Haploinsufficiency of Endothelial Nitric Oxide Synthase Mitigates Beneficial Effects of Maternal Exercise on Fetal Heart Development During Pregestational Diabetes

Figures

• 

  Figure 1. Effects of maternal exercise on blood glucose, body weight, litter size, offspring mortality rate, and congenital heart defects in the offspring of pregestational diabetes. A, Fasting blood glucose levels in eNOS+/− dams with or without PGD or exercise at E0.5 to 18.5. B, Maternal body weight changes during gestation in eNOS+/− dams with or without PGD or exercise. C, Running distance of eNOS+/+ and eNOS+/− dams with PGD during gestation. D, Litter size of eNOS+/− dams with or without PGD or exercise at E18.5. E, Mortality rate of fetuses of eNOS+/− dams with or without PGD /or exercise at E18.5. F, Number of CHD and healthy fetuses of eNOS+/− dams with or without PGD or exercise at E18.5. The numbers are a combination of both eNOS+/+ and eNOS+/− fetuses in each maternal condition. Data represent mean±SEM and were analyzed using 2‐way ANOVA followed by Tukey’s test (A–E). a indicates P<0.0001 for PGD dams (with or without exercise) vs eNOS+/− control dams (A). b indicates P<0.0001 for eNOS+/++PGD+exercise vs eNOS+/−+PGD+exercise dams (A). Fetal CHD numbers were analyzed using a χ2 test. +/− indicates heterozygous; CHD, congenital heart defect; E, embryonic day; eNOS, endothelial…

• 

  Figure 2. Representative images of cardiac septation defects, myocardial hypertrophy, outflow tract and valve defects in E18.5 eNOS+/+ and eNOS+/− offspring exposed to PGD and maternal exercise. A–C, Normal cardiac septation morphology from eNOS+/− control dams; D, Normal myocardial wall thickness; E, ASD type 1; F, Perimembranous VSD; G, AVSD with 1 common valve; H, Myocardial hypertrophy; I, Normal right ventricle and aorta morphology; J, K, Normal aorta morphology (tricuspid); L, Normal aorta connected to LV;, M, Aorta connected to RV as part of DORV (position more than 50% above RV); N, BAV; O, thickened aortic leaflets; P, thickened pulmonary valve leaflets. Scale bar is 500 μm in heart images and 100 μm in valve images. +/− indicates heterozygous; Ao indicates aorta; E, embryonic day; eNOS, endothelial nitric oxide synthase; EX, exercise; LA, left atrium; LCC, left coronary cusp; LV, left ventricle; NCC, noncoronary cusp; PA, pulmonary artery; PGD, pregestational diabetes; RA, right atrium; RCC, right coronary cusp; and RV, right ventricle.

• 

  Figure 3. Cardiac rupture and hemopericardium CHD observed in an E18.5 eNOS+/− offspring of an eNOS+/− diabetic dam and maternal exercise. A, Normal pericardium morphology represented by the arrow. B and D, Cardiac rupture at atrioventricular junction and blood‐filled pericardium (hemopericardium) represented by the arrow. Scale bar represents 500 μm. C and E are enlarged areas of (B) and (D), respectively, showing rupture sites and thrombus formation. Scale bar represents 100 μm. D and E are 25 μm lower than images in (B) and (C). CHD indicates coronary heart defect; E, embryonic day; eNOS, endothelial nitric oxide synthase; LA, left atrium; LV, left ventricle; RA, right atrium; and RV, right ventricle.

• 

  Figure 4. Craniofacial defect incidence and types observed in E18.5 eNOS+/+ and eNOS+/− exposed to eNOS+/− dams with PGD with and without maternal exercise. A, Incidence of craniofacial defects in offspring exposed to eNOS+/− control dams (n=44), eNOS+/−+PGD (n=42), eNOS+/−+PGD+exercise (n=48), and eNOS+/++PGD+exercise (n=36). eNOS+/− dams with PGD and maternal exercise produced offspring with the highest risk of craniofacial defect development. B, Normal facial and snout morphology. C, Cleft lip: failure of the skin to close over the mouth, Encephalocele: brain matter begins to grow outside the skull. D, The normal brain is encapsulated by meninges, skull and skin. E, Exencephaly: the brain is located entirely outside of the skull. There was no statistical significance by Fisher’s exact test (P=0.0714). +/− indicates heterozygous; E, embryonic day; eNOS, endothelial nitric oxide synthase; EX, exercise; and PGD, pregestational diabetes.

