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. 2015 Jun;183(6):610-9.
doi: 10.1667/RR13979.1. Epub 2015 May 26.

Effects of Local Heart Irradiation in a Glutathione S-Transferase Alpha 4-Null Mouse Model

Marjan Boerma  1 , Preeti Singh  2 , Vijayalakshmi Sridharan  1 , Preeti Tripathi  1 , Sunil Sharma  3 , Sharda P Singh  2
Affiliations

Effects of Local Heart Irradiation in a Glutathione S-Transferase Alpha 4-Null Mouse Model

Marjan Boerma et al. Radiat Res. 2015 Jun.

Abstract

Glutathione S-transferase alpha 4 (GSTA4-4) is one of the enzymes responsible for the removal of 4-hydroxynonenal (4-HNE), an electrophilic product of lipid peroxidation in cellular membranes during oxidative stress. 4-HNE is a direct activator of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor with many target genes encoding antioxidant and anti-electrophile enzymes. We have previously shown that Gsta4-null mice on a 129/Sv background exhibited increased activity of Nrf2 in the heart. Here we examined the sensitivity of this Gsta4-null mouse model towards cardiac function and structure loss due to local heart irradiation. Male Gsta4-null and wild-type mice were exposed to a single X-ray dose of 18 Gy to the heart. Six months after irradiation, immunohistochemical staining for respiratory complexes 2 and 5 indicated that radiation exposure had caused most pronounced alterations in mitochondrial morphology in Gsta4-null mice. On the other hand, wild-type mice showed a decline in cardiac function and an increase in plasma levels of troponin-I, while no such changes were observed in Gsta4-null mice. Radiation-induced Nrf2-target gene expression only in Gsta4-null mice. In conclusion, although loss of GSTA4-4 led to enhanced susceptibility of cardiac mitochondria to radiation-induced loss of morphology, cardiac function was preserved in Gsta4-null mice. We propose that this protection against cardiac function loss may occur, at least in part, by upregulation of the Nrf2 pathway.

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Figures

FIG. 1
FIG. 1
Echocardiographic parameters and plasma troponin I (cTnI) levels measured 6 months after local heart irradiation. Exposure to radiation caused a significant reduction in stroke volume (panel A), fractional shortening (panel B) and cardiac output (panel C), together with a significant increase in plasma cTnI levels (panel D) only in wild-type mice. Average ± SEM, n = 5–8. *P < 0.05 when compared to wild-type sham-irradiated mice.
FIG. 2
FIG. 2
Histological parameters at 6 months after local heart irradiation. Panel A: Irradiation significantly reduced the capillary density in wild-type mice. Panel B: Cardiac mast cell numbers were significantly reduced only in irradiated Gsta4-null mice. Average ± SEM, n = 5–8. *P < 0.05 when compared to wild-type sham-irradiated mice. ϕP < 0.05 when compared to Gsta4-null sham-irradiated mice.
FIG. 3
FIG. 3
Cardiac markers of apoptosis determined 6 months after local heart irradiation. Panel A: Representative result of caspase-3 Western blot analysis. Row 1 shows caspase-3; row 2 shows cleaved caspase-3; and row 3 shows GAPDH loading control. In 3 out of 5 irradiated Gsta4-null mice, an increase was observed in left ventricular levels of cleaved caspase-3. Panel B: Numbers of apoptotic nuclei per heart section, determined from CardioTACS™ staining. Reduced numbers of apoptotic cells were seen in both sham-irradiated and irradiated Gsta4-null mice when compared to wild-type mice. Average ± SEM, n = 4–5. *P < 0.05 when compared to wild-type sham-irradiated mice.
FIG. 4
FIG. 4
Left ventricular protein expression of SOD2 and Gpx1. Increased mRNA levels of the Nrf2 target genes Sod2 (panel A) and Gpx1 (panel B) in irradiated Gsta4-null mice (Table 1) coincided with increased left ventricular protein expression in these mice. Average ± SEM, n = 4–5. *P < 0.05 when compared to wild-type sham-irradiated mice. ϕP < 0.05 when compared to Gsta4-null sham irradiated. ΔP < 0.05 when compared to wild-type irradiated mice.
FIG. 5
FIG. 5
Cardiac tissue expression patterns of mitochondrial respiratory complexes 2 and 5 at 6 months after local heart irradiation. Representative images of complexes 2 and 5 immunohistochemistry of the left ventricles and atria of sham-irradiated and irradiated Gsta4-null and wild-type mice. Staining patterns were reduced in irradiated Gsta4-null mice. Scale bars indicate 100 μm.
FIG. 6
FIG. 6
Oxygen flux measured in left ventricular tissue samples isolated at 6 months after local heart irradiation. In sham-irradiated mice, oxygraph measurements revealed a significantly higher oxygen flux through complexes 1, 2 and 3 in Gsta4-null compared to wild-type mice. Although exposure to radiation caused a reduction in oxygen flux through complexes 2 and 3 in Gsta4-null mice, the oxygen flux was still significantly higher compared to that in irradiated wild-type mice. Average ± SEM, n = 4. *P < 0.05 when compared to wild-type sham-irradiated mice. ϕP < 0.05 when compared to Gsta4-null sham-irradiated mice. ΔP < 0.05 when compared to wild-type irradiated mice.
FIG. 7
FIG. 7
Mouse embryonic fibroblast survival in response to irradiation in culture. A clonogenic assay revealed a significantly higher postirradiation survival in mouse embryonic fibroblasts isolated from Gsta4-null mice compared to those isolated from wild-type mice. Average ± SEM, n = 3. ΔP < 0.05 when compared to wild-type mice.
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