Satellite cells say NO to radiation

Abstract

Skeletal muscles are commonly exposed to radiation for diagnostic procedures and the treatment of cancers and heterotopic bone formation. Few studies have considered the impact of clinical doses of radiation on the ability of satellite cells (myogenic stem cells) to proliferate, differentiate and contribute to recovering/maintaining muscle mass. The primary objective of this study was to determine whether the proliferation of irradiated satellite cells could be rescued by manipulating NO levels via pharmacological approaches and mechanical stretch (which is known to increase NO levels). We used both SNP (NO donor) and PTIO (NO scavenger) to manipulate NO levels in satellite cells. We observed that SNP was highly effective in rescuing the proliferation of irradiated satellite cells, especially at doses less than 5 Gy. The potential importance of NO was further illustrated by the effects of PTIO, which completely inhibited the rescue effect of SNP. Mechanical cyclic stretch was found to produce significant increases in NO levels of irradiated satellite cells, and this was associated with a robust increase in satellite cell proliferation. The effects of both radiation and NO on two key myogenic regulatory factors (MyoD and myogenin) were also explored. Irradiation of satellite cells produced a significant increase in both MyoD and myogenin, effects that were mitigated by manipulating NO levels via SNP. Given the central role of myogenic regulatory factors in the proliferation and differentiation of satellite cells, the findings of the current study underscore the need to more fully understand the relationship between radiation, NO and the functionality of satellite cells.

FIG. 1

The effects of an NO donor (SNP) and scavenger (PTIO) on nitric oxide-based DAF fluorescence in satellite cells. Measures of DAF signal intensity were also made under conditions in which neither SNP nor PTIO was present. The DAF signal intensity under this null condition was zero and is not shown. Note that 1 μM of SNP produced a significant (P < 0.001) increase in NO levels that remained elevated throughout the 90-min experiment. When PTIO was added in combination with 1 μM of SNP, there was an initial rise in NO levels, but over 90 min the NO levels progressively declined to baseline levels (P < 0.001). When SNP was increased to 100 μM, there was a large and continuous increase in NO levels (P < 0.001). Although PTIO was initially effective in suppressing the effects of 100 μM SNP, at the later times PTIO did not quench the SNP effect. Each data point represents the mean ± SE.

FIG. 2

Rescue of satellite cells from the effects of γ radiation. Cell count data were collected 48, 72 and 96 h after irradiation. All data were normalized to the cell count measured under control conditions (first bar in each panel; dashed horizontal line; 0 Gy; 0 μM SNP). SNP significantly increased cell counts at each time. Furthermore, SNP was capable of rescuing proliferation in satellite cells receiving <5 Gy. All data are reported as means + SE. Data for each time were analyzed using two-way ANOVA with group variables of radiation dose and SNP concentration. The main effects (radiation dose, SNP concentration and interaction effect) were all significant at P < 0.05 at each time.

FIG. 3

The rescue effect of NO can be blocked using the NO scavenger PTIO. Data were collected 72 and 96 h after γ irradiation. The effects of SNP shown at each time are consistent with the concept that NO donors can be used to rescue satellite cell proliferation after irradiation. However, to provide further evidence of the importance of NO, we used the NO scavenger PTIO to blunt/block the rescue effect of SNP. At each time and each dose of γ radiation, PTIO was highly effective in blocking the rescue effect of SNP. A PTIO-only treated group was also included at each time as a negative control, and the mean value was only 2% greater than the control cell counts (data not shown). All results are reported as means ± SE. Data for each time and each dose of γ radiation were analyzed using two-way ANOVA. The interaction effect (SNP × PTIO main effect) was highly significant (P = 5 0.001) at each dose and time.

FIG. 4

Effects of mechanical stretch on satellite cell proliferation after γ irradiation. Satellite cells exposed to 5 Gy were subjected to a cyclic stretch over 24 h. Assessment of NO levels and proliferation showed that mechanical stretch resulted in significantly higher levels of NO (P < 0.01) and increased numbers of cells (P < 0.01) compared to unstretched controls. Cells were assayed in triplicate, and results are shown as means ± SE.

FIG. 5

Effects of 2 Gy γ radiation and SNP on expression of MyoD and myogenin. All results are expressed relative to the null condition (0 Gy and 0 μmM SNP; horizontal dashed line). SNP did not affect either MyoD or myogenin levels. A dose of 2 Gy produced significant increases in MyoD (P < 0.01) and myogenin levels (P < 0.01). This radiation effect on these myogenic regulatory factors was consistently suppressed when irradiated cells were treated with SNP. All assays were run in triplicate. All results are reported as means ± SE.