Lifetime Evaluation of Left Ventricular Structure and Function in Male C57BL/6J Mice after Gamma and Space-Type Radiation Exposure

Figure 1. Physiological response of C57BL/6J male mice to gamma and simGCRsim-IR. (A) Three-month-old age-matched C57BL/6J wild-type mice were irradiated with 137Cs-γ-IR (100, 200 cGy) and simGCRsim (50, 100 cGy). We assessed LV function via transthoracic echocardiography at 14, 28, 365, 440, and 660 days post IR. No-IR normal diet (ND)-fed mice served as negative control. In addition to transthoracic echocardiography, blood and hearts were collected at each time point. Created with BioRender.com. (B) Heart rates were obtained during transthoracic echocardiography, while mice were anesthetized using isoflurane (3–4% induction, 1–2% maintenance). (C) Animal bodyweights. (D) Heart weights post exsanguination. For acute time points (14 and 28 days), n = 3–10 animals (50 cGy simGCRsim and 200 cGy gamma-IR at 14 days, n = 3; the rest of the samples for 14 and 28 days, n = 5–10). For the degenerative time points (365, 440, and 660 days), n= 10–15 animals. p-values were calculated using two-way ANOVA, ** p < 0.01, * p < 0.05. Note, due to technical reasons, heart weights were not collected at the 14-day timepoint.

Figure 2. Longitudinal echocardiography results in C57BL/6J male mice at 14 and 28 days after gamma (γ) and simGCRsim irradiation. Three-month-old male C57BL/6J mice were irradiated with simGCRsim-IR (50 and 100 cGy) or gamma (γ)-IR (100 and 200 cGy). Left ventricular (LV) function is represented by (A) ejection fraction, (B) fractional shortening, (C) stroke volume (SV), and (D) radial wall strain (DWS). Parameters of LV dimensions and remodeling are represented by (E) LV end diastolic diameter, (F) intraventricular septal thickness, (G) LV posterior wall thickness, (H) LV internal cavity diameter, (I) relative wall thickness, and (J) LV mass. No-IR normal-diet (ND)-fed mice served as controls. n = 3–10 mice per treatment condition and time point (50 cGy simGCRsim and 200 cGy gamma-IR at 14 days, n = 3; the rest of samples for 14 and 28 days, n = 5–10); p-values were calculated using one-way ANOVA. ** p < 0.01, * p < 0.05.

Figure 3. Longitudinal echocardiography results in C57BL/6J male mice at 365, 440, and 660 days after gamma (γ) and simGCRsim irradiation. Left ventricular (LV) function is represented by (A) ejection fraction, (B) fractional shortening, (C) stroke volume (SV), and (D) radial wall strain (DWS). Parameters of LV dimensions and remodeling are represented by (E) LV end diastolic diameter, (F) intraventricular septal thickness, (G) LV posterior wall thickness, (H) LV internal cavity diameter, (I) relative wall thickness, and (J) LV mass. n = 10–15 mice per treatment condition and time point; p-values were calculated using one-way ANOVA. **** p < 0.0001, ** p < 0.01, * p < 0.05.

Figure 4. Myocardial fibrosis at 660 days post IR. Transverse sections of hearts at basal and midventricular levels (four heart tissue sections taken 80 µm apart from each other) were stained with Masson’s Trichrome to assess fibrotic burden. (A) Representative images of heart sections from three different animals show fibrosis, highlighted in bright blue, in contrast with dark, red-stained myocardium. Scale 100 µm, 200× magnification. (B) Fibrosis burden in heart tissue was quantified using the ImageJ program, with the fraction of the blue stained area being normalized to the total red area. n = 3 mice per treatment condition; p-values were calculated using one-way ANOVA. *** p < 0.001.

Figure 5. Gene expression in left ventricular tissue was assessed 660 days after simGCRsim and gamma (γ) irradiation. Relative mRNA expression of (A) cardiac fibrosis (Tgfβ1, Col1a1, and Col3a1), (B) calcium handling (Ncx, and Serca2a), (C) inflammation (TNFα, and MCP1), (D) cardiac remodeling (Mmp9, Gals3, and βmhc), and (E) hemodynamic stress (Anp, and Bnp) markers within the left ventricle (LV) of mice irradiated with 50 cGy simGCRsim or 100 cGy γ at 660 days post-irradiation. Tgfβ1 indicates transforming growth factor beta 1; Col1a1, type I fibrillar collagen; Col3a, type III fibrillar collagen; Ncx, cardiac sodium-calcium exchanger; Serca2a, Sarcoplasmic Reticulum Ca2+ ATPase; Tnfα, tumor necrosis factor alpha; Mcp1, monocyte chemoattractant protein-1; Mmp9, matrix metalloproteinase-9; Gals3, galectin 3; βmhc, cardiac beta myosin heavy chain; Anp, atrial natriuretic peptide; Bnp, brain natriuretic peptide. Each dot represents an individual mouse. Expression was normalized to GAPDH. Data reported as mean ± SEM. n = 5 animals/treatment group; p-values were calculated using Kruskal-Wallis H test, ** p < 0.01, * p < 0.05.

Figure 6. Select cardiac serum markers assessed 660 days after gamma and simGCRsim irradiation. Peripheral blood serum was processed using standard ELISA to assess serum levels of (A) brain natriuretic peptide (BNP). Remaining samples were used for Multiplex assay to assess markers of (B) cardiac remodeling: plasminogen activator inhibitor-1 (PAI-1) and pro-matrix metalloprotease-9 (proMMP-9), (C) inflammation: Monocyte Chemoattractant Protein-1 (MCP-1), (D) Thrombosis: thrombomodulin, and (E) endothelial dysfunction: sE-Selectin, sP-Selectin, sICAM, and PECAM-1. All values are reported as pg/mL as mean ± SEM. n = 5 animals/treatment group. p-values were calculated using Kruskal-Wallis H test.

Figure 7. Modeling and comparative effect measures of C57Bl/6J mice 365 days after exposure to gamma and simGCRsim irradiation. Comparison of (A) LVEF, (D) LVFS, and (G) DWS mean dose-response data (SEM error bars) superimposed on BMD dose-response models for gamma (orange triangles) and simGCRsim (blue circles) irradiation. To the right are the corresponding RBE calculations within the viable ranges (B,E,H), and the corresponding RER calculation (C,F,I) for LVEF, LVFS, and DWS, respectively.