• 

  Figure 5. Maternal exercise in eNOS+/− dams with PGD improves coronary artery abundance in E18.5 eNOS+/+ and eNOS+/− hearts. A–D, Representative images of coronary arteries in E18.5 fetal hearts. Sections are immunostained with α‐SMA to label coronary arteries as indicated by arrows. D’ shows a coronary artery stained with α‐SMA in the RV free wall of the heart. E, Average number of coronary arteries counted in 3 sections for eNOS+/+ and eNOS+/− fetuses combined (n=10 hearts per group). F, Average coronary artery abundance in eNOS+/+ fetuses only (n=5 hearts per group). G, The average coronary artery abundance in eNOS+/− fetuses (n=5 hearts per group). Vessels were deemed coronary arteries with a diameter 15–60 μm. Data represent means±SEM and were analyzed using 1‐way ANOVA followed by Tukey’s test. +/− indicates heterozygous; α‐SMA, α‐smooth muscle actin; E, embryonic day; eNOS, endothelial nitric oxide synthase; EX, exercise; PGD, pregestational diabetes; and RV, right ventricle.

• 

  Figure 6. Maternal exercise in eNOS+/+ and eNOS+/− dams with PGD does not alter capillary density in E18.5 eNOS+/+ and eNOS+/− hearts. A–D, Representative images of ventricular capillaries at the 4‐chamber level of E18.5 fetal hearts. Sections are immunostained with lectin‐1 to label endothelial cells. E, Average number of capillaries counted in 3 sections per ventricular structure for eNOS+/+ and eNOS+/− fetuses combined (n=10 hearts per group). F, Average capillary density in eNOS+/+ fetuses only (n=5 hearts per group). G, Average capillary density in eNOS+/− fetuses only (n=5 hearts per group). Data represent means±SEM and were analyzed using 1‐way ANOVA followed by Tukey’s test. +/− indicates heterozygous; E, embryonic day; eNOS, endothelial nitric oxide synthase; EX, exercise; and PGD, pregestational diabetes.

• 

  Figure 7. Maternal exercise increased cellular proliferation in E12.5 eNOS+/+ and eNOS+/− hearts exposed to PGD. A, B, Representative images of pHH3 immunofluorescence in eNOS+/+ and eNOS+/− hearts of E12.5 fetuses. C, Rates of cellular proliferation per 1000 cells were not significantly different between eNOS+/+ and eNOS+/− fetuses. D–F, Representative images of pHH3 immunofluorescence in E12.5 fetal hearts from eNOS+/− control, eNOS+/−+PGD, and eNOS+/−+PGD+exercise dams. G, Rates of proliferation per 1000 cells were reduced by PGD and rescued by maternal exercise in eNOS+/+ and eNOS+/− fetal hearts. The same trend is observed in eNOS+/+ hearts (H) and eNOS+/− hearts (I). Data represent mean±SEM of n=4 to 10 hearts per group and were analyzed using unpaired Student’s t test (C) or 1‐way ANOVA followed by Tukey’s test (G–I). +/− indicates heterozygous; E, embryonic day; eNOS, endothelial nitric oxide synthase; EX, exercise; PGD, pregestational diabetes; and pHH3, phosphorylated histone H3.

