Effect of ionizing radiation on human skeletal muscle precursor cells

Abstract

Background: Long term effects of different doses of ionizing radiation on human skeletal muscle myoblast proliferation, cytokine signalling and stress response capacity were studied in primary cell cultures.

Materials and methods: Human skeletal muscle myoblasts obtained from muscle biopsies were cultured and irradiated with a Darpac 2000 X-ray unit at doses of 4, 6 and 8 Gy. Acute effects of radiation were studied by interleukin - 6 (IL-6) release and stress response detected by the heat shock protein (HSP) level, while long term effects were followed by proliferation capacity and cell death.

Results: Compared with non-irradiated control and cells treated with inhibitor of cell proliferation Ara C, myoblast proliferation decreased 72 h post-irradiation, this effect was more pronounced with increasing doses. Post-irradiation myoblast survival determined by measurement of released LDH enzyme activity revealed increased activity after exposure to irradiation. The acute response of myoblasts to lower doses of irradiation (4 and 6 Gy) was decreased secretion of constitutive IL-6. Higher doses of irradiation triggered a stress response in myoblasts, determined by increased levels of stress markers (HSPs 27 and 70).

Conclusions: Our results show that myoblasts are sensitive to irradiation in terms of their proliferation capacity and capacity to secret IL-6. Since myoblast proliferation and differentiation are a key stage in muscle regeneration, this effect of irradiation needs to be taken in account, particularly in certain clinical conditions.

Keywords: apoptosis; interleukin 6; irradiation; muscle regeneration; myoblasts; proliferation.

FIGURE 1.

The effect of selected therapeutic doses of ionizing radiation (4 Gy, 6 Gy, 8 Gy) on the proliferation of human skeletal myoblasts assessed 72 h after irradiation. Columns and bars represent means ± SD (n= 8). Means are expressed as arbitrary units which are relative units of absorbance measurement at dual wavelengths of 450–540 nm. The control was used as the predetermined reference measurement. Statistically significant differences (p < 0.001) are indicated by ***. Control (CTR) – non-irradiated myoblast; Inhibitor – myoblasts treated with 10 μM AraC, an inhibitor of cell proliferation; 4 Gy – myoblasts irradiated with 4 Gy; 6 Gy - myoblasts irradiated with 6 Gy; 8 Gy – myoblasts irradiated with 8 Gy.

FIGURE 2.

The effect of selected therapeutic doses of ionizing radiation (4 Gy, 6 Gy, 8 Gy) on cell death immediately after irradiation (0 h after IR.), 36 h after irradiation (36 h after IR.) and 72 h after irradiation (72 h after IR). Columns and bars represent means ± SD (n=4). (A) The assessment of myoblasts membranes integrity at different time points after irradiation is shown as the activity of lactate dehydrogenase (LDH) released in the medium. Statistically significant differences (p ≤ 0,001) are indicated by ***. (B) The cleavage activity of the amino acid sequence DEVD (recognised and cleaved by apoptotic caspase 3 and 7) is shown at different time points after irradiation. There were no statistically significant differences among groups.

FIGURE 3.

Effects of different doses of ionizing radiation on constitutive level of IL-6. Level of constitutive IL-6 secretion was estimated by ELISA in control myoblast cultures and compared with level of IL-6 secretion in myoblast cultures 24 hours after exposure to 4 Gy, 6 Gy and 8 Gy dose of irradition. Data are means ± SD (n = 18 in each independent experiment). * p < 0.05 (t-test) denotes difference in level of IL-6 in exposed cultures vs. respective level of control cultures, # significant difference between groups indicated p < 0.05 (t-test).

FIGURE 4.

The effect of different doses of ionizing radiation on the HSP 27 and HSP 70 level in myoblasts 24 h after exposure. Representative Western blots for HSP 27 and HSP 70 (A). Relative levels of HSP 27 and HSP 70 shown as % of control level of proteins in myoblasts not exposed to irradiation (B), * p < 0.05 denotes significant difference in level of HSP in exposed myoblasts vs. level in control non-exposed myoblasts.