Age-dependent musculoskeletal changes during mechanical unloading with bisphosphonate treatment.

Abstract

Mechanical unloading (disuse) leads to reductions in bone and muscle mass. Bone and muscle adaptation are often studied together in terms of mechanical stimulus, but non-mechanical (biological) crosstalk during periods of disuse and how this is affected by age and bone-preserving pharmaceuticals has not been assessed. This study aimed to determine how mechanical unloading and concurrent bisphosphonate treatment affect bone and muscle structure and function in young, middle-aged, and old mice. We hypothesized that unloading would cause bone loss in untreated mice, but bisphosphonate treatment would prevent this loss. Additionally, we expected that unloading would result in muscle atrophy and reduced contraction force, but we hypothesized that these reductions would be partially mitigated by bisphosphonate treatment due to decreased release of osteokines, and that this mitigation would decrease with age. To investigate these hypotheses, young (3-mo, n = 40), middle-aged (12-mo, n = 40), and old (20-m, n = 40) male C57BL/6 J mice received biweekly subcutaneous bisphosphonate injections (0.03 mg alendronate/mouse) or vehicle injections starting one week before unloading. Mice underwent hindlimb unloading (HLU) via tail suspension for 14 days. Maximum force production of the hind limb was measured after 14 days of unloading. Femurs were imaged with micro-computed tomography (μCT 35, SCANCO Medical AG); cortical bone was analyzed at the mid-diaphysis, and trabecular bone was analyzed at the distal femur to determine bone microstructural outcomes. Muscle fiber cross-sectional area (CSA) and fiber type were analyzed via IHC. Muscle myostatin and serum TGF-β1 levels were measured via ELISA. Achilles tendon mechanical properties were also assessed using tensile testing. We found that HLU decreased the mass of the triceps surae muscles, and this loss was not recovered during bisphosphonate treatment. Muscle mass in old mice decreased during HLU with bisphosphate treatment. Maximum hindlimb force production and respective force to muscle mass ratio differed between all age groups and did not correlate with bone or muscle changes. Muscle myostatin concentrations increased with age (p = 0.040), bisphosphonate treatment (p = 0.003), and unloading (p = 0.002), as well as due to the interaction of treatment and unloading (p = 0.0239). Serum levels of TGF-β1 increased with age (p < 0.0001) and unloading (p = 0.012), as well as with interactions between both age and treatment (p = 0.006) and age and unloading (p = 0.013). Age impacted muscle, tendon, and bone responses to unloading and/or bisphosphonate treatment. Bone and muscle adaptation to unloading are different across the lifespan, as are the effects of bisphosphonate treatment. Characterizing these changes is essential for understanding clinical outcomes related to periods of disuse and clinical bone and muscle preserving treatments during bedrest, immobilization, or even spaceflight across different age groups.