Intrauterine inflammation exacerbates maladaptive remodeling of the immature myocardium after preterm birth in lambs

Figures

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  Fig. 1. Myocardial collagen content. Levels of interstitial myocardial collagen in the left ventricle + septum (LV + S) and right ventricle (RV) of postconceptional age-matched fetal lambs (gray circles, fetal control, n = 7) and 7-day-old preterm lambs exposed antenatally to saline (blue squares, Preterm SAL, n = 9) or LPS (orange triangles, preterm LPS, n = 9). Statistical analyses were performed using a one-way ANOVA followed by a Bonferroni post hoc test. Data are presented as mean ± SD, ** denotes p < 0.01, *** denotes p < 0.001.

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  Fig. 2. CD45+ immunostaining for myocardial immune cell infiltration. Representative images of immunohistochemical labeled CD45+ cells in the left ventricle + septum (LV + S; a–c) and right ventricle (RV; d–f) from postconceptional aged-matched fetal lambs (fetal control, n = 7) and preterm lambs exposed antenatally to saline (preterm SAL, n = 9) or LPS (preterm LPS, n = 9). Scale bar = 100 μm. Graphs show immune cell infiltration of the LV + S (g) and RV (h) in each experimental group (gray circles = fetal control; blue squares = preterm SAL; orange triangles = preterm LPS). Statistical analyses were performed using a one-way ANOVA followed by Bonferroni post hoc test. Data are presented as mean ± SD, ** denotes p < 0.01, *** denotes p < 0.001.

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  Fig. 3. Ki67 immunostaining of proliferating cardiomyocytes. Representative images of Ki67 immunohistochemical staining in the left ventricle + septum (LV + S; a–c) and right ventricle (RV; d–f) from aged-matched fetal lambs (fetal control, n = 7) and preterm lambs exposed antenatally to saline (preterm SAL, n = 9) or LPS (preterm LPS, n = 9). Scale bar = 100 μm. Graphs show the proportion of Ki67 positively-labeled cardiomyocytes in the LV + S (g) and RV (h) of each experimental group (gray circles = fetal control; blue squares = preterm SAL; orange triangles = preterm LPS). Statistical analyses were performed using a one-way ANOVA followed by a Bonferroni post hoc test. Data are presented as mean ± SD, * denotes p < 0.05, *** denotes p < 0.001.

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  Fig. 4. Cardiomyocyte endowment. Total number of cardiomyocytes in the left ventricle + septum (LV + S; a) and right ventricle (RV; b) of postconceptional age-matched fetal lambs (gray circles, fetal control, n = 7) and 7-day-old preterm lambs exposed antenatally to saline (blue squares, preterm SAL, n = 9) or LPS (orange triangles, preterm LPS, n = 9). Statistical analyses were performed using a one-way ANOVA followed by a Bonferroni post hoc test. Data are presented as mean ± SD, ** denotes p < 0.01, *** denotes p < 0.001.

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  Fig. 5. Changes in mRNA expression within the left ventricular myocardium. Genes involved in cardiac metabolism (a, b), calcium handling (c, d), and inflammation/ cardiac remodeling (e) were differentially expressed in the left ventricle + septum from aged-matched fetal lambs (gray circles, fetal control, n = 7) and preterm lambs exposed antenatally to saline (blue squares, preterm SAL, n = 9) or LPS (orange triangles, preterm LPS, n = 9). Expression of all genes was represented as the calibrated normalized relative quantity (CNRQ); normalized to YWHAZ and expressed relative to the fetal control group normalized at 1.00. Statistical analyses were performed using a one-way ANOVA followed by Tukey–Kramer post hoc test. Data are presented as mean ± SD, * denotes p < 0.05.

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  Fig. 6. Changes in relative mRNA expression within the right ventricular myocardium. Genes involved in cardiac metabolism (a), calcium handling (b–d), and inflammation/ cardiac remodeling (e, f) were differentially expressed in the right ventricle from aged-matched fetal lambs (gray circles, fetal control, n = 7) and preterm lambs exposed antenatally to saline (blue squares, preterm SAL, n = 9) or LPS (orange triangles, preterm LPS, n = 9). Expression of all genes was represented as the calibrated normalized relative quantity (CNRQ); normalized to YWHAZ and expressed relative to the fetal control group normalized at 1.00. Statistical analyses were performed using a one-way ANOVA followed by Tukey–Kramer post hoc test. Data are presented as mean ± SD, * denotes p < 0.05.