Sex differences in a mouse model of radiation-induced cardiomyopathy

Figures

• 

  Fig. 1. Study plan and dosimetric protocol. (a) Experimental timeline for male and female BALB/c mice. (b) A representative plan for whole heart radiation using 22 Gy for female (left) and male (right) BALB/c mice showing the axial section. Females and males were analyzed in separate experiments using sex-specific dosimetric plans to account for differences in heart size between sexes. The heart (purple for females, green for males) was irradiated using a parallel opposed method in the axillary region (beams are 180 degrees apart and parallel to each other so that the beam converges to the isocenter). Mice were placed in a feet-first prone position in relation to the gantry which was equipped with a 5-millimeter circular collimator (C5). The isocenter of radiation is aimed medial to the left and right ventricles and superior to the apex of the heart. (c) Representative image of the dose-volume histogram (DVH) showing the percentage of heart (purple for females, green for males) and lung (pink for females, blue for males) receiving 22 Gy radiation

• 

  Fig. 2. Sex differences in dsDNA breaks at day 10 post radiation. Male and female BALB/c mice were irradiated with sex-specific dosimetric plans and IHC performed on hearts to measure dsDNA breaks using antibodies against γH2A.x. (a) Representative images of γH2A.x IHC staining of 0 and 22 Gy (bars = 200 μm). Positive staining is brown on a blue background (arrows). dsDNA breaks were quantified at day 10 from γH2A.x IHC and shown as the number of strong positives (NSP) normalized to the total area (TA) for female and male mice (b) combined or (c) separated by sex that received 0 Gy (0Gy) vs. 22 Gy (22Gy) (n = 8–10/group). b) Data shown as the mean ± SEM for n = 8–10 mice/group using an unpaired Mann-Whitney rank test. c) 2-way ANOVA, radiation p < 0.001, sex p < 0.001, interaction p = 0.001, p values obtained from Sidak’s multiple comparisons test

• 

  Fig. 3. Radiation induces vascular damage at day 10 and 35 in females and males. Male and female BALB/c mice were irradiated with sex-specific dosimetric plans and vessel size determined from Masson’s trichrome stained slides. a) Representative photos of vessels for females (pink) and males (blue) are shown using Masson’s trichrome stain. Green lines indicate inner and outer vessel tracings used to determine vessel area while 4 lines were used/vessel to determine vessel wall thickness (VWT). Female and male BALB/c mice received 0 Gy (0Gy) or 22 Gy (22Gy) at day 0 and were examined at b-e) day 10 (d10) and f-i) day 35 (d35) after irradiation for vascular damage. Vessel area and VWT are shown for males and females combined (b, d, f, h) or separated by sex (c, e, g, i). Data show the mean ± SEM for 8–10 mice/group. b, d, f, h) P values obtained using unpaired Mann-Whitney rank tests. c, e, g, i) A two-way ANOVA was used to assess the interaction between radiation and sex, with p values obtained using Sidak’s multiple-comparisons test

• 

  Fig. 4. Irradiated males gain less body weight by day 10, with similar weight gain to controls by day 35 and no change in heart weight vs. controls at day 35. Mice received 0 Gy (0Gy) vs. 22 Gy (22Gy) irradiation at day 0 and were assessed at a-c) day 10 (d10) and d-f) day 35 (d35) for body weight, change in body weight, and heart weight (HW) to tibia length (TL). Data shown as mean ± SEM for 8–10 mice/group. A two-way ANOVA was used to assess the interaction between radiation and sex, with p values obtained using Sidak’s multiple-comparisons test

• 

  Fig. 5. Males develop DCM by day 35. The cardiac function of female and male BALB/c mice was assessed at day 0 (baseline, 0Gy/d0) and day 35 after receiving 22 Gy (22Gy/d35) for the following parameters in separate experiments for females and males: (a) left ventricular (LV) ejection fraction (LVEF), (b) LV end systolic volume (LVESV) and diameter (LVESD), (c) fractional shortening (FS), (d) LV end diastolic volume (LVEDV) and diameter (LVEDD), (e) representative M-mode images of myocardium illustrating regions of the heart and where measurements were obtained showing 0 and 22 Gy females and males. (f) Average global longitudinal strain (GLS) measurements (left) and (g) representative images of GLS desynchrony using speckle tracking (blue lines) of 48 locations of the endocardial wall inside of the LV over three cardiac cycles in females (top) and males (bottom) are shown at day 35. a-d,f) Data show the mean ± SEM for n = 8–13 mice/group, p values obtained using Wilcoxon matched-pairs signed rank test. Abbreviations: FS, fractional shortening; GLS, global longitudinal strain; LVAW, left ventricular anterior wall; LV, left ventricular; LVEDD, LV end-diastolic diameter; LVEDV, LV…

