Sex differences in left-ventricular strain in a murine model of coxsackievirus B3 myocarditis

Figures

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  Figure 1. Males develop worse myocarditis compared to females (A) Myocarditis severity (Inflammation %) between male (M) and female (F) uninfected controls (C, n = 37) and myocarditis (M, n = 47) (unpaired t-test; violin plots depict mean and interquartile range); (B) Myocarditis severity by sex: F-C (n = 17), F-M (n = 23), M-C (n = 20), M-M (n = 22) (one-way ANOVA with Tukey’s multiple comparison test; scatterplots show mean ± SEM); (C) Representative heart sections (scale bars, 2 mm and 200 μm for low and high-power images, respectively). A, B ∗∗∗∗p < 0.0001.

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  Figure 2. Proinflammatory markers are increased in male hearts during myocarditis (A) Percent positive staining of mouse myocardium controls (F-CON n = 17, M-CON n = 19) and myocarditis (F-M n = 25, M-M n = 22; bar graphs show mean ± SEM) with (B) representative images for CD45, CD11b, F480, and CD3 (scale bars, 200 μm). One-way ANOVA with Tukey’s multiple comparison tests for each immune marker. (A) ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Figure 3. Males have higher heart weight than females during myocarditis (A) Body weight of mice at baseline (B, n = 50) compared to myocarditis (M, n = 48) (violin plots depict mean and interquartile range); (B) scatterplots show change in body weight for F-B (n = 25), M-B (n = 25), F-M (n = 25) and M-M (n = 23); C, heart weight (HW); (D) HW/BW ratio (arbitrary units = a.u.); (E) HW/tibia length (HW/TL) (scatterplots show mean ± SEM); (A–E) ∗∗∗∗p < 0.0001.

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  Figure 4. Traditional 2D M-mode shows no difference in LVEF during myocarditis (A–O) Standard LV motion (M)-mode parameters comparing female and male mice at baseline (B, n = 44) compared to myocarditis (M, n = 42) (unpaired t-test; violin plots depict mean and interquartile range); (P) representative short-axis (SAX) and M-mode images at baseline and during myocarditis. Abbreviations: CO, cardiac output; FS, fractional shortening; LVAW; d, LV anterior wall thickness at diastole; LVAW; s, LV anterior wall thickness at systole; LVEDD, LV end diastolic diameter; LVEDV, LV end diastolic volume; LV, left ventricular; LVEF, LV ejection fraction; LVESD, LV end systolic diameter; LVESV, LV end systolic volume; LVPW; d, LV posterior wall thickness at diastole; LVPW; s, LV posterior wall thickness at systole; SV, stroke volume. (A–O) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001.

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  Figure 5. 4D mode LV parameters reveal worse myocarditis but no sex differences (A–F) LV functional parameters from 4D mode compare mice at baseline (B, n = 21) to myocarditis (M, n = 20) (unpaired t-test; violin plots depict mean and interquartile range); (G–L) females (F-B) vs. males (M-B) at baseline (Mann-Whitney; scatterplots show mean ± SEM); (M–R) females (F-M) vs. males (M-M) during myocarditis (Mann-Whitney; scatterplots show mean ± SEM). Abbreviations: CO, cardiac output; dV/dT = rate of change of LV volume; LV, left ventricular; LVEDV, LV end diastolic volume; LVEF, LV ejection fraction; LVESV, LV end systolic volume; SV, stroke volume. (A–R) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Figure 6. 4D strain software reveals decreased cardiac function during myocarditis in males (A–E) LV functional parameters from 4D strain software analysis compare baseline (B, n = 8) and myocarditis (M, n = 10) (Mann-Whitney); (F–J) compare females (F-C, n = 3) vs. males (M-B, n = 5) at baseline (Mann-Whitney); (K–O) compare females (F-M, n = 5) vs. males (M-M, n = 5) during myocarditis (Mann-Whitney). Abbreviations: LV, left ventricular; LVEDV, LV end diastolic volume; LVEF, LV ejection fraction; LVESV, LV end systolic volume; SV, stroke volume (scatterplots show mean ± SEM). (A–O) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

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  Figure 7. 4D GCS reveals decreased cardiac function during myocarditis in males (A–D) GCS and CS compare baseline (B, n = 8) vs. myocarditis (M, n = 10) (Mann-Whitney); (F–I) compare females (F-C, n = 3) vs. males (M-B, n = 5) at baseline (Mann-Whitney); (J–M) compare females (F-M, n = 5) vs. males (M-M, n = 5) during myocarditis (Mann-Whitney) (scatterplots show mean ± SEM), (E) representative images show transmural strain with bullseye plots and 3D renderings at peak strain for baseline (BASE) and during myocarditis (MYO). Abbreviations: ANT, anterior wall; CS, regional circumferential strain; FW, free wall; GCS, global circumferential strain; SW, septal wall. (A–M) ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Figure 8. 2D LAX strain reveals worse GLS in myocarditis in males (A–G) LV functional parameters from 2D LAX strain analysis compare baseline (B, n = 43) and myocarditis (M, n = 37) (unpaired t-test; violin plots depict mean and interquartile range); (H–N) Compare females (F-B, n = 22) vs. males (M-B, n = 21) at baseline (Mann-Whitney); (O–U) Compare females (F-M, n = 17) vs. males (M-M, n = 20) during myocarditis (Mann-Whitney) (scatterplots show mean ± SEM). Abbreviations: CO, cardiac output; FS, fractional shortening; GLS, global longitudinal strain; LV, left ventricular; LVEDV, LV end diastolic volume, LVEF, LV ejection fraction; LVESV, LV end systolic volume; SV, stroke volume. (A–U) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

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  Figure 9. Representative 2D long axis strain images Representative images showing endocardial global longitudinal strain (GLS) (mean ± 95% confidence intervals) among 3 contraction cycles of female and male mice at baseline and during myocarditis.

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  Figure 10. GLS correlates to CD11b and CD3 inflammation Global longitudinal strain (GLS) correlation to (A) H&E score, or immunohistochemistry for (B) CD45; (C) CD11b; (D) F4/80; or (E) CD3 during myocarditis. Black, females and males, pink, females only, turquoise, males only. Data are represented as linear regressions with one-way Pearson correlations.

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  Figure 11. Greater reduction in cardiac function in males during myocarditis (A) Sex differences in stroke volume as measured by 2D GLS software and 4D mode (two-way ANOVA mixed effects model with Tukey’s multiple comparisons) (B) Sex differences in cardiac output as measured by 2D GLS software and 4D mode (two-way ANOVA mixed effects model with Tukey’s multiple comparisons; scatterplots show mean ± SEM). Abbreviations: CO; cardiac output, GLS; global longitudinal strain, SV; stroke volume. (A–B) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.