. There were

3 min in between eccentric contractions. The highest single force produced during the series of

contractions was considered the peak eccentric force. This usually occurred on the first or second

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eccentric contraction of the protocol. Peak eccentric force was not obtained for 8 of the 99

muscles studied; for two, this was because of technical problems and for six, i.e., the

OVX+Tamoxifen+60d Run mice, another set of measurements was substituted.

Following the eccentric contraction protocol, the muscle was removed from the glass

chamber and it was trimmed, blotted dry, weighed, and frozen in liquid N2. The muscle was

stored at -80 oC until total protein content was assayed. For measurement of total protein content,

a muscle was homogenized in 10 mM phosphate buffer (pH 7.0). Sodium dodecyl sulfate was

added to a final volume of 0.01% to solubilize myofibrils. Muscle homogenates were assayed in

triplicate for total noncollagenous protein content using the bicinchoninic acid protein assay with

albumin standards (Pierce Biotechnology; Rockford, IL). Total protein content assays were not

performed on muscles from OVX+Tamoxifen+60d Run mice because they were used for another

purpose.

To provide an estimate of muscle quality, Po was normalized by total protein content per

muscle fiber length instead of by physiological cross-sectional area of the muscle. This was done

because OVX increases muscle fluid content which makes the physiological cross-sectional area

artifactually large (26, 27). Thus, Po normalized by physiological cross-sectional area is

inaccurate and is not reported.

Micro-CT of tibial mid-diaphysis. A micro-CT system (Scanco Medical µCT 40;

Basserdorf, Switzerland) was used to non-destructively image and quantify the morphology and

volumetric bone mineral density (vBMD) of each excised tibia. The scanner was set to a voltage

of 55 kVp and a current of 145 µA and the bones scanned using a 16-µm voxel size with a 200-

ms integration time. Fifty 2D slices were obtained at the mid-diaphysis, the portion of the bone

subjected to load during the three-point bending mechanical test (described below). Cortical

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cross-sectional area, cortical wall thickness, principal cross-sectional moments of inertia (CSMI),

and vBMD were determined for each slice and then averaged across the 50 slices. Because the

minimum principal CSMI (CSMImin) best corresponded to the CSMI about the bone bending axis

during the 3-point bending test (described below), only it was analyzed statistically. After

completion of imaging, the bones were re-frozen in phosphate-buffered saline and stored at -80

oC until the time they underwent mechanical testing.

Mechanical testing of tibial mid-diaphysis. The procedures used to assess the functional

capacities of the mouse tibia have been described in detail previously (14, 37). After thawing to

room temperature, the medial-lateral periosteal diameter of the tibia at mid-diaphysis was

measured using calipers. Tibial bone functional and intrinsic material properties were then

determined by a three-point bending test on an Instron 1125 machine at the mid-diaphysis. The

tibia was placed lateral side down on a nonadjustable set of supports (1 cm apart) and quasi-static

loading was applied to the medial surface of the tibia midway between the lower supports at a

displacement rate of 5.08 mm/min. Bones were placed lateral side down to create the most stable

position possible during testing. Displacement of the Instron crosshead was measured by a linear

variable differential transformer and the applied load was measured by a load cell with 0.05-N

resolution. The load and displacement analog outputs were sampled at 10 Hz using a computer

and software (Laboratory Technologies LabTech Notebook Pro version 8.01; Wilmington, MA).

The load-displacement curves were analyzed using a custom-written TestPoint program.

The program defined ultimate load as the highest load obtained prior to fracture (Fig. 1A). Once

the data point in the load-displacement curve corresponding to the occurrence of ultimate load

was determined, deflection and energy absorbed to ultimate load were calculated. For

determination of stiffness, the program searched the linear portion of the load-displacement

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curve prior to fracture for the highest slope; for a portion of the curve to be considered as having

the highest slope, it had to contain a minimum of 15 data points and the correlation of load and

displacement in that region had to exceed 0.99.

Intrinsic material properties for the tibia were estimated using classical beam theory.

Ultimate stress was calculated using the equation: ultimate stress = (UL.d.L)/(8.CSMImin), where

UL, d, and L are ultimate load, medial-lateral periosteal diameter, and bottom support span

length (1 cm), respectively. Modulus of elasticity was calculated using the equation: modulus of

elasticity = (k.L3)/(48.CSMImin), where k equals stiffness.

Calculation of bone:muscle functional relationships. The functional capacity

measurements considered most important for bone, i.e., ultimate load and stiffness, were divided

by those for muscle, i.e., Po and peak eccentric force. These bone:muscle ratio calculations were

done on an animal by animal basis, meaning that if an animal did not have measurements for

both its tibial bone and soleus muscle the ratio was not calculated.

Plasma E2 analysis. Plasma samples from mice in the OVX (n=21 out of 39), OVX+E2

(n=16 out of 24), and Sham (n=14 out of 30) groups were randomly selected for analysis of E2

concentration. As described previously (26), the plasma E2 concentration was determined using

commercially-available enzyme-linked immunosorbent assays (Diagnostic Systems Labs DSL-

10-4300; Webster, TX or Immuno Biological Laboratories RE50241; Minneapolis, MN). The

Immuno Biological Laboratories kit was used only because of a recall on the Diagnostic Systems

Labs kit and it was used for ~25% of the assays.

