Sapropterin Treatment Prevents Congenital Heart Defects Induced by Pregestational Diabetes Mellitus in Mice

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  Figure 1. Experimental design to examine the effects of sapropterin (tetrahydrobiopterin) on congenital heart defects induced by pregestational diabetes mellitus. A study flow chart illustrates timelines of saline or streptozotocin injection, breeding, sapropterin or insulin treatment, and assessments of fetal hearts in 5 groups of mice. BG indicates blood glucose; E, embryonic day; eNOS, endothelial NO synthase; IP, intraperitoneal injection; OFT, outflow tract; PO, oral administration; ROS, reactive oxygen species.

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  Figure 2. Blood glucose levels of pregnant mice, percentage of successful plugs, litter size, fetal body weight, and biopterin levels. A, Nonfasting blood glucose levels from before mating (basal) to embryonic day (E) 18.5 during pregnancy in streptozotocin‐treated and control female mice with and without sapropterin (tetrahydrobiopterin) administration (n=4–8 mice per group). B, The percentage of successful pregnancies. The numbers in the parentheses indicate the number of successful pregnancies at the day of fetus harvest/total plugs. C, The offspring litter size measured at the day of fetus harvest (n=5–11 litters per group). D, Fetal body weight at E18.5 (n=5–8 fetuses per group). E through G, Levels of 7,8‐dihydrobiopterin in maternal plasma, and fetal 7,8‐dihydrobiopterin and tetrahydrobiopterin levels at E12.5 with or without tetrahydrobiopterin treatment in the diabetic dams (n=3–9 mice per group). Two‐way repeated‐measures ANOVA (A) or two‐way ANOVA (C and D), followed by Bonferroni post hoc test, was used. B was analyzed by Fisher's exact test. E through G were analyzed by unpaired Student t test. Data are means±SEM (A and C through G). *P<0.05, ***P<0.001 vs corresponding…

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  Figure 3. Effects of sapropterin (tetrahydrobiopterin) on congenital heart defects (CHDs) induced by pregestational diabetes mellitus. Representative histological sections of embryonic day (E) 18.5 hearts from offspring of diabetic mothers with and without tetrahydrobiopterin treatment. A through C, Heart sections from controls. Pregestational diabetes mellitus resulted in atrial septal defect (D), ventricular septal defect (E), double‐outlet right ventricle (F), truncus arteriosus (G), and thickened aortic (H) and pulmonary (I) valves. J through O, Tetrahydrobiopterin treatment in diabetic dams shows normal heart morphological characteristics. P through R, Diabetic dams receiving insulin therapy to control blood glucose levels show normal fetal heart morphological characteristics. Bar=200 μm. S, Incidence of CHDs as percentage of offspring separated by sex at E18.5 from diabetic dams with or without tetrahydrobiopterin treatment. *P<0.05 vs streptozotocin group of corresponding sex; † P<0.05 vs corresponding males. T through W, Left ventricular (LV) fractional shortening and ejection fraction and anterior wall thickness during systole (LVAWTs) and diastole (LVAWTd), assessed by…

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  Figure 4. Effects of sapropterin (tetrahydrobiopterin) on fetal heart wall thickness of pregestational diabetes mellitus at embryonic day 18.5. A, Representative images of myocardial wall thickness in control and pregestational diabetes mellitus with and without tetrahydrobiopterin treatment. Arrows indicate compact myocardium boundary from which measurements were obtained. B and C, Quantification of left ventricular (LV) and right ventricular (RV) myocardial thickness, respectively. n=6 per group from 3 to 6 litters. Bar=200 μm. Data are means±SEM and analyzed using 2‐way ANOVA, followed by Bonferroni post hoc test (B and C). ***P<0.01 vs untreated control; † P<0.01 vs untreated diabetes mellitus.

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  Figure 5. Effects of sapropterin (tetrahydrobiopterin) on aortic, pulmonary, and mitral valve defects induced by pregestational diabetes mellitus at embryonic day (E) 18.5. A through C, Representative images of toluidine blue staining of glycosaminoglycans in aortic, pulmonary, and mitral valves in E18.5 hearts. D, The ratio of glycosaminoglycan‐positive area/total valve leaflet area. E, Pulmonary valve leaflet thickness. F, Mitral valve leaflet thickness. G, Total aortic orifice area. H, Pulmonary artery luminal diameter at the base of the orifice. Bars=50, 20, and 20 μm (A, B, and C, respectively). n=4 to 7 hearts per group from 3 to 4 litters. Data are means±SEM and analyzed using 2‐way ANOVA, followed by Bonferroni post hoc test (B and C). LC indicates left cusp; LCC, left coronary cusp; NCC, noncoronary cusp; RC, right cusp; RCC, right coronary cusp. **P<0.01 vs controls; † P<0.01, †† P<0.01 vs untreated STZ group.

