Hindlimb Skeletal Muscle Function and Skeletal Quality and Strength in +/G610C Mice With and Without Weight-Bearing Exercise

Abstract

Osteogenesis imperfecta (OI) is a heterogeneous heritable connective tissue disorder associated with reduced bone mineral density and skeletal fragility. Bone is inherently mechanosensitive, with bone strength being proportional to muscle mass and strength. Physically active healthy children accrue more bone than inactive children. Children with type I OI exhibit decreased exercise capacity and muscle strength compared with healthy peers. It is unknown whether this muscle weakness reflects decreased physical activity or a muscle pathology. In this study, we used heterozygous G610C OI model mice (+/G610C), which model both the genotype and phenotype of a large Amish OI kindred, to evaluate hindlimb muscle function and physical activity levels before evaluating the ability of +/G610C mice to undergo a treadmill exercise regimen. We found +/G610C mice hindlimb muscles do not exhibit compromised muscle function, and their activity levels were not reduced relative to wild-type mice. The +/G610C mice were also able to complete an 8-week treadmill regimen. Biomechanical integrity of control and exercised wild-type and +/G610C femora were analyzed by torsional loading to failure. The greatest skeletal gains in response to exercise were observed in stiffness and the shear modulus of elasticity with alterations in collagen content. Analysis of tibial cortical bone by Raman spectroscopy demonstrated similar crystallinity and mineral/matrix ratios regardless of sex, exercise, and genotype. Together, these findings demonstrate +/G610C OI mice have equivalent muscle function, activity levels, and ability to complete a weight-bearing exercise regimen as wild-type mice. The +/G610C mice exhibited increased femoral stiffness and decreased hydroxyproline with exercise, whereas other biomechanical parameters remain unaffected, suggesting a more rigorous exercise regimen or another exercise modality may be required to improve bone quality of OI mice.

Keywords: BONE; COLLAGEN CROSS-LINKS; EXERCISE; MUSCLE; OSTEOGENESIS IMPERFECTA.

Fig. 1.

Male and female +/G610C mice do not exhibit altered levels of locomoter activity relative to age-matched Wt mice. Mean ± SD of distance traveled (cm)/60 min for 3 consecutive days by Wt (solid black) and +/G610C (white diagonal) mice at 1 and 4 months of age (n = 8–13 per group). ^ap ≤ 0.05 versus 4-month-old mice.

Fig. 2.

(A) Hematoxylin and eosin-stained cross sections of 4-month-old male Wt (left) and +/G610C (right) tibialis anterior muscles do not show evidence of necrosis, regeneration, or inflammatory cell infiltration. Male (B) and female (C) Wt (solid black) and +/G610C mice (white diagonal) exhibit similar myofiber cross-sectional areas of their soleus (S),plantaris (P), gastrocnemius (G), or tibialis anterior (TA) muscles (n = 8–10 per group). Mean ± SD, ^ap ≤ 0.05 versus Wt mice.

Fig. 3.

Four-month-old +/G610C mice tibias have reduced trabecular bone geometry relative to Wt mice tibias. (A) Trabecular bone volume per total bone volume (BV/TV); (B) trabecular bone thickness; (C) trabecular bone number; (D) trabecular bone separation; (E) connectivity density; (F) trabecular bone density of male and female Wt control (solid black), Wt treadmill-exercised (solid gray), +/G610C control (white diagonal), and +/G610C treadmill-exercised (gray diagonal) mice (n = 7–9 per group). Mean ± SD, ^ap ≤ 0.05 versus Wt mice; ^bp ≤ 0.05 versus control mice.

Fig. 4.

Four-month-old +/G610C femora exhibit compromised bone biomechanical properties compared with Wt femora. Treadmill exercise increased stiffness in both whole-bone and material properties. (A) Torsional ultimate strength (T_max), (B) tensile strength (Su), (C) torsional stiffness, (D) shear modulus of elasticity, (E) energy to failure (U) in femora of male and female Wt control (solid black), Wt treadmill-exercised (solid gray), +/G610C control (white diagonal), and +/G610C treadmill-exercised (gray diagonal) mice (n = 8–10 per group). Mean ± SD, ^ap ≤ 0.05 versus Wt mice; ^bp ≤ 0.05 versus control mice.

Fig. 5.

By Raman spectroscopy the crystallinity (A), mineral/matrix ratios, PO₄/CH₂ (B) and CO₃/CH₂ (C), and CO₃/PO₄ (D) were evaluated in tibias of 4-month-old male and female Wt control (solid black), Wt treadmill-exercised (solid gray), +/G610C control (white diagonal), and +/G610C treadmill-exercised (gray diagonal) mice (n = 5–6 per group). Mean ± SD, ^ap ≤ 0.05 versus Wt mice.

Fig. 6.

Treadmill exercise reduces hydroxyproline content in female mouse femora. Hydroxyproline (mg/g of bone) in male and female 4-month-old Wt control (solid black), Wt treadmill-exercised (solid gray), +/G610C control (white diagonal), and +/G610C treadmill-exercised (gray diagonal) mice (n = 710 per group). Mean ± SD, ^bp ≤ 0.05 versus control mice.

Fig. 7.

+/G610C mouse tibias have increased hydroxylysylpyridinoline (HP), HP + lysylpyridinoline (LP), and HP/LP ratio relative to control Wt mouse tibias. (A) HP, (B) LP, (C) HP + LP, and (D) HP/LP content in tibias of 4-month-old male (circle) and female (triangle) Wt control (Wt CTRL; black), Wt treadmill-exercised (WtTRD; light gray), +/G610Ccontrol (+/G610C CTRL; dark gray), and +/G610C treadmill-exercised (+/G610C TRD; white) mice (n = 4 per group). Mean ± SD, ^ap ≤ 0.05 versus Wt mice; ^bp ≤ 0.05 versus control mice.