Statistical analyses. To analyze the effects of surgical condition (OVX vs. Sham),

activity (Run vs. Sed), and time of sacrifice after surgery (30d vs. 60d), 3-way ANOVAs were

utilized. When an interaction was found to be significant, Holm-Sidak post-hoc tests were used

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to determine which combination of conditions were different from another. To analyze the

effectiveness of E2 supplementation, the OVX+E2 mice were compared against OVX and Sham

mice also sacrificed at the 60d time point; thus, 2-way AVOVAs were used with one factor being

condition (OVX+E2 vs. OVX vs. Sham) and the other activity (Run vs. Sed). When a condition

main effect or interaction was found to be significant, Holm-Sidak post-hoc tests were used. To

compare the effect of tamoxifen supplementation to that of E2, i.e., OVX+Tamoxifen+60d Run

vs. OVX+E2+60d Run, t-tests were used; when assumptions of normality and/or equal variance

were violated, Mann-Whitney U tests were substituted. Differences among OVX+E2, OVX, and

Sham mice in plasma E2 concentration and daily running distance were analyzed using one-way

ANOVAs and Holm-Sidak post-hoc tests; when assumptions of normality and/or equal variance

were violated, Kruskal-Wallis tests along with Dunn's multiple comparison post hoc tests were

substituted. To determine if the often-observed abnormal bone mechanical test responses

occurred in one group of mice more than another, Chi-square tests were employed. Statistical

analyses were performed using SigmaStat version 3.5 (Systat Software; Point Richmond, CA)

with an α level of 0.05. Values are reported as means (SD) except when comparisons among

combinations of conditions are reported. In these exceptions, the values are expressed as least-

square-mean values (SD) or relative changes between least-square-mean values.

Results

Effectiveness of surgical and activity manipulations. The effectiveness of the OVX

surgeries and E2 supplementation were assessed by plasma E2 concentration. Plasma E2

concentrations for OVX, OVX+E2, and Sham mice were 8.3 (3.9), 28.9 (9.3), and 18.1 (6.4)

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pg/ml, respectively. The OVX value was significantly less than those of the other two groups and

there was no significant difference between the OVX+E2 and Sham values.

The effectiveness of the voluntary wheel run training was assessed by the average daily

distance covered. Because the time between surgery and sacrifice had no effect on the daily

distance ran by the OVX and Sham mice, the 30d Run and 60d Run groups for each type of mice

were collapsed and compared to the OVX+E2+60d Run mice. The average daily distance ran by

the OVX, OVX+E2, and Sham mice in the Run groups were 1.45 (1.21), 2.91 (2.39), and 7.35

(2.83) km, respectively, with the only significant differences being between the Sham mice and

the other two groups. The daily distance covered during normal cage activity by the Sed mice

was markedly less. The OVX+60d Sed and OVX+E2+60d Sed mice on average covered 0.44

(0.09) and 0.61 (0.23) km, respectively, every 24 h and these values were not significantly

different (p=0.072); data were not obtained for Sham Sed mice.

Effect of surgical condition and activity at 30- and 60-d post-surgery

Comparing body weights among OVX+30d Run, OVX+30d Sed, OVX+60d Run,

OVX+60d Sed, Sham+30d Run, Sham+30d Sed, Sham+60d Run, and Sham+60d Sed groups

indicated that there was a significant effect of surgical condition, but not for activity. OVX mice

weighed 17% more than did Sham mice (Table 1).

Tibial bone. In general, the functional capacities of the tibial bone were affected by both

surgical condition and activity but more strongly by surgical condition. Ultimate load was

however only affected by surgical condition (p=0.002). Ultimate load for Sham mice was 7%

greater than that for OVX mice (Fig. 2A). For stiffness, there were both significant surgical

condition (p=0.002) and activity (p=0.015) effects and these effects were independent of each

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other, i.e., no significant interaction. Stiffness for Sham mice was 12% greater than that for OVX

mice while it was 9% greater for Run mice compared to Sed mice (Fig. 2B). For energy absorbed

to ultimate load, there was a significant effect of activity (p<0.001) with the Sed mouse bones

absorbing 25% more energy than those for the Run mice (Table 1). The greater energy

absorption by the Sed mice can be explained mostly by a greater deflection to ultimate load for

those mice. This effect was statistically significant only for the Sham mice however. The

deflection for the Sham Sed mice was 34% greater than that for the Sham Run mice whereas the

Sed vs. Run difference for the OVX mice was only 10% (n.s.). The greater deflection and energy

absorbed to ultimate load for the Sed mice appears to be related to a partial failure of the

proximal lateral tibia occurring early in many mechanical tests (Fig. 1B). The partial failures

appeared as a splintering of the bone that was located 3-4 mm away from the eventual failure

site, which was commonly at the center of the span beneath the upper load contact. These partial

failures were much more likely (p=0.014) to occur in tests of bones from Sed mice (51% of the

mechanical tests for those bones) compared to Run mice (25%). It is important to point out that

these partial failures had no adverse effect on the ultimate load measured for those bones. There

was no significant difference observed in ultimate load between bones with and without the

partial failures (11.50 (0.99) vs. 11.41 (0.96); p=0.71).

