Abstract Body (Do not enter title and authors here): Introduction:
Exposure of the heart to ionizing radiation (IR) leads to myocardial damage. Our group has previously shown that cardiac targeted irradiation (20 Gy; CTI) caused left ventricular (LV) dilation in male but not female wild-type (WT) mice monitored for 9 months. Additionally, sex-dependent changes in mitochondrial complex I and II activities were observed. Furthermore, CTI in Nitric Oxide Synthase 1 haploinsufficient (Nos1^+/-) mice demonstrated decreased LV size, ejection fraction (EF), and PR intervals, without sex differences. In this study, we analyzed the expression of 3-nitrotyrosine (3-NT), a protein nitrosylation byproduct of nitric oxide (NO), to evaluate the role of NO in CTI-induced cardiac injury.

Methods:
10-week-old WT C57B6 male and female mice were exposed to a single dose of CTI (20 Gy) or sham irradiation, monitored to 9 months, and euthanized. Hearts were zinc-formalin fixed, embedded in paraffin blocks, and cut parallel to the long axis to provide transmural sections of the right ventricular (RV) free wall and the interventricular septum. Tissues were stained for immunohistochemistry using an antibody to 3-NT (06-284; MilliporeSigma; Burlington, MA) with DAPI counterstaining of the nuclei. Images of each heart were analyzed using Olympus Image Viewer (OlyVIA) software. 3-NT expression was quantified using ImageJ by region [1, RV epicardium; 2, RV mid-myocardium; 3, RV free wall & septal endocardium; 4, septal mid-myocardium; 5, LV endocardium] and normalized against region 3.

Results:
Decreased expression of 3-NT was observed in the mid-myocardial region of the septum (Region 4) relative to the endocardium (Region 3) in sham mice (p ≤ .01; Figure). Additionally, marked decreases in 3-NT expression in the mid-myocardial regions of both the RV (Region 2) and septum (Region 4) compared to the endocardium (Region 3) occurred in mice exposed to CTI (p ≤ .001). These findings were similar in the hearts of both male and female mice.

Conclusions:
CTI exacerbates the decrease in nitrosylated protein expression in the mid-myocardium of the mouse RV and septum, the regions of the heart responsible for pressure development and subjected to maximal oxidative stress. These findings suggest that changes in NO expression may play a role in sex-dependent chronic cardiac damage following CTI.