Effects of low-dose oxygen ions and protons on cardiac function and structure in male C57BL/6J mice

Abstract

Purpose: Astronauts traveling beyond low-Earth orbit will be exposed to high linear-energy transfer charged particles. Because there is concern about the adverse effects of space radiation on the cardiovascular system, this study assessed cardiac function and structure and immune cell infiltration in a mouse model of charged-particle irradiation.

Materials and methods: Male C57BL/6 J mice were exposed to oxygen ions (¹⁶O, 600 MeV/n at 0.25-0.26 Gy/min to a total dose of 0, 0.05, 0.1, 0.25, or 1 Gy), protons (150 MeV, 0.35-0.55 Gy/min to 0, 0.5, or 1 Gy), or protons (150 MeV, 0.5 Gy) followed by ¹⁶O (600 MeV/n, 0.1 Gy). Separate groups of mice received ¹³⁷Cs γ-rays (1 Gy/min to 0, 0.5, 1, or 3 Gy) as a reference. Cardiac function and blood velocity were measured with ultrasonography at 3, 5, 7, and 9 months after irradiation. At 2 weeks, 3 months, and 9 months, cardiac tissue was collected to assess apoptosis, tissue remodeling, and markers of immune cells.

Results: Ejection fraction and fractional shortening decreased at 3 and 7 months after ¹⁶O. These parameters did not change in mice exposed to γ-rays, protons, or protons followed by ¹⁶O. Each of the radiation exposures caused only small increases in cleaved caspase-3 and numbers of apoptotic nuclei. Changes in the levels of α-smooth muscle cell actin and a 75-kDa peptide of collagen type III in the left ventricle suggested tissue remodeling, but there was no significant change in total collagen deposition at 2 weeks, 3 months, and 9 months. Increases in protein amounts of cluster of differentiation (CD)2, CD68, and CD45 as measured with immunoblots at 2 weeks, 3 months, and 9 months after exposure to protons or ¹⁶O alone suggested immune cell infiltration. For type III collagen, CD2 and CD68, the efficacy in inducing protein abundance of CD2, CD68, and CD45 was ¹⁶O > protons > γ-rays > protons followed by ¹⁶O.

Conclusions: Low-dose, high-energy charged-particle irradiation caused mild changes in cardiac function and tissue remodeling in the mouse.

Keywords: Cardiovascular system; Degenerative tissue effects; Immune cell infiltration; Mouse model; Space radiation.

Figure 1.. Bodyweight at time of sacrifice.

Bodyweight was measured after exposure to protons (A), ¹⁶O (0–1 Gy) (B), ¹⁶O (0 and 0.05 Gy) and protons (0.5 Gy) followed by ¹⁶O (0.1 Gy) (C), and after γ-rays (D). Graphs indicate average bodyweight and standard error of the mean (SEM); n = 9–15/group. *p<0.05 compared to time-matched sham controls.

Figure 2.. Effects of radiation on cleaved and full-length caspase-3 after exposure to ¹⁶O.

(A) Representative image of immunoblot for cleaved and full-length caspase-3 at 9 months after ¹⁶O irradiation. (B) Left ventricle protein levels of cleaved caspase-3 after exposure to ¹⁶O (0.1 – 1 Gy). (C) Left ventricle protein levels of full length caspase-3 after exposure to ¹⁶O (0.1 – 1 Gy). Left ventricular protein levels were normalized to GAPDH and then calculated relative to time-matched sham-irradiated controls (arbitrary units). Graphs indicate average values and SEM. *p<0.05 compared to time-matched sham controls. n = 6/group.

Figure 3.. Effects of radiation on apoptosis after exposure to protons or ¹⁶O.

Number of apoptotic nuclei per tissue area after exposure to ¹⁶O (A) or protons (B). n = 9–15/group. Graphs indicate average values and SEM. *p<0.05 compared to time-matched sham controls.

Figure 4.. Immunoblot analysis of type III collagen in left ventricle.

(A) Representative image of an immunoblot indicating a 75-kDa band recognized by the anti-collagen III antibody, in samples at 3 months after exposure to ¹⁶O. (B) – (E) Left ventricle protein levels of the type III collagen 75-kDa peptide. Left ventricular peptide levels were normalized to GAPDH and then calculated relative to time-matched sham-irradiated controls (arbitrary units). Graphs indicate average values and SEM. n = 5–6/group. *p<0.05 compared to time-matched sham controls.

Figure 5.. Effects of radiation on mast cell tryptase in the heart.

(A) Representative image of immunoblot for mast cell tryptase at 9 months after ¹⁶O exposure. (B) – (E) Mast-cell tryptase abundance in the left ventricle. Left ventricular protein levels were normalized to GAPDH and then calculated relative to time-matched sham-irradiated controls (arbitrary units). Graphs indicate average values and SEM. n = 5–6/group. *p<0.05 compared to time-matched sham controls.

Figure 6.. CD2 protein levels in the left ventricle.

(A) Representative image of immunoblot for CD2 at 3 months after ¹⁶O exposure. (B) CD2 abundance in the left ventricle. Left ventricular protein levels were normalized to GAPDH and then calculated relative to time-matched sham-irradiated controls (arbitrary units). Graphs indicate average values and SEM. n = 5–6/group. *p<0.05 compared to time-matched sham controls.

Figure 7.. CD68 protein levels in the left ventricle.

(A) Representative image of immunoblot for CD68 at 9 months after ¹⁶O exposure. (B) CD68 abundance in the left ventricle. Left ventricular protein levels were normalized to GAPDH and then calculated relative to time-matched sham-irradiated controls (arbitrary units). Graphs indicate average values and SEM. n = 5–6/group. *p<0.05 compared to time-matched sham controls.

Figure 8.. Effects of radiation on CD45-positive cells in the heart after ¹⁶O and γ-rays.

(A) Representative immunoblot of CD45 at 3 months after ¹⁶O. Please note that the 0 Gy samples had been loaded on the gel as the second group from the left, to the right of the 0.1 Gy samples. (B) Representative image of CD45 immunohistochemistry, 3 months after sham-irradiation, 40× magnification; Scale bar: 60 μm. (C) Representative image of CD45 immunohistochemistry, 3 months after ¹⁶O (0.25 Gy), 40× magnification; Scale bar: 60 μm. (D) Left ventricle protein levels of CD45 after ¹⁶O. (E) Number of CD45-positive cells per tissue area at 9 months after ¹⁶O. (F) Left ventricle protein levels of CD45 after γ-rays. (G) Number of CD45-positive cells per tissue area at 9 months after γ-rays. In immunoblotting, left ventricular protein levels were normalized to GAPDH and then calculated relative to time-matched sham-irradiated controls (arbitrary units). All graphs indicate average values and SEM. n = 6/group. *p<0.05 compared to time-matched sham controls.