Micro-CT-determined properties reflecting the size and geometry of the tibial mid-

diaphysis were affected by surgical condition but not activity (Table 1). There was a significant

effect of surgical condition on both cortical wall thickness and cortical cross-sectional area but

only for mice sacrificed at the 60d time point; at this time point, these measures were 11-15%

greater for the Sham mice compared to OVX mice. For CSMImin, there was a significant effect of

surgical condition (p=0.004) with the value for the Sham condition being 10% greater.

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Intrinsic material properties, which reflect bone quality, were modestly affected by

surgical condition or activity (Table 1). For vBMD, there was a small but significant effect of

surgical condition (p=0.005). Bones from Sham mice had a 1.3% greater vBMD than that of

OVX mice. There was also a significant effect of activity but only at the 30d time point.

Unexpectedly, vBMD at that time point was 2.4% greater for Sed mice compared to Run mice.

Ultimate stress was affected by surgical condition effect (p=0.048) with the value for OVX mice

bones being 4% greater than that for Sham. On the other hand, the modulus of elasticity was not

affected by surgical condition but was by activity, and then, only at the 30d time point; the value

at that time point for the Run condition was 21% greater compared to that for the Sed condition.

Soleus muscle. The functional capacities of the soleus muscle were affected by both

surgical condition and activity but in contrast to the tibia, the soleus muscle was more strongly

affected by activity. For Po, there were both significant surgical condition (p=0.011) and activity

(p<0.001) effects and these effects were independent of each other. Po was 9% greater for Sham

mice compared to OVX mice and 21% greater for Run mice compared to Sed mice (Fig. 3A).

For peak eccentric force, there was no surgical condition effect, only an activity effect (p=0.016).

Peak eccentric force was 10% greater in the Run condition compared to Sed (Fig. 3B).

The properties reflecting muscle size, i.e., extrinsic properties, were somewhat

contradictory (Table 2). There was a significant surgical condition effect on soleus muscle

weight (p<0.001) but contrary to what one might predict from the Po data above, muscle weight

was greater for the OVX condition by 14%. This is most likely due to an increased fluid content

in those muscles as previously described (26, 27). There was also a significant effect of activity

at the 60d time point with the muscles from Run mice weighing 18% more than those from Sed

mice. For total protein content, there was no effect of surgical condition (p=0.637) but there was

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a significant activity effect (p<0.001); total protein content in muscles from Run mice was 14%

greater than that from Sed mice. For the property reflecting muscle quality, i.e., maximal

isometric tetanic force normalized to total protein content per fiber length, there was a significant

surgical condition effect (p=0.047) with the Sham condition being greater by 14% compared to

OVX (Table 2). There was no effect of activity on normalized Po.

Bone:muscle functional relationships. The ratio of tibial bone ultimate load to soleus

muscle Po within an animal was not affected by surgical condition but was by activity (p<0.001);

the ratio was higher for the Sed condition compared to the Run condition, i.e., 70.4 (10.2) vs.

57.1 (10.2) (Fig. 4). The statistical findings were the same for the ratios of ultimate load to peak

eccentric force and stiffness to Po with no effect of surgical condition but a significant activity

effect (p≤0.036); the ratios were 11-12% higher for the Sed condition (Table 2). There were no

statistically significant effects on the ratio of bone stiffness to peak eccentric force.

Effect of E2 supplementation

E2 supplementation delivered via the slow-release pellets was totally effective in

restoring the bone and muscle functional capacities of OVX mice to those like for Sham mice

(Fig. 5 and 6; Tables 3 and 4). There were only three measures, all bone measures, for which the

OVX+E2 mice did not respond exactly like Sham mice, i.e., CSMImin, vBMD, and ultimate

stress. For CSMImin, the OVX+E2 value was intermediate to those for the OVX and Sham groups

and not significantly different from either group (compare Tables 1 and 3); however, the Sham

value was significantly greater than the OVX value. E2 supplementation was ineffective in

restoring vBMD. vBMD for the OVX+E2 mice was significantly less than that of the Sham mice

(compare Tables 1 and 3). In fact, vBMD for the OVX+E2 mice was less than or the same as that

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for the OVX mice. For ultimate stress, the OVX+E2 bones behaved more like OVX bones than

Sham bones (compare Tables 1 and 3). The OVX+E2 ultimate stress value was significantly

greater than the Sham value but was not different from the OVX value. As for the bone:muscle

functional relationships analyses presented above under Effect of surgical condition and activity

at 30- and 60-d post-surgery, these were found to be affected only by activity (Fig. 7, Table 4).

There were no differences among OVX+E2, OVX, and Sham mice.

Comparison of tamoxifen and E2 supplementation

In contrasting the OVX+Tamoxifen+60d Run and OVX+E2+60d Run groups, no

significant differences were found between the tamoxifen and E2 supplementation in the bone or

muscle functional capacities or in the ratios of those capacities (Fig. 5, 6, and 7; Tables 3 and 4).