• 

  Fig. 6. Radiation induces cardiac inflammation at day 35 in males. (a) In separate experiments female (pink) and male (blue) BALB/c mice received 0 (0Gy) or 22 (22Gy) Gy on day 0 and hearts examined at 24 h (24H), day 10 (d10) or day 35 (d35) after irradiation by qPCR for relative gene expression (RGE) of CD45, a common leukocyte marker, normalized to the housekeeping gene Hprt. (b) Representative H&E histology are shown for a female and male heart at day 35 after irradiation (bar = 60 μm). Arrow indicates inflammation. (c) Cardiac inflammation at day 35 after irradiation was quantified using a microscope eye-piece grid normalized to the size of the heart. (d) Hearts at day 35 after irradiation were analyzed by qPCR for mRNA expression of immune-cell-associated markers: CD45 (leukocytes), CD11b (myeloid cells and some mast cells), F4/80 (macrophage-associated), CD4 (T helper-cell-associated), and cKit (mast-cell-associated). a, c, d) Data shown as mean ± SEM for 5–10 mice/group. c, d) 2-way ANOVA, p values obtained using Sidak’s multiple comparisons test

• 

  Fig. 7. Radiation induces remodeling and fibrosis at day 35 in males and cardiac cytokine changes at day 10. Female and male BALB/c mice received 0 Gy (0Gy) or 22 Gy (22Gy) at day 0 and were examined at day 35 (d35) after irradiation for fibrosis and at day 10 (d10) for cardiac cytokine levels by ELISA. (a) Representative images of collagen deposition in the myocardium using Masson’s trichrome stain (Bar = 70 μm). (b) Quantification of fibrosis from Masson’s trichrome stained hearts using a microscope eye-piece grid normalized to the size of the heart. (c) Representative images of perivascular fibrosis from Masson’s trichrome slides (Bar = 50 μm). Pink (female, left) and blue (male, right) illustrate how the perivascular area of fibrosis was calculated. Quantification of perivascular fibrosis for (d) males and females together and (e) individually are shown. (f) ELISA of IL-1β levels in the heart at day 10 after radiation normalized to heart weight. Data shows the mean ± SEM for 8–10 mice/group. d) P values obtained using unpaired Mann-Whitney rank tests. b, e, f) 2-way ANOVA, p values obtained using Sidak’s multiple comparisons test

• 

  Fig. 8. Characterization of gene changes in the heart of irradiated females and males vs. controls at day 10. Female and male BALB/c mice received 0 Gy (0Gy) or 22 Gy (22Gy) at day 0 and bulk RNA sequencing was performed on the heart at day 10 after irradiation in separate experiments (n = 8–9/group). Heatmap, principal component analysis (PCA) and volcano plots of females a-c) and males d-f) at day 10 after irradiation. A p value of 0.01 was used as the cutoff for genes shown in the volcano plots for c) females 857 down- and 762 up-regulated genes and f) males 411 down- and 486 up-regulated genes

• 

  Fig. 9. Weighted gene co-expression network analysis reveals radiation-induced mitochondrial dysfunction in females and males at day 10, but upregulation of Pgc1α, master regulator of mitochondrial function, in females. Female (a-d) and male (e-h) BALB/c mice received 0 Gy (0Gy) or 22 Gy (22Gy) at day 0 and bulk RNA sequencing was performed on the heart at day 10 after irradiation in separate experiments (n = 8–9/group). a, e) Module color assignments from high dimensional Weighted Gene Co-Expression Network Analysis comparing 0 to 22 Gy by sex revealed the top 12 up- (red) or down- (blue) regulated genes. Significantly down-regulated gene modules that related to mitochondrial function for b) females (yellow module, correlation = 0.56, p = 8.3 e-12, n = 126 genes) and f) males (pink module, correlation = 0.54, p = 5.3 e-49, n = 644 genes). Modules represent clusters of co-expressed genes associated with distinct transcriptional programs. Top 10 significant changed gene ontology (GO) pathways related to mitochondrial function in the c) yellow module in females and g) the pink module in males with oxidative phosphorylation and aerobic respiration being the top two GO pathways for yellow…

• 

  Fig. 10. Graphical representation illustrating proposed mechanisms of sex differences in radiation-induced cardiomyopathy. Radiation directed to the heart leads to dsDNA breaks, mitochondrial dysfunction, vacuolation and vessel damage by day 10 after radiation in both males and females. Mitochondrial damage is known to upregulate TLR4 which leads to IL-1β production in the heart of males. Females may repair mitochondrial dysfunction by upregulating the master regulator of mitochondrial function, Pgc1α. Female hearts have less cardiac profibrotic cytokine IL-1β, less fibrosis and less chronic cardiac dysfunction than males. In contrast, males do not upregulate Pgc1α in response to mitochondrial damage. Instead, males have higher acute IL-1β that leads to fibrosis and iDCM after radiation