Genetic variation influences the skeletal response to hindlimb unloading in the eight founder strains of the diversity outbred mouse population

During disuse, mechanical unloading causes extensive bone loss, decreasing bone volume and strength. Variations in bone mass and risk of osteoporosis are influenced by genetics; however, it remains unclear how genetic variation affects the skeletal response to unloading. We previously found that genetic variation affects the musculoskeletal response to 3 weeks of immobilization in the 8 Jackson Laboratory J:DO founder strains: C57Bl/6J, A/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ. Hindlimb unloading (HLU) is the best model for simulating local and systemic contributors of disuse and therefore may have a greater impact on bones than immobilization. We hypothesized that genetic variation would affect the response to HLU across the eight founder strains. Mice of each founder strain were placed in HLU for 3 weeks, and the femurs and tibias were analyzed. There were significant HLU and mouse strain interactions on body weight, femur trabecular BV/TV, and femur ultimate force. This indicates that unloading only caused significant catabolic effects in some mouse strains. C57BL/6 J mice were most affected by unloading while other strains were more protected. There were significant HLU and mouse strain interactions on gene expression of genes encoding bone metabolism genes in the tibia. This indicates that unloading only caused significant effects on bone metabolism genes in some mouse strains. Different mouse strains respond to HLU differently, and this can be explained by genetic differences. These results suggest the outbred J:DO mice will be a powerful model for examining the effects of genetics on the skeletal response to HLU.


| INTRODUCTION
Disuse from physical inactivity, 1 paralysis, 2,3 or spaceflight 4,5 causes extensive loss of bone mass and strength.This bone loss greatly increases the risk of bone fracture.There is a need to better understand the mechanisms involved in regulating bone loss from disuse.7][8][9] However, it remains unclear how genetics affects the response to disuse in bone.
Outbred mice have the potential to reveal how genetic variation influences the response to disuse.To determine the feasibility for using J:DO mice to study how genetics influences the response to disuse, we first investigated how the eight inbred founder strains responded to immobilization.We found strong evidence that the magnitude of bone and skeletal muscle loss from immobilization is a heritable trait. 13,14mobilization is one of several animal models that are used for studying the effects of disuse on bone.Other models include hindlimb unloading (HLU), neurectomy, and bed rest. 5Amongst models of disuse, HLU best recapitulates the effects of microgravity on the skeleton experienced during spaceflight. 15In this study, we sought to determine how HLU affects the skeletal response to disuse in the eight founder strains of the J:DO mice.We hypothesized that genetic variation would affect the skeletal response to HLU, causing some mouse strains to have greater magnitudes of bone loss than other mouse strains.

| Animals
All animal procedures were done with the approval of the Virginia Commonwealth University Institutional Animal Care and Use Committee.Mice were purchased from the Jackson Laboratory (Jackson Labs) between 4 and 14 weeks of age.Sixteen male mice of each of the founder strains of the DO mice were used-C57BL/ 6J (stock #000664), PWK/PhJ (stock #003715), WSB/EiJ (stock #001145), A/J (stock #000646), 129S1/SvImJ (stock #002448), NOD/ShiLtJ (stock #001976), NZO/HlLtJ (stock #002105), and CAST/EiJ (stock #000928).Mouse strain, age, sex, sample size, and duration of interventions were determined based on our previous work studying the effects of single-limb immobilization in these mouse strains. 14All mice except WSB/EiJ mice were kept in group housing upon arrival, and all mice were kept on a 12-h light/dark cycle and given Teklad LM-485 chow (Envigo) and water ad libitum.At 15 weeks of age, mice were transferred to cages with wire mesh floors and housed two per cage for acclimation.WSB/EiJ mice were single-housed from arrival through acclimation.After acclimation, mice underwent 3 weeks of HLU.Then mice were killed, and femurs and tibias were harvested for analysis.

| Hindlimb unloading
At 16 weeks of age, mouse cages were randomly assigned to either control or HLU groups (n = 8 mice per group) for 3 weeks.HLU was performed as we have done previously. 16The mice were suspended with their tails attached to a bar at the top of the cage, and their tails were held at a 30°angle of elevation.Mice were able to navigate and rotate around a limited space in the cages using their forelimbs to grab on to the wire mesh floor.Each cage had two suspended mice, facing away from each other and unable to make physical contact with each other.Control mice were housed in the same style of cage as HLU, but they did not have their tails attached to the bar.Control mice were free to roam around the cages unrestricted.The health of all animals was assessed daily, and mice were removed from the study based on their health scores and consultations with veterinary staff.