There were only three bone measures for which the two groups differed significantly on, i.e.,

cortical wall thickness, vBMD, and ultimate stress. Cortical wall thickness and vBMD for the

tamoxifen-supplemented mice were greater by 8% and 4%, respectively. On the other hand,

ultimate stress was higher for the E2-supplemented mice by 7%.

Discussion

Five main findings resulted from this study. First, the effect of surgical condition on bone

functional capacity was greater than that of activity. There was no significant effect of activity on

ultimate load and its effect on bone stiffness was relatively small compared to that of surgical

condition. Second, the effect of surgical condition on muscle functional capacity was less than

that of activity. There was no significant effect of surgical condition on peak eccentric force and

its effect on maximal isometric tetanic force was less than half that of activity’s. Third, the ratio

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19

of bone functional capacity to that of muscle was only affected by activity. This is because

surgical condition’s effects on the two tissues were relatively the same so that the effects

canceled each other out when one measure was divided by the other. For example, ultimate load

and maximal isometric tetanic force for Sham mice were greater than those for OVX mice by

similar percentages, i.e., 7% and 9%, respectively. Conversely, activity improved bone

functional capacities marginally (i.e., 0-9%) while improving muscle’s more substantially (i.e.,

10-21%) resulting in reduced bone:muscle ratios. Fourth, the functional differences observed

between Sham and OVX mice can be attributed to E2. E2 supplementation given to OVX mice

restored muscle and bone functional capacities to Sham levels and resulted in bone:muscle

functional relationships like those for Sham and OVX mice. Finally, there were no differences

between tamoxifen- and E2-supplemented OVX mice observed in muscle and bone functional

capacities. We interpret this to indicate that E2’s effects on muscle and bone function are

probably mediated via E2 receptors and not via its antioxidant or cell membrane-stabilizing

properties because there is no evidence for tamoxifen-related antioxidant or membrane effects.

This study’s findings indicate that the means by which E2 improves the functional

capacity of bone differs from that for muscle. E2 improved ultimate load and stiffness by an

effect on bone size and geometry and not via an effect on bone tissue quality. E2 increased

cortical wall thickness, cortical bone cross-sectional area, and CSMImin but had no beneficial

effect on any measure of bone quality, i.e., ultimate stress, elastic modulus, or vBMD. It was

surprising that there was no beneficial effect of E2 supplementation on bone mineral density.

This might be explained by the relatively short period of supplementation used in this study, i.e.,

30 days but E2 supplementation at a slightly lower dosage over a shorter duration (24 days) has

been demonstrated to increase vBMD in the femora of mice (32). On the other hand, the means

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by which E2 enhanced muscle functional capacities, specifically maximal isometric tetanic force,

appears to have been by improving muscle quality and not by increasing muscle size. In fact, E2

supplementation to OVX mice resulted in smaller muscles compared to OVX mice supplemented

with placebo pellets. There was also no effect of E2 supplementation on total protein content,

another indicator of muscle size. Though the effect of E2 supplementation on normalized Po did

not quite reach significance (p=0.052), the percentage difference between OVX+E2 and OVX

(19%) suggests, along with the muscle weight and total protein content data, that E2

supplementation enhanced muscle quality. This enhancement is in accord with our previous

studies that showed muscle and myosin quality were detrimentally altered from the loss of

ovarian hormones (27) and favorably affected in ovariectomized mice supplemented with E2

(26).

The data from this study also indicate that activity affects bone and muscle but differently

from E2. Though the effects of activity on bone functional capacities were less than those of E2,

those effects appeared to have been mediated by an improvement in bone quality and not by

altered extrinsic properties. Activity had no effect on cortical wall thickness, cortical bone cross-

sectional area, or CSMImin but did increase the elastic modulus, at least at the 30d time point. An

increased elastic modulus could explain the activity-elicited increase in stiffness that was

observed. Activity’s effect on the energy absorbed and deflection to ultimate load also appears to

have been mediated via an effect on bone quality. Bones from mice that exercised on the running

wheels were less likely to undergo a partial failure of the proximal lateral tibia during

mechanical testing. This suggests a qualitative difference between bones from Run and Sed

mice. However, it is possible that bone size and/or geometry in the proximal lateral tibia region

may have differed between the two groups of mice. We only assessed bone size and geometry in

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the mid-diaphysis. As a final comment on this topic, even though the occurrence of a partial

failure was associated with an increase in energy absorbed and deflection to ultimate load, it is

not obvious that this is a beneficial change in bone function. Somewhat unexpectedly, activity

had a small but significant adverse effect on bone mineral density. vBMD was 2.3% less for

bones from Run mice compared to Sed mice at the 30d time point and vBMD for OVX+E2+60d

Run mice was 2.7% less than that for the OVX+E2+60d Sed mice. Similar findings have been

reported for the femora of male mice following 3 weeks of treadmill run training (34) and the rat

tibia following intensive run training (4). Despite our findings, there is no evidence that the

lower vBMD in the Run mice had any detrimental effect on bone function. For muscle, activity

had a strong effect on functional capacity that appears to be explained mostly by an effect on

muscle size. Total protein content and muscle weight were greater for the Run mice. These

findings are consistent with other reports of soleus muscle hypertrophy in response to wheel

running by rodents (38, 39).

