Simulated space radiation sensitizes bone but not muscle to the catabolic effects of mechanical unloading

Abstract

Deep space travel exposes astronauts to extended periods of space radiation and mechanical unloading, both of which may induce significant muscle and bone loss. Astronauts are exposed to space radiation from solar particle events (SPE) and background radiation referred to as galactic cosmic radiation (GCR). To explore interactions between skeletal muscle and bone under these conditions, we hypothesized that decreased mechanical load, as in the microgravity of space, would lead to increased susceptibility to space radiation-induced bone and muscle loss. We evaluated changes in bone and muscle of mice exposed to hind limb suspension (HLS) unloading alone or in addition to proton and high (H) atomic number (Z) and energy (E) (HZE) (16O) radiation. Adult male C57Bl/6J mice were randomly assigned to six groups: No radiation ± HLS, 50 cGy proton radiation ± HLS, and 50 cGy proton radiation + 10 cGy 16O radiation ± HLS. Radiation alone did not induce bone or muscle loss, whereas HLS alone resulted in both bone and muscle loss. Absolute trabecular and cortical bone volume fraction (BV/TV) was decreased 24% and 6% in HLS-no radiation vs the normally loaded no-radiation group. Trabecular thickness and mineral density also decreased with HLS. For some outcomes, such as BV/TV, trabecular number and tissue mineral density, additional bone loss was observed in the HLS+proton+HZE radiation group compared to HLS alone. In contrast, whereas HLS alone decreased muscle mass (19% gastrocnemius, 35% quadriceps), protein synthesis, and increased proteasome activity, radiation did not exacerbate these catabolic outcomes. Our results suggest that combining simulated space radiation with HLS results in additional bone loss that may not be experienced by muscle.

Fig 1. Effect of HLS and radiation on trabecular parameters measured with MicroCT scans at distal femur.

Measures are shown as absolute values at conclusion of study for trabecular bone volume fraction (Tb.BVTV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), trabecular connectivity density (Conn.Dens) and trabecular tissue mineral density (Tb.TMD). Values are means ± SEM; n = 14–18 bones/group. Values with line and asterisk indications are significantly different (p<0.05).

Fig 2. Representative images of trabecular samples from GC mice (top) and HLS mice (bottom) on day 14.

Images represent a 72 slice region (756 μm) of distal femur.

Fig 3. Effect of HLS and radiation on cortical parameters measured with MicroCT scans at distal femur.

Measures are shown as absolute values at conclusion of study for cortical total volume, cortical bone volume, cortical bone volume fraction (Ct. BVTV), cortical thickness, cortical bone mineral density, and cortical porosity. Values are means ± SEM; n = 14–18 bones/group. Values with line and asterisk indications are significantly different (p<0.05).

Fig 4. Effect of HLS and radiation on mechanical properties of femurs observed during 3 point bending until failure.

Measures are shown as absolute values at conclusion of study for max force during testing (A), bone stiffness (B), and energy absorbed during testing (C). Values are means ± SEM; n = 14–18 bones/group. Values with line and asterisk indications are significantly different (p<0.05).

Fig 5

Effect of HLS and radiation on wet muscle weight of gastrocnemius (A), quadriceps (B), and protein synthesis (C) measured with the SUnSET method. Representative Western blot of Puromycin incorporation into muscle protein for each of the six experimental groups (D left), and representative Ponceau stain demonstrating equal protein loading per lane (D right). Measures are absolute values normalized to body weight at conclusion of study (GC in protein synthesis). Values are means ± SEM; n = 14-18/group (p<0.05).

Fig 6. Effect of HLS and radiation on phosphorylation of mTOR downstream regulators 4EBP1 and S6K1 measured via Western blot analysis.

Top panel (A) is a representative Western blot for phosphorylated and total S6K1 (B) and 4E-BP1 (C) in muscle from each of the six experimental groups; tubulin is also included to demonstrate equal protein loading all groups. All samples were run on the same gel. Bar graphs represent average values at conclusion of study and are normalized to GC group. Values are means ± SEM; n = 14-18/group, (p<0.05).

Fig 7. Effect of HLS and radiation on 20S proteasome(A), atrogin-1 (B), MuRF-1 (C), and insulin growth factor (D) expression.

Measures are absolute values at conclusion of study. Values are means ± SEM; n = 14-18/group, Values with line and asterisk indications are significantly different (p<0.05).