Superoxide Dismutase 1 In Vivo Ameliorates Maternal Diabetes Mellitus-Induced Apoptosis and Heart Defects Through Restoration of Impaired Wnt Signaling - PubMed (original) (raw)

Superoxide Dismutase 1 In Vivo Ameliorates Maternal Diabetes Mellitus-Induced Apoptosis and Heart Defects Through Restoration of Impaired Wnt Signaling

Fang Wang et al. Circ Cardiovasc Genet. 2015 Oct.

Abstract

Background: Oxidative stress is manifested in embryos exposed to maternal diabetes mellitus, yet specific mechanisms for diabetes mellitus-induced heart defects are not defined. Gene deletion of intermediates of Wingless-related integration (Wnt) signaling causes heart defects similar to those observed in embryos from diabetic pregnancies. We tested the hypothesis that diabetes mellitus-induced oxidative stress impairs Wnt signaling, thereby causing heart defects, and that these defects can be rescued by transgenic overexpression of the reactive oxygen species scavenger superoxide dismutase 1 (SOD1).

Methods and results: Wild-type (WT) and SOD1-overexpressing embryos from nondiabetic WT control dams and nondiabetic/diabetic WT female mice mated with SOD1 transgenic male mice were analyzed. No heart defects were observed in WT and SOD1 embryos under nondiabetic conditions. WT embryos of diabetic dams had a 26% incidence of cardiac outlet defects that were suppressed by SOD1 overexpression. Insulin treatment reduced blood glucose levels and heart defects. Diabetes mellitus increased superoxide production, canonical Wnt antagonist expression, caspase activation, and apoptosis and suppressed cell proliferation. Diabetes mellitus suppressed Wnt signaling intermediates and Wnt target gene expression in the embryonic heart, each of which were reversed by SOD1 overexpression. Hydrogen peroxide and peroxynitrite mimicked the inhibitory effect of high glucose on Wnt signaling, which was abolished by the SOD1 mimetic, tempol.

Conclusions: The oxidative stress of diabetes mellitus impairs Wnt signaling and causes cardiac outlet defects that are rescued by SOD1 overexpression. This suggests that targeting of components of the Wnt5a signaling pathway may be a viable strategy for suppression of congenital heart defects in fetuses of diabetic pregnancies.

Keywords: Wnt signaling pathway; apoptosis; congenital; diabetes mellitus; heart defects; oxidative stress; pregnancy.

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Conflict of interest statement

Conflict of Interest Disclosure: None.

Figures

Figure 1

SOD1 overexpression abrogates maternal diabetes-induced oxidative stress in the developing heart. A. E12.5 hearts were sectioned in a frontal plane as shown. B, representative images of DHE staining in E12.5 heart sections. DHE reacts with cellular superoxide producing red fluorescence product 2-hydroxyethidium. All cell nuclei were stained with DAPI (Blue). Three hearts from embryos of three different dams (n = 3) per group were used and similar results were obtained. Bars = 150 µm. C, levels of lipid hydroperoxide (LPO) in E12.5 hearts, expressed as µM per gram of protein. Hearts from embryos of five different dams (n = 5) per group were analyzed. * indicates significant differences compared to the other three groups. ND: nondiabetic; DM: diabetic mellitus; WT: wild-type hearts; SOD1: SOD1 overexpressing hearts.

Figure 2

Maternal diabetes induces outflow tract and ventricular septal defects (VSDs). A), H&E stained of E17.5 hearts from anterior to posterior: normal heart (left column) from WT embryos of nondiabetic (ND) dam, heart with an isolated VSD (middle column) representative images of hematoxylin and eoxin-stained sections whereas B were representative images of india ink dye injections in E17.5 embryonic hearts. A represented morphologically normal heart (left column) from WT embryos of nondiabetic (ND) dam, a typical VSD heart (middle column) and aorta-overriding VSD heart (right column) from WT embryos of diabetic (DM) dam; B, E17.5 heart and blood vessels imaged in whole mount after injection of India Ink: a normal heart from WT embryos of nondiabetic dam and a heart with PTA from WT embryos of diabetic dam. Scale bars = 300