For all of the activity effects observed on muscle and bone, there was only one where the

magnitude of the effect was dependent on the surgical condition, i.e., deflection to ultimate load.

This is remarkable when considering that there was great variability (5-fold) among the OVX,

OVX+E2, and Sham groups in the daily distance ran on the running wheels. The data suggest that

running as little as 1.45 km daily on average, the amount ran by the OVX mice, is sufficient to

induce the activity effects observed on the two tissues. When this number is contrasted against

the distance normally covered in a cage by Sed mice in a day, i.e., ~0.5 km, the minimum

amount of additional activity necessary for an effect is even more impressive. However, it should

be pointed out that one cannot equate distances covered in a running wheel to those in a standard

cage. The metabolic costs and forces exerted per km are most likely different. Furthermore, for

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the Run mice, we do not know the distance covered in their cage while not in the running wheel.

It may have been greater or less than that for the Sed mice during normal cage activity. As a final

comment, the observation that activity’s effect on most bone measures was statistically

independent of E2 status is noteworthy. It has been argued that the E2 alters the

mechanosensitivity of bone as well as the remodeling threshold (9, 15). Superficially, it appears

that our data do not support either of these hypotheses. However, for the mice with access to

running wheels, the Sham mice were much more active than were the OVX mice yet had similar

activity effects on bone. This could be explained if the presence of E2 offset the greater activity

in the Sham mice by decreasing the mechanosensitivity of bone.

The impact and limitations of the present study’s findings are as follows. The present

study’s data confirm our previous findings (14, 37) using younger mice that E2 deficiency does

not induce a bone:muscle functional capacity mismatch. However, bone:muscle functional

capacity mismatches can occur using a physiologically-relevant run training regimen in female

mice. Though with voluntary wheel running, the muscle became relatively strong compared to

the bone, there is no evidence that this did or would lead to a stress fracture or other skeletal

injury. Bone function for the Run mice was the same as or higher than that for the Sed mice.

There is a need for follow-up studies to investigate the relationships of tibial bone function to

those of the posterior and anterior crural muscle groups and not just one muscle in response to

voluntary wheel running. These two muscle groups probably account for most of the forces

exerted on the tibia and absorbed from the tibia. The use of voluntary wheel running could also

be considered a limitation of our study particularly in regards to modeling stress fractures that

occur in women in the U.S. Armed Forces. For this situation, forced treadmill running by mice

may be more applicable. Alternatively, loaded wheel running causes different muscle adaptations

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relative to unloaded, i.e., minimal-resistance, wheel running (18) as was used in our study and

loaded wheel running would likely result in greater bone adaptations as well since the peak

strains experienced by bone would presumably be higher.

Perspectives and Significance. Bones and muscles are biomechanically linked. Thus,

evaluating bone-muscle as a functional unit is valuable, particularly in situations where one or

both of the tissues are adversely or beneficially affected. The results of our study indicate that E2

status equally affects the functional capacities of bone and its associated muscle but that run

training may have greater beneficial effects on muscle than bone leading to a mismatch in the

functional properties of the two linked tissues. This mismatch did not appear to be detrimental in

this short term study but the outcomes of more prolonged periods of mismatching are not known

and need to be investigated. Future studies also need to investigate if and when bone functional

capacity catches up to that of muscle during longer duration wheel running in mice. One would

speculate that the longer a bone:muscle functional capacity mismatch exists, the greater the

chance for occurrence of a stress fracture. Finally, there is a need to test whether having a

relatively strong muscle compared to the bone is detrimental or beneficial when the two tissues

are subjected to a standardized set of intense and/or prolonged loadings, e.g., running at a high

speed over a prolonged period over consecutive days.

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Acknowledgements

We thank Sarah Greising, Rachel Landisch, and Steven Nelson for assistance on this project.

Grants

The research was supported by National Institutes of Health (NIH) Grants AG-20990 and AG-

25861 to D. A. Lowe, and A. L. Moran was supported by NIH Training Grant T32-AR-07612

and a University of Minnesota Dissertation Grant.

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Figure Legends

Fig. 1 - Representative load-displacement data obtained during 3-point bending mechanical tests

of two tibial bones. A: data obtained during a test without a partial failure of the bone

prior to the occurrence of ultimate load. The procedures for determining ultimate load,

stiffness, energy to ultimate load, and deflection to ultimate load are illustrated. B: data

obtained during a test in which a partial failure occurred.

Fig. 2 - Effect of surgical condition (OVX vs. Sham), activity (Run vs. Sed), and time of

sacrifice after surgery (30 days vs. 60 days) on ex vivo measurements of tibial bone

ultimate load (panel A) and stiffness (panel B). OVX: ovariectomy. Sham: sham

ovariectomy. Run: mice given access to running wheels. Sed: mice housed in standard

cages. Values are means (SD). Statistically significant effects (p<0.05) are indicated

above the bars in each panel.

