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 whether 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 group were 7–12% greater than those for OVX animals (P = 0.002), whereas only stiffness was greater for Run than for Sed animals (9%; P = 0.015). The muscle functional capacities were affected by both surgical condition and activity; however, in contrast to the bone, the muscle was more affected by activity. Po and peak eccentric force were 10–21% greater for Run than for Sed animals (P ≤ 0.016), whereas only Po was greater in sham than in OVX animals (9%; P = 0.011). The bone-to-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.
- functional capacity
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. However, a stress fracture may 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 US 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 than in those with regular menses (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 from 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 the rate of stress fractures is elevated by 2–3 wk into a training program, with peak incidences occurring at 5–8 wk (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 on 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 with the bone, whereas the latter theory depends on the muscle becoming relatively weak compared with 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. Our group has 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. Although 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 1 mo of greatly reduced muscle and bone loading, the muscle becomes relatively strong compared with the bone during the second week of recovery and remains so for 6–10 wk (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.
Three-month-old female C57BL/6 mice (n = 99) were purchased from the National Institute on Aging aging colony. Initially, four or five mice were housed per cage and subjected to a 12:12-h light-dark cycle at 20–23°C. Mice were given phytoestrogen-free rodent chow (2019 Teklad Global 19% rodent diet; Harland Teklad, Madison, WI) and water ad libitum. After 2 wk, 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).
Mice were randomly assigned into one of two surgical conditions [ovariectomy (OVX) or sham ovariectomy (sham group)] and one of two activity conditions [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 and because this training mode is considered less stressful on the animal. The Sed condition meant that a mouse was housed in a standard mouse cage (30 × 18 × 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 death, 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 whether 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 killed 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 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-to-muscle ratios of those capacities would indicate that an E2 effect is mediated via the E2 receptor.
OVX and sham surgical procedures were conducted when the mice were 3.75–4 mo 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 with 6-0 silk suture, and skin incisions were closed with 7-mm wound clips. After 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 by 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 an ∼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 each mouse received ∼3 μg/day. This dosage was based on 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 that received 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.
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, Brookfield, WI), which was capable of storing the number of revolutions per 24-h period for 8 days. The Run mice were allowed to acclimate to the running wheels for 1 wk before the OVX or sham surgery. Four to five days after the surgery, the mice were reintroduced to the wheels.
To estimate the activity levels of sedentary mice, the average daily distance covered in a cage was measured with 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 eight OVX+60d Sed and eight OVX+E2+60d Sed mice over 24 h during the last 1–2 days before they were killed. Before the measurements were made, mice were placed in mock activity chambers for 24 h to acclimate to the new environment. Mock chambers were exactly the same as the activity monitor chambers except there were no infrared beams.
At the time of death, mice were first anesthetized with pentobarbital sodium (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°C until assay of E2 concentration as described below. After blood collection, the mouse was killed by exsanguination, and the left tibia was dissected free. The tibia was placed in vial containing PBS and stored at −80°C until microcomputed 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. First, 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 the muscle was mounted in a glass chamber at optimal muscle length (Lo), the muscle was equilibrated in Krebs-Ringer buffer (pH 7.4) for 15 min at 25°C. 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 with 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 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 minutes after the second isometric stimulation, the muscle began a series of 10 eccentric contractions. For these contractions, the muscles 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; muscles were 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 eccentric contraction of the protocol. Peak eccentric force was not obtained for 8 of the 99 muscles studied; this was because of technical problems for 2 mice and because another set of measurements was substituted for six OVX+tamoxifen+60d Run mice.
After the eccentric contraction protocol was completed, the muscle was removed from the glass chamber and trimmed, blotted dry, weighed, and frozen in liquid N2. The muscle was stored at −80°C until total protein content was assayed. For measurement of total protein content, a muscle was homogenized in 10 mM phosphate buffer (pH 7.0). SDS 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 nondestructively image and quantify the morphology and volumetric bone mineral density (vBMD) of each excised tibia. The scanner was set to a voltage of 55 kV and a current of 145 μA, and the bones were scanned using a 16-μm voxel size with a 200-ms integration time. Fifty two-dimensional 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 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 three-point bending test (described below), only it was analyzed statistically. After completion of imaging, the bones were refrozen in PBS and stored at −80°C 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 samples were thawed to room temperature, the medial-lateral periosteal diameter of the tibia at mid-diaphysis was measured with 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 by a computer and software (Laboratory Technologies LabTech Notebook Pro version 8.01; Wilmington, MA).
The load-displacement curves were analyzed with a custom-written TestPoint program. The program defined ultimate load as the highest load obtained before 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 curve before 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 by classical beam theory. Ultimate stress was calculated with the following 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 with the following 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-to-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 of 39), OVX+E2 (n = 16 of 24), and sham (n = 14 of 30) groups were randomly selected for analysis of E2 concentration. As described previously (26), the plasma E2 concentration was determined by commercially available ELISAs (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 Laboratories kit, and it was used for ∼25% of the assays.