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  Figure 6. Effects of sapropterin (tetrahydrobiopterin) on outflow tract (OFT) development and cell proliferation in embryonic day (E) 10.5 hearts of pregestational diabetes mellitus. A, Length of the OFT was measured via sagittal sections according to the line. B through D, Immunostaining for phosphohistone H3 (pHH3), marking proliferating cells in the OFT (B through C) and ventricular myocardium (D). C is the magnification of boxed area in B. E, Quantification of OFT length. F, Quantification of pHH3+ cells in the OFT. G, Quantification of pHH3+ cells in the ventricle. n=3 to 6 hearts per group from 2 to 4 litters. Bars=50 (in A and B) and 20 μm (in C and D). Data are means±SEM and analyzed using 2‐way ANOVA, followed by Bonferroni post hoc test. **P<0.01, ***P<0.001 vs untreated control; † P<0.05, †† P<0.01 vs untreated diabetes mellitus.

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  Figure 7. Effects of pregestational diabetes mellitus on second heart field (SHF) progenitor contribution to embryonic day (E) 9.5 and E12.5 hearts. Fate mapping using the mT/mG reporter showing green fluorescent protein–positive (GFP +) cells expressing Cre recombinase under the control of the anterior heart field–specific Mef2c transcription factor. Representative sections of outflow tract (OFT) of E9.5 (A) and E12.5 (B) hearts from control and diabetic mothers. C, Representative sections showing the cell layers of the right ventricular (RV) myocardium from E12.5 hearts. Quantification of SHF GFP + cells (D) and total number of cells (E) in the OFT cushions at E9.5. Quantification of SHF GFP + cells in the OFT cushions (F) and number of cell layers in the RV myocardium (G) at E12.5. N=3 per group from 2 litters (D and E). N=5 per group from 2 to 4 litters (F and G). Bar=50 μm. Data are means±SEM and analyzed using unpaired Student t test (D‐G). *P<0.05, **P<0.01, *** P<0.001 vs control.

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  Figure 8. Effects of sapropterin (tetrahydrobiopterin) on molecular regulators of heart development at embryonic day (E) 12.5. Real‐time reverse transcription–polymerase chain reaction (RT‐PCR) of cardiac transcription factors and regulators in E12.5 hearts of offspring from control and diabetic mothers. A through F, The expression levels of Gata4, Tbx5, Nkx2.5, Gata5, Bmp10, and Notch1 were significantly decreased under maternal diabetes mellitus and restored with tetrahydrobiopterin treatment. G and H, The expression levels of Gch1 and Dhfr were significantly decreased with maternal diabetes mellitus. Tetrahydrobiopterin treatment rescues expression of DHFR. n=4 to 7 hearts per group. Data are means±SEM and analyzed using 2‐way ANOVA, followed by Bonferroni post hoc test. *P<0.05, **P<0.01 vs untreated control; † P<0.05, †† P<0.01 vs untreated diabetes mellitus.

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  Figure 9. Effects of sapropterin (tetrahydrobiopterin) on superoxide production and endothelial NO synthase (eNOS) dimer/monomer protein levels in embryonic day (E) 12.5 hearts. A, Representative images of dihydroethidium staining in the ventricular myocardium of E12.5 hearts from control and diabetic dams with and without tetrahydrobiopterin administration. A subset of heart sections from diabetic dams were pretreated with Nω‐nitro‐l‐arginine methyl ester (L‐NAME; 300 μmol/L) or superoxide dismutase (SOD; 100 U/mL) for 30 minutes before dihydroethidium probing. B, Quantification of dihydroethidium fluorescence signals. C, Representative Western blotting showing eNOS dimer and monomer bands of E12.5 hearts from control and diabetic dams with and without tetrahydrobiopterin treatment. Denatured endothelial cell lysate validates the size of the eNOS monomer band. D, Densitometric analysis of eNOS dimer/monomer ratios. n=5 to 6 hearts per group from 2 to 4 litters. Data are means±SEM and analyzed using 2‐way ANOVA, followed by Bonferroni post hoc test. EC indicates endothelial cell. *P<0.05 vs untreated control; † P<0.05, †† P<0.01 vs untreated diabetes mellitus.

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  Figure 10. Schematic summary of endothelial NO synthase (eNOS) uncoupling and congenital heart defects induced by pregestational diabetes mellitus and the inhibitory effects by sapropterin treatment. Abnormalities of both second heart field (SHF) and non‐SHF derived structures are prevented by sapropterin treatment. LV indicates left ventricle; OFT, outflow tract; ROS, reactive oxygen species; and RV, right ventricle.