Fig. 3 - Effect of surgical condition (OVX vs. Sham), activity (Run vs. Sed), and time of

sacrifice after surgery (30 days vs. 60 days) on in vitro contractile properties of soleus

muscles. A: maximal isometric tetanic force (Po). B: peak eccentric force. Values are

means (SD). Statistically significant effects (p<0.05) are indicated above the bars in

each panel.

Fig. 4 - The ratio of bone ultimate load to muscle maximal isometric tetanic force as a function

of surgical condition (OVX vs. Sham), activity (Run vs. Sed), and time of sacrifice after

surgery (30 days vs. 60 days). Values are means (SD) and are unitless. Statistically

significant effects (p<0.05) are indicated above the bars.

Fig. 5 - Effects of E2 and tamoxifen supplementation on ex vivo measurements of tibial bone

ultimate load (panel A) and stiffness (panel B). All measurements were made on bones

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31

taken from mice sacrificed 60 days after surgery. Values are means (SD). For the six

leftmost bars, statistically significant effects (p<0.05) among those groups are indicated

above the bars. There were no significant differences between OVX+Tamoxifen+60d

Run and OVX+E2+60d Run groups.

Fig. 6 - Effects of E2 and tamoxifen supplementation on in vitro contractile properties of soleus

muscles. A: maximal isometric tetanic force (Po). B: peak eccentric force. All

measurements were made on muscles taken from mice sacrificed 60 days after surgery.

Values are means (SD). For the six leftmost bars, statistically significant effects

(p<0.05) among those groups are indicated above the bars. Peak eccentric force

measurements were not made on muscles from OVX+Tamoxifen+60d Run mice. n.s.:

not significant.

Fig. 7 - The ratio of bone ultimate load to muscle maximal isometric tetanic force as a function

of E2 and tamoxifen supplementation. Values are means (SD) and are unitless. For the

six leftmost bars, statistically significant effects (p<0.05) among those groups are

indicated above the bars. There were no significant differences between

OVX+Tamoxifen+60d Run and OVX+E2+60d Run groups.

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32Table 1. Effect of surgical condition and activity at 30- and 60-d post-surgery on mouse weight and tibial bone properties.

Sham+30d

Run

Sham+30d

Sed

Sham+60d

Run

Sham+60d

Sed

OVX+30d

Run

OVX+30d

Sed

OVX+60d

Run

OVX+60d

Sed

Statistical findings

Mouse weight (g) 20.9 (0.9) 21.6 (1.4) 22.8 (0.8) 22.9 (1.4) 22.9 (1.1) 25.0 (1.4) 28.7 (3.5) 26.5 (3.1) OVX > Sham

60d Run > 30d Run

60d Run > 30d Sed

60d Sed > 30d Run

Tibial bone

Energy to ultimate

load (mJ)

2.25 (0.26) 2.83 (0.48) 2.16 (0.41) 3.16 (0.56) 2.06 (0.42) 2.56 (0.64) 2.54 (0.61) 2.72 (0.56) Sed > Run

Deflection to

ultimate load (mm)

0.33 (0.04) 0.43 (0.03) 0.33 (0.04) 0.46 (0.03) 0.33 (0.09) 0.40 (0.08) 0.40 (0.10) 0.41 (0.06) Sham Sed > Sham Run

Sham Sed > OVX Run

OVX Sed > Sham Run

Cortical wall

thickness (mm)

0.200

(0.006)

0.199

(0.009)

0.213

(0.013)

0.207

(0.005)

0.190

(0.011)

0.194

(0.011)

0.183

(0.030)

0.183

(0.009)

Sham 60d > OVX 60d

Sham 30d > OVX 60d

Sham 60d > OVX 30d

Cortical cross-

sectional area

(mm2)

0.574

(0.028)

0.573

(0.034)

0.618

(0.040)

0.603

(0.024)

0.539

(0.033)

0.555

(0.038)

0.552

(0.022)

0.548

(0.032)

Sham 60d > OVX 60d

Sham 60d > Sham 30d

Sham 60d > OVX 30d

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33CSMImin (102.mm4) 5.10 (0.57) 5.08 (0.48) 5.64 (0.74) 5.59 (0.76) 4.42 (0.50) 5.01 (0.78) 4.91 (0.49) 5.19 (0.72) Sham > OVX

60d > 30d

vBMD

(mg hydroxyapatite

/cm3)

1367 (13) 1410 (21) 1439 (25) 1427 (22) 1370 (23) 1392 (46) 1412 (16) 1399 (22) Sham > OVX

30d Sed > 30d Run

60d Run > 30d Run

60d Sed > 30d Run

60d Run > 30d Sed

Ultimate stress

(MPa)

311 (18) 292 (25) 272 (17) 277 (23) 314 (17) 307 (32) 297 (25) 283 (29) OVX > Sham

30d > 60d

Elastic modulus

(GPa)

19.6 (1.9) 17.0 (2.1) 17.5 (1.9) 16.9 (2.0) 20.1 (2.1) 15.8 (2.2) 17.8 (2.9) 16.3 (1.8) 30d Run > 30d Sed

30d Run > 60d Run

30d Run > 60d Sed

Values are means (SD). The Statistical findings column indicates the significant (p<0.05) main effects and interactions of surgical condition (OVX

vs. Sham), activity (Run vs. Sed), and time of sacrifice after surgery (30 days vs. 60 days), OVX: ovariectomy. Sham: sham ovariectomy. Run: mice

given access to running wheels. Sed: mice housed in standard cages. 30d: sacrificed 30 days after surgery. 60d: sacrificed 60 days after surgery.