| Femur morphology
All femurs were scanned by micro-CT (Bruker Skyscan 1276) at the end of the study.Scans were done using 7.0 µm voxel size, 60 kV, 200 µA, 730 ms exposure, and a 0.5 mm Al filter.Left femur bone morphology was analyzed at three locations-the mid-diaphysis, distal metaphysis, and distal epiphysis.Bones were reoriented along anatomical locations, and measurements were taken as we have done previously. 17Cortical bone measurements were taken of a 180-µm section of the mid-diaphysis located 4 mm distal to the lesser trochanter.Trabecular bone measurements were taken of a 750-µm section of the distal metaphysis located 200 µm proximal to the growth plate.Trabecular bone measurements were also taken of a 520-µm section of the distal epiphysis located immediately distal to the growth plate.

| Femur mechanical properties
During dissection, femurs were cleaned of soft tissue, placed in calcium buffer comprised of phosphate-buffered saline plus calcium chloride, and stored at −20°C.Mechanical properties of the femur were measured by loading the bones in three-point bending to failure at 1.0 mm/min.Left femurs were loaded with the anterior side in tension and with support spans of 6 mm.Results were analyzed, and mechanical properties were calculated as we have done previously. 17e fracture site was determined, and micro-CT morphology measurements from that location were used to calculate tissue-level mechanical properties. 18IEDMAN ET AL.  19

| Statistical analysis
All analyses were done in GraphPad Prism.Mixed effects analysis models were used to identify significant main effects (p < 0.05) of mouse strain or unloading for each bone property.A repeated measures two-way analysis of variance (ANOVA) was used to calculate changes in body weight from Day 1 to Day 22. Sidak's multiple comparisons test was used post hoc to test for significant group differences.

| RESULTS
Several mice were removed from the study during acclimation and during HLU for reasons including excessive weight loss of more than 20% initial body weight, difficulty adapting to the wire mesh floors, signs of distress, and escaping the cage.All control and HLU groups finished the study with n = 3-8 mice per group.Thus, power to detect significant effects of HLU was limited for some mouse strains.Body weight declined from baseline in all HLU groups, and it was significantly decreased in C57BL/6J, A/J, and NZO/HiLtJ mice (Table 1).Control groups for each mouse strain displayed less change in body weight, and there were no significant differences between final day and initial body weight.
There was a significant main effect of unloading and a significant main effect of mouse strain on femur mid-diaphyseal cortical area and cortical area/total area (Figure 1A,B, p < 0.05, two-way ANOVA).
There was a significant interaction between unloading and mouse strain on cortical thickness.CAST/EiJ mice had the largest magnitude of difference between control and HLU groups in all cortical bone properties measured.
There was a significant unloading and mouse strain interaction on bone volume/total volume (BV/TV) of the distal femur metaphysis (Figure 2A, p < 0.05, two-way ANOVA).Metaphyseal BV/TV was T A B L E 1 Mean mouse body weight on Day 1 and after 3 weeks of treatments.significantly decreased by HLU only in C57BL/6J mice.NOD/ShiLtJ HLU mice had significantly greater BV/TV than controls.There was a significant main effect of unloading and a significant main effect of mouse strain on trabecular thickness (Tb Th) in the distal femur metaphysis (Figure 2B).There was a significant unloading and mouse strain interaction on BV/TV and Tb Th of the distal femur epiphysis (Figure 2C,D).Five of the eight mouse strains had significantly lower epiphyseal BV/TV or Tb Th in HLU groups, compared to controls.
Similar effects were seen on trabecular number and separation.
There was a significant unloading and mouse strain interaction on femur ultimate force (Figure 3A, p < 0.05, two-way ANOVA) and elastic modulus (Figure S2).There was a significant main effect of unloading on yield force, yield stress, and ultimate stress.Mouse strain had a significant main effect on yield force and stiffness (Figure S2).C57BL/6J and CAST/EiJ HLU mice had significantly lower ultimate force than their controls.PWK/PhJ HLU mice had significantly lower ultimate stress than their controls (Figure 3B).
C57BL/6J mice had significantly greater Tnfsf11/Tnfrsf11b ratio in HLU mice than controls.WSB/EiJ mice had significantly greater Tnfsf11 and Tnfrsf11b in HLU mice than controls.A/J mice had significantly lower Tnfsf11 in HLU than in controls.129S1/SvImJ mice had significantly lower Sost in HLU than in controls.