• 

  Figure 8. Rates of apoptosis in E12.5 fetal hearts from eNOS+/− dams without and with PGD and maternal exercise. A, B, Representative images of cleaved caspase‐3 immunostaining in E12.5 eNOS+/+ and eNOS+/− hearts. C, Ratio of apoptotic cells to heart area in fetal eNOS+/+ and eNOS+/− hearts of eNOS+/− dams. D–F, Representative images of cleaved caspase‐3 immunostaining in E12.5 eNOS+/+ and eNOS+/− hearts from eNOS+/− control, eNOS+/−+PGD, and eNOS+/−+PGD+exercise dams. G, The ratio of apoptotic cells to heart area of eNOS+/+ and eNOS+/− fetal hearts. H, eNOS+/+ fetuses of diabetic dams with exercise showed elevated rates of apoptosis. I, The ratio of apoptotic cells to heart area of eNOS+/− fetal hearts. Data represent mean±SEM of n=4 to 10 hearts per group and were analyzed using unpaired Student’s t test (C) or 1‐way ANOVA followed Tukey’s test (F). +/− indicates heterozygous; CC3, cleaved caspase‐3; E, embryonic day; eNOS, endothelial nitric oxide synthase; EX, exercise; and PGD, pregestational diabetes.

• 

  Figure 9. Oxidative stress levels were similar in E12.5 eNOS+/+ and eNOS+/− hearts. A, B, Representative images of DHE staining in eNOS+/+ (A) and eNOS+/− (B) fetal hearts of eNOS+/− dams. C, DHE fluorescence intensity of eNOS+/+ and eNOS+/− fetal hearts. D, E, Representative images of 4‐HNE immunostaining with nuclear DAPI in eNOS+/+ (D) and eNOS+/− (E) fetal hearts of eNOS+/− dams. F, 4‐HNE fluorescence intensity in eNOS+/+ and eNOS+/− fetal hearts. Data represent mean±SEM of n=5 hearts per group and were analyzed using unpaired Student’s t test. +/− indicates heterozygous; 4‐HNE, 4‐hydroxynonenal; DHE, dihydroethidine; E, embryonic day; eNOS, endothelial nitric oxide synthase; EX, exercise; and PGD, pregestational diabetes.

• 

  Figure 10. Oxidative stress was elevated by PGD and unchanged by maternal exercise in E12.5 eNOS+/+ and eNOS+/− hearts. A–C, Representative images of DHE staining in fetal hearts exposed to eNOS+/− dams (A), eNOS+/− dams with PGD (B), and eNOS+/− dams with PGD and maternal exercise (C). D, DHE fluorescence intensity in eNOS+/+ and eNOS+/− fetal hearts. E, DHE fluorescence intensity in eNOS+/+ fetal hearts. F, DHE fluorescence intensity in eNOS+/− fetal hearts. G–I, Representative images of 4‐HNE immunostaining and DAPI nuclear stain in fetal hearts exposed to eNOS+/− dams (G), eNOS+/− dams with PGD (H), and eNOS+/− dams with PGD and maternal exercise (I). J, 4‐HNE fluorescence intensity in eNOS+/+ and eNOS+/− fetal hearts. K, 4‐HNE fluorescence intensity in eNOS+/+ fetal hearts. L, 4‐HNE fluorescence intensity in eNOS+/− fetal hearts. Data represent mean±SEM of n=5 to 10 hearts per group and were analyzed using 1‐way ANOVA followed by Tukey’s test. +/− indicates heterozygous; 4‐HNE, 4‐hydroxynonenal; DHE, dihydroethidine; E, embryonic day; eNOS, endothelial nitric oxide synthase; EX, exercise; and PGD, pregestational diabetes.

• 

  Figure 11. eNOS protein levels in E12.5 fetal hearts of control dams and dams exposed to PGD and maternal exercise. A, Representative Western blot depicting eNOS protein levels in eNOS+/+, eNOS+/−, and eNOS−/− E12.5 hearts of control dams. B, Densitometry quantification of the ratio of eNOS to α‐actinin. C, Representative protein levels from eNOS+/+ or eNOS+/− fetal hearts of dams exposed to PGD without and with maternal exercise. D, Densitometry quantification of the ratio of eNOS to α‐actinin. E and F, Immunostaining and quantification of eNOS serine 1177 phosphorylation. Data represent mean±SEM of n=3 to 6 hearts per group and were analyzed using 1‐way ANOVA followed by Tukey’s test. +/− indicates heterozygous; E, embryonic day; eNOS, endothelial nitric oxide synthase; EX, exercise; and PGD, pregestational diabetes.