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34Table 2. Effect of surgical condition and activity at 30- and 60-d post-surgery on soleus muscle and bone:muscle properties.

Sham+30d

Run

Sham+30d

Sed

Sham+60d

Run

Sham+60d

Sed

OVX+30d

Run

OVX+30d

Sed

OVX+60d

Run

OVX+60d

Sed

Statistical findings

Soleus muscle

Muscle weight (mg) 7.78 (0.39) 6.82 (0.58) 8.76 (0.41) 7.18 (0.23) 8.53 (1.24) 8.30 (0.79) 9.59 (0.83) 8.43 (0.84) OVX > Sham

60d Run > 60d Sed

60d Run > 30d Run

60d Run > 30d Sed

Total protein content

(mg)

1.43 (0.06) 0.97 (0.18) 1.43 (0.25) 1.05 (0.20) 1.44 (0.14) 1.07 (0.21) 1.39 (0.32) 1.10 (0.16) Run > Sed

Normalized Po

(N.cm/mg)

0.122

(0.021)

0.148

(0.038)

0.128

(0.030)

0.131

(0.031)

0.106

(0.020)

0.112

(0.045)

0.119

(0.032)

0.128

(0.030)

Sham > OVX

Bone:muscle

Ultimate load / peak

eccentric force

32.0 (4.0) 35.2 (4.0) 31.8 (6.2) 34.4 (6.4) 29.1 (4.0) 35.5 (8.2) 33.8 (6.9) 36.3 (10.3) Sed > Run

Stiffness / Po (mm-1) 217 (30) 240 (41) 233 (51) 276 (45) 219 (28) 240 (80) 233 (42) 246 (48) Sed > Run

Stiffness / peak

eccentric force (mm-1)

125 (19) 126 (20) 128 (29) 130 (31) 115 (14) 121 (47) 129 (29) 129 (41) n.s.

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35Values are means (SD). The Statistical findings column indicates the significant (p<0.05) main effects and interactions of surgical condition (OVX

vs. Sham), activity (Run vs. Sed), and time of sacrifice after surgery (30 days vs. 60 days), OVX: ovariectomy. Sham: sham ovariectomy. Run: mice

given access to running wheels. Sed: mice housed in standard cages. 30d: sacrificed 30 days after surgery. 60d: sacrificed 60 days after surgery. n.s.:

no significant effects or interactions.

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36Table 3. Effect of E2 and tamoxifen supplementation on mouse weight and tibial bone properties.

OVX+E2+

60d Run

OVX+E2+

60d Sed

OVX+

Tamoxifen+

60d Run

Statistical findings for comparisons

among OVX+E2+60d, OVX+60d and

Sham+60d groups

Statistical findings for comparison

of OVX+Tamoxifen+60d Run

against OVX+E2+60d Run

Mouse weight (g) 24.0 (1.8) 23.0 (1.8) 23.9 (1.6) OVX > OVX+E2

OVX > Sham

n.s.

Tibial bone

Energy to ultimate

load (mJ)

2.76 (0.48) 3.10 (0.61) 2.39 (0.22) Sed > Run n.s.

Deflection to ultimate

load (mm)

0.37 (0.05) 0.44 (0.07) 0.34 (0.03) OVX+E2+60d Sed > OVX+E2+60d Run

Sham+60d Sed > Sham+60d Run

n.s.

Cortical wall thickness

(mm)

0.223

(0.013)

0.221

(0.028)

0.240

(0.015)

OVX+E2 > OVX

Sham > OVX

OVX+Tamoxifen+60d Run >

OVX+E2+60d Run

Cortical cross-

sectional area (mm2)

0.623

(0.035)

0.621

(0.047)

0.646

(0.021)

OVX+E2 > OVX

Sham > OVX

n.s.

CSMImin (102.mm4) 5.40 (0.63) 5.42 (0.50) 5.37 (0.25) Sham > OVX n.s.

vBMD

(mg hydroxyapatite

/cm3)

1366 (19) 1404 (25) 1421 (15) OVX+60d Run > OVX+E2+60d Run

Sham+60d Run > OVX+60d Run

Sham+60d Sed > OVX+60d Sed

OVX+Tamoxifen+60d Run >

OVX+E2+60d Run

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37OVX+E2+60d Sed > OVX+E2+60d Run

Sham+60d Run > OVX+E2+60d Run

Sham+60d Sed > OVX+E2+60d Sed

Ultimate stress (MPa) 303 (18) 290 (26) 283 (20) OVX+E2 > Sham OVX+E2+60d Run >

OVX+Tamoxifen+60d Run

Elastic modulus (GPa) 18.5 (2.1) 17.8 (2.6) 18.1 (1.5) n.s. n.s.