To analyze the effects of surgical condition (OVX vs. sham), activity (Run vs. Sed), and time of death 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 to determine which combination of conditions were different from another. To analyze the effectiveness of E2 supplementation, the OVX+E2 mice were compared with OVX and sham mice also killed 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 being 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 concentrations and daily running distances were analyzed by 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 whether the often-observed abnormal bone mechanical test responses occurred in one group of mice more than another, χ2-tests were employed. Statistical analyses were performed with 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. For these exceptions, the values are expressed as least-square mean values (SD) or relative changes between least-square mean values.
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) 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 death had no effect on the daily running distance of OVX and sham mice, the 30d Run and 60d Run groups for each type of mouse were collapsed and compared with the OVX+E2+60d Run mice. The average daily running distances of 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 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 30d and 60d Postsurgery
When 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 were compared, a significant effect of surgical condition, but not of activity, was found. OVX mice weighed 17% more than sham mice (Table 1).
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 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 other, i.e., no significant interaction. Stiffness for sham mice was 12% greater than that for OVX mice, whereas it was 9% greater for Run mice than for 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 Run mouse bones (Table 1). The greater energy absorption by the Sed mice can be explained mostly by a greater deflection to ultimate load for those mice. However, this effect was statistically significant only for the sham mice. 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% (not significant). The greater deflection and energy absorbed to ultimate load for the Sed mice appear 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) than in those from 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 by 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 killed at the 60d time point; at this time point, these measures were 11–15% greater for the sham mice than for 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.
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 those 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 than for Run mice. Ultimate stress was affected by surgical condition effect (P = 0.048), with the value for bones of OVX mice being 4% greater than that for bones of sham mice. 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 than that for the Sed condition.
The functional capacities of the soleus muscle were affected by both surgical condition and activity; however, 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 than for OVX mice and 21% greater for Run mice than for 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 than in the 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); however, 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 because of 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 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 Po 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% vs. 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 than for 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 levels shown in sham mice (Figs. 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 result was significantly greater than the OVX result. E2 supplementation was ineffective in restoring vBMD. vBMD for the OVX+E2 mice was significantly less than that shown for sham mice (compare Tables 1 and 3). In fact, vBMD for the OVX+E2 mice was less than or the same as that shown for 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-to-muscle functional relationship analyses presented above under Effect of Surgical Condition and Activity at 30d and 60d Postsurgery, these were found to be affected only by activity (Fig. 7 and Table 4). There were no differences among OVX+E2, OVX, and sham mice.
Comparison of Tamoxifen and E2 Supplementation
When the OVX+tamoxifen+60d Run and OVX+E2+60d Run groups were compared, 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 (Figs. 5–7 and Tables 3 and 4). There were only three bone measures for which the two groups differed significantly: cortical wall thickness, vBMD, and ultimate stress. Cortical wall thickness and vBMD results 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%.
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 with 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 Po was less than half that of activity's effect. Third, the ratio of bone functional capacity to that of muscle was only affected by activity. This is because the effects of surgical condition on the two tissues were relatively the same, and thus the effects canceled each other out when one measure was divided by the other. For example, ultimate load and maximal Po for sham mice were greater than those for OVX mice by similar percentages (7% and 9%, respectively). Conversely, activity marginally improved bone functional capacities (i.e., 0–9%) but more substantially improved those in muscle (i.e., 10–21%), resulting in reduced bone-to-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 observed differences between tamoxifen- and E2-supplemented OVX mice 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. However, 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 by which E2 enhanced muscle functional capacities, specifically maximal Po, 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 than found in OVX mice supplemented with placebo pellets. There was also no effect of E2 supplementation on total protein content, another indicator of muscle size. Although the effect of E2 supplementation on normalized Po did not quite reach significance (P = 0.052), the percent 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 qualities 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, although differently from E2. Although 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 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 than for those from 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 after 3 wk of treadmill run training (34) and the rat tibia after 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, which 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 run 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 the Run mice, we do not know the distance covered in their cage while not on the running wheel. This distance 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 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 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) in younger mice that E2 deficiency does not induce a bone-to-muscle functional capacity mismatch. However, bone-muscle functional capacity mismatches can occur in a physiologically relevant run training regimen in female mice. Although with voluntary wheel running the muscle became relatively strong compared with 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 regard to modeling stress fractures that occur in women in the US Armed Forces. For this situation, forced treadmill running by mice may be more applicable. Alternatively, loaded wheel running causes different muscle adaptations 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 on 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 whether 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 with 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.
The research was supported by National Institute on Aging Grants AG-20990 and AG-25861 to D. A. Lowe. A. L. Moran was supported by National Institutes of Health Training Grant T32-AR-07612 and a University of Minnesota Dissertation Grant.
We thank Sarah Greising, Rachel Landisch, and Steven Nelson for assistance on this project.
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- Copyright © 2007 the American Physiological Society