PMC

Maternal exercise reduces oxidative stress in hearts of offspring of maternal diabetes (OMD). (A), Representative images of E14.5 hearts stained with dihydroethidine (DHE) probe for superoxide content imaged at 20‐ms exposure. (B), Quantification of relative fluorescence by densitometry normalized to area (n = 5 hearts per group). (C), Representative images of E14.5 embryonic hearts immunostained with anti‐4‐HNE to measure lipid peroxidation. Images were taken at 100‐ms exposure. (D), Quantification of relative fluorescence by densitometry normalized to area (n = 3‐4 hearts per group). **P < 0.01 vs control. ^†† P < 0.01 vs OMD without exercise. Data are means ± SEM

Maternal exercise restores gene expression and eNOS phosphorylation in E12.5 hearts of offspring of maternal diabetes (OMD). (A‐H), Cardiac mRNA levels of Gata4, Hif‐1α, Cyclin D1, Notch1, Snail1, Vegf‐A, Nkx2.5 and Bmp10 in OMD with and without maternal exercise (n = 5‐8 hearts per group). (I), Representative Western blot depicting p‐eNOS and total eNOS at 130 kDA and α‐actinin at 100 kDa. (J), Densitometry quantification of the ratio of p‐eNOS to total eNOS (n = 3 hearts per group) and (K), ratio of total eNOS protein to α‐actinin quantified by densitometry (n = 3 hearts per group). *P < 0.05 vs control without exercise. ^† P < 0.05 vs OMD without exercise. Data are means ± SEM

Maternal exercise improves cell proliferation and epithelial‐to‐mesenchymal transition in E12.5 hearts of offspring of maternal diabetes (OMD). (A‐C), Sections of E12.5 hearts immunostained with anti‐phosphorylated histone H3 (pHH3) antibody‐marking proliferating cells. (B, C), Representative images of ventricular wall thickness in E12.5 hearts. Red arrow heads indicate pHH3‐positive cells (dark brown). (D), E12.5 epicardium immunostained with anti‐WT1 antibody. Red arrow heads indicate WT1‐positive cells. E, Number of pHH3‐positive cells, (F), RV wall thickness and (G), LV wall thickness (n = 3‐6 per group). (H), Quantification of the number of WT1‐positive cells normalized to epicardial length (n = 4‐5 per group). *P < 0.05 vs control. ^† P < 0.05 vs OMD without exercise. Data are means ± SEM

Maternal exercise prevents pulmonary and aortic valve defects in offspring of maternal diabetes (OMD). Representative images of pulmonary and aortic valves from E18.5 hearts of normal controls (A, B, E, F) and OMD (C, D, G, H) with and without maternal exercise. Abnormalities of pulmonary (C) and aortic (G) valves in offspring of diabetic dams are prevented with maternal exercise (D, H). (I), Pulmonary valve size to orifice diameter ratio. (J), Ratio of aortic valve area to total aortic area. (K), Total aortic area. *P < 0.05, **P < 0.01 vs control. ^†† P < 0.01 vs OMD without exercise. Data are means ± SEM. n = 6‐7 hearts per group. NCC, non‐coronary cusp; RCC, right coronary cusp; LCC, left coronary cusp. Scale bars are 50 µm

Maternal exercise prevents coronary artery and capillary abnormalities in offspring of maternal diabetes (OMD). Representative images of coronary artery size (A) and abundance (B) in E18.5 hearts stained with anti‐α‐smooth muscle actin antibody. (C), Three‐dimensional reconstruction of coronary arteries. (D), Lectin‐1 staining shows capillaries (brown colour) in the ventricular myocardium of E18.5 hearts. Quantification coronary artery diameter (E) and abundance (F) in E18.5 hearts of control and OMD with and without maternal exercise (n = 4‐7 hearts per group). (G), AMIRA software was used to quantify coronary artery and myocardial volume of the heart using 5 µm sections 25 µm apart, from the level of the pulmonary valves to the apex of the heart (n = 3 per group). (H), Capillary density is normalized to the area of RV and LV myocardium of OMD with and without exercise (n = 4‐6 per group). Data are means ± SEM. *P < 0.05, **P < 0.01 vs control. ^† P < 0.05, ^†† P < 0.01 vs OMD without exercise

Effects of maternal exercise on blood glucose, litter size and congenital heart defects in the offspring of maternal diabetes (OMD). (A), Non‐fasting blood glucose levels during gestation in streptozotocin (STZ)‐treated and control female mice with and without exercise (n = 3‐5). Basal indicates before STZ injection. (B), Running distance per day of gestation from E0.5‐17.5 (n = 4‐7 per group). (C), Offspring litter size. (D), Average number of absorbed offspring per litter. (E), Maternal body weight at E0.5 (n = 7‐11 per group). (F), Per cent congenital heart defects in males and females of OMD. Data are means ± SEM. *P < 0.05, **P < 0.01 vs control. Without maternal exercise, E18.5 hearts of OMD show ASD (G), VSD (H), AVSD (I), DORV (M), HLHS and HRHS (N and O) with ASD, aortic stenosis and hypoplasia of LV, RV, mitral and tricuspid valves. Corresponding E18.5 normal hearts of OMD with maternal exercise are shown in J‐L and P‐R RA: right atrium, LA: left atrium, Ao: aorta, PA: pulmonary artery. Scale bars are 200 µm