Values are means (SD). The Statistical findings for comparisons among OVX+E2+60d, OVX+60d and Sham+60d groups column indicates the

significant (p<0.05) main effects and interactions of condition (OVX+E2 vs. OVX vs. Sham) and activity (Run vs. Sed). Refer to Table 1 for the

OVX+60d and Sham+60d groups’ data. OVX+E2: ovariectomy followed by E2 supplementation. OVX: ovariectomy. Sham: sham ovariectomy. Run:

mice given access to running wheels. Sed: mice housed in standard cages. 60d: sacrificed 60 days after surgery. n.s.: no significant effects or

interactions.

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38Table 4. Effect of E2 and tamoxifen supplementation on soleus muscle and bone:muscle properties.

OVX+E2+

60d Run

OVX+E2+

60d Sed

OVX+

Tamoxifen+

60d Run

Statistical findings for comparisons

among OVX+E2+60d, OVX+60d and

Sham+60d groups

Statistical findings for comparison of

OVX+Tamoxifen+60d Run against

OVX+E2+60d Run

Soleus muscle

Muscle weight (mg) 8.70 (0.93) 7.32 (0.63) 9.10 (0.79) OVX > OVX+E2

OVX > Sham

Run > Sed

n.s.

Total protein content

(mg)

1.27 (0.29) 1.07 (0.14) - Run > Sed -

Normalized Po

(N.cm/mg)

0.148 (0.032) 0.145 (0.029) - n.s. (p=0.052 for surgical condition

effect)

-

Bone:muscle

Ultimate load / peak

eccentric force

33.0 (6.2) 36.6 (5.4) 34.1 (4.9) n.s. n.s.

Stiffness / Po (mm-1) 225 (22) 260 (45) 235 (29) Sed > Run n.s.

Stiffness / peak

eccentric force (mm-1)

126 (25) 135 (24) 132 (19) n.s. n.s.

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39Values are means (SD). The Statistical findings for comparisons among OVX+E2+60d, OVX+60d and Sham+60d groups column indicates the

significant (p<0.05) main effects and interactions of condition (OVX+E2 vs. OVX vs. Sham) and activity (Run vs. Sed). Refer to Table 2 for the

OVX+60d and Sham+60d groups’ data. OVX+E2: ovariectomy followed by E2 supplementation. OVX: ovariectomy. Sham: sham ovariectomy. Run:

mice given access to running wheels. Sed: mice housed in standard cages. 60d: sacrificed 60 days after surgery. n.s.: no significant effects or

interactions. Total protein content measurements were not made on muscles from OVX+Tamoxifen+60d Run mice and thus normalized Po cannot be

calculated for that group.

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0.0 0.2 0.4 0.6 0.8 1.0 1.2

Displacement (mm)

0

2

4

6

8

10

12

14Lo

ad (N

)

Ulti

mat

e lo

ad

Deflection toultimate load

Shaded area = Energy absorbed to ultimate load

Slope of line = Stiffness

A

0.0 0.3 0.6 0.9 1.2 1.5 1.8

Displacement (mm)

0

2

4

6

8

10

12

14

Load

(N)

Partial failure

B

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30 days 60 days 30 days 60 days0

3

6

9

12

15U

ltim

ate

load

(N)

Run Sed

Sham OVX

Sham > OVX


10

20

30

40

50

60

Stif

fnes

s (N

/mm

)

Sham OVX

Sham > OVXRun > Sed

A

B

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50

100

150

200

250M

axim

al is

omet

ric te

tani

c fo

rce

(mN

)

Run Sed

Sham OVX

Sham > OVXRun > Sed


100

200

300

400

500

Pea

k ec

cent

ric fo

rce

(mN

)

Sham OVX

Run > Sed

A

B

of 46


20

40

60

80

100U

ltim

ate

load

/ m

axim

al is

omet

ric te

tani

c fo

rce

Run Sed

Sham OVX

Sed > Run

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Sham OVX OVX+E OVX+0

3

6

9

12

15U

ltim

ate

load

(N)

Run Sed

Sham > OVX, OVX+E2 > OVX

2 Tamoxifen


10

20

30

40

50

60

Stif

fnes

s (N

/mm

)

2

Sham > OVX, OVX+E2 > OVX

Tamoxifen

A

B

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50

100

150

200

250M

axim

al is

omet

ric te

tani

c fo

rce

(mN

)

Run Sed

OVX+E2 > OVXRun > Sed

2 Tamoxifen


100

200

300

400

500

Pea

k ec

cent

ric fo

rce

(mN

)

n.s.

2 Tamoxifen

A

B

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20

40

60

80

100U

ltim

ate

load

/ m

axim

al is

omet

ric te

tani

c fo

rce

Run Sed

Sed > Run

2 Tamoxifen

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Voluntary run training but not estradiol deficiency alters the tibial bone-soleus muscle functional...

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