| DISCUSSION
This is the first study to evaluate how bones of the founder strains of the Diversity Outbred mouse population respond to HLU.To our knowledge, except C57BL/6J, these mouse strains have not been previously studied using this model of unloading.There was a wide variety of response to unloading with some mouse strains having large magnitudes of bone loss and other strains appearing to be protected from the catabolic effects of unloading.These results suggest J:DO mice will be an excellent mouse model for studying the effects of genetic variation on the skeletal response to HLU.Additionally, since HLU is the most accurate Earth-based model of spaceflight effects on bone, J:DO mice could also be used for studying the effects of spaceflight on a genetically diverse population.
Similar to what was seen when we exposed these mice to singlelimb immobilization, the magnitude of bone loss varied by mouse strain. 14C57BL/6 J mice were the most sensitive to unloading, having the greatest magnitudes of bone loss in femoral metaphyseal and epiphyseal trabecular bone volume.CAST/EiJ were also very sensitive to unloading, with the greatest magnitudes of loss of femoral cortical area and ultimate force.Variance in the data when these mice were exposed to HLU here was also similar to variance seen using single limb immobilization.HLU mice had coefficients of variation for body weight, cortical area, and ultimate load of 36%, 20%, and 25%, respectively.Immobilized mice had coefficients of variation of 34%, 20%, and 32%, respectively.Wergedal et al.
performed a large study using 29 different inbred mouse strains and found coefficients of variation for these properties to be 23%, 18%, and 26%, respectively. 8The founder strains of the J:DO mice had more variance in properties due to the extreme difference in body weight and bone size of the NZO/HiLtJ strain.There were regional differences in the effects of unloading on the femur.As expected, after only 3 weeks of unloading, trabecular bone was more sensitive to unloading than cortical bone. 14,20All mouse strains in HLU had decreased epiphyseal BV/TV compared to controls.Genetic differences between mouse strains only influenced the magnitude of bone loss in this region.In the distal metaphysis, however, not every strain had bone loss from HLU. NOD/ShiLtJ HLU mice even had greater BV/TV than NOD/ShiLtJ controls.
Genes that are associated with bone metabolism were mostly unaffected by HLU.One exception, however, was that C57BL/6J had (A) (B) F I G U R E 3 Mouse left femoral strength (mean ± standard deviation) measured by three-point bending to failure after 3 weeks of hindlimb unloading.# Significant main effect of unloading (p < 0.05, two-way ANOVA).^Significant main effect of mouse strain.& Significant unloading × mouse strain interaction.**p < 0.01 group difference.***p < 0.001 group difference.
significantly elevated expression of the Tnfsf11/Tnfrsf11b ratio.
These genes encode RANKL and OPG, respectively and are representative of bone resorption and formation, respectively.This increase in RANKL/OPG has been observed in HLU in C57BL/6J mice before, 21 and here, we did not notice this increase in RANKL/ OPG in other mouse strains.C57BL/6J, the mouse strain most sensitive to unloading, had the highest RANKL/OPG gene expression ratio in HLU mice.This is partly due to these mice having the lowest OPG gene expression of the eight strains studied here.OPG is an antagonist of RANKL and protects against bone resorption.Some limitations of this study include lack of intra-animal controls such as in vivo micro-CT, changes in mouse cage activity, food consumption, or blood biomarkers.Only male mice were used to reduce the number of animals needed.Previous studies indicated male mice were more sensitive to unloading. 23,24Future work using J:DO mice should include male and female mice to investigate sex differences in the role of genetic variation on the skeletal response to unloading.
Genetic variation influences bone mass and strength.Here | 139 magnitude of bone loss in response to unloading.This could lead to novel therapies that target mechanisms involved in regulating the response to unloading.

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we have shown that genetic variation also influences the skeletal response to unloading via HLU.Similar effects of genetics and unloading were seen when the J:DO founder strains were exposed to single limb immobilization, indicating that each model can be used effectively for studying how genetic variation affects the response to disuse or spaceflight in bone.Since HLU was more stressful than immobilization resulting in a high rate of attrition among some strains, we recommend using immobilization in future studies in bone.Future work should use J:DO mice to uncover which genes regulate the Mouse left tibial gene expression (mean ± standard deviation) measured by RT-qPCR on diaphyseal cortical bone after 3 weeks of hindlimb unloading.& Significant unloading × mouse strain interaction (p < 0.05, two-way ANOVA).*p < 0.05 group difference.**p < 0.01 group difference.***p < 0.001 group difference.****p < 0.0001 group difference.FRIEDMAN ET AL.