Divergent modification of low-dose ⁵⁶Fe-particle and proton radiation on skeletal muscle

Abstract

It is unknown whether loss of skeletal muscle mass and function experienced by astronauts during space flight could be augmented by ionizing radiation (IR), such as low-dose high-charge and energy (HZE) particles or low-dose high-energy proton radiation. In the current study adult mice were irradiated whole-body with either a single dose of 15 cGy of 1 GeV/n ⁵⁶Fe-particle or with a 90 cGy proton of 1 GeV/n proton particles. Both ionizing radiation types caused alterations in the skeletal muscle cytoplasmic Ca²⁺ ([Ca²⁺]i) homeostasis. ⁵⁶Fe-particle irradiation also caused a reduction of depolarization-evoked Ca²⁺ release from the sarcoplasmic reticulum (SR). The increase in the [Ca²⁺]i was detected as early as 24 h after ⁵⁶Fe-particle irradiation, while effects of proton irradiation were only evident at 72 h. In both instances [Ca²⁺]i returned to baseline at day 7 after irradiation. All ⁵⁶Fe-particle irradiated samples revealed a significant number of centrally localized nuclei, a histologic manifestation of regenerating muscle, 7 days after irradiation. Neither unirradiated control or proton-irradiated samples exhibited such a phenotype. Protein analysis revealed significant increase in the phosphorylation of Akt, Erk1/2 and rpS6k on day 7 in ⁵⁶Fe-particle irradiated skeletal muscle, but not proton or unirradiated skeletal muscle, suggesting activation of pro-survival signaling. Our findings suggest that a single low-dose ⁵⁶Fe-particle or proton exposure is sufficient to affect Ca²⁺ homeostasis in skeletal muscle. However, only ⁵⁶Fe-particle irradiation led to the appearance of central nuclei and activation of pro-survival pathways, suggesting an ongoing muscle damage/recovery process.

FIG. 1

Gamma radiation increases resting [Ca²⁺] within 1 h. Analysis of relative resting cytoplasmic [Ca²⁺] in dissociated muscle fibers from control and γ-irradiated mice. Cells were prepared 1 h and 7 days after irradiation (*P < 0.05; n-mice_IR = 3, n-mice_control = 3).

FIG. 2

Low-dose ⁵⁶Fe-particle radiation increases [Ca²⁺]_i within 24 h and decreases Ca²⁺ transient amplitude up to 48 h after irradiation. Panel A: Analysis of relative resting cytoplasmic [Ca²⁺] in dissociated muscle fibers from control and ⁵⁶Fe-particle (15 cGy, 1 GeV/n) irradiated mice. Cells were prepared 24, 48 h and 7 days after irradiation, (*P < 0.05; n-mice_IR = 3, n-mice_control = 3). Panels B and C: Analysis of electrical depolarization-evoked Ca²⁺ release in dissociated muscle fibers from control and ⁵⁶Fe-particle (15 cGy, 1 GeV/n) irradiated mice at 24 and 48 h time point. In panel B, amplitude of transients from irradiated cells were normalized to the peak amplitude of transients in the control, nonirradiated cells obtained at each respective time point (*P < 0.05; n-mice_IR = 3, n-mice_control = 3).

FIG. 3

Low-dose ¹H-particle radiation increases [Ca²⁺]_i and decreases Ca²⁺ transient amplitude only by 72 h after irradiation. Panel A: Analysis of relative resting cytoplasmic [Ca²⁺] in dissociated muscle fibers from control and proton (90 cGy, 1 GeV/n) irradiated mice. Cells were prepared 24 and 72 h after irradiation (*P < 0.05; n-mice_IR = 3, n-mice_control = 3). Panel B: Analysis of electrical depolarization-evoked Ca²⁺ release in dissociated muscle fibers from control and proton (90 cGy, 1 GeV/n) irradiated mice at 24 and 72 h time point (*P < 0.05; n-mice_IR = 3, n-mice_control = 3).

FIG. 4

⁵⁶Fe irradiation increases the number of central nuclei in skeletal muscle tissue. Panel A: Representative images of H&E stained skeletal muscle tissue obtained 7 days after whole-body low-dose high-energy proton (90 cGy, 1 GeV/n) or iron (15 cGy, 1 GeV/n) irradiation at 40× magnification to show central nuclei clearly. Individual muscle fibers that contain central nucleus or nuclei are outlined by white dotted lines. Panel B: Graphic representation of the number of central nuclei per ~85 ± 7 individual muscle fibers at 20× magnification from three different animals (at least 20 images/animal) per radiation group. Statistical significance was assigned when P < 0.05 (n-mice_IR = 3, n-mice_control = 3).

FIG. 5

⁵⁶Fe-particle radiation increases survival and proliferation signaling in skeletal muscle tissue one week after irradiation. Panels A, C, E: Representative images of phospho-Erk1/2 (p-Erk1/2) and total-Erk1/2 (T-Erk1/2), p-Akt and T-Akt, p-rpS6k and T-rpS6k, and corresponding for each protein GAPDH Western blot analyses in skeletal muscle tissue homogenates in control, iron and proton whole-body irradiated mice. Panels B, D, F: Quantification of Erk1/2, Akt and rpS6k protein levels and phosphorylation using densitometric analysis of p-Erk1/2, p-Akt and p-rpS6k band intensities after adjusting for corresponding GAPDH and T-Erk1/2, T-Akt and T-rpS6k band intensities. Graphs represent data from three biological samples per radiation group. Statistical significance was assigned when P < 0.05 (n-mice_IR = 3, n-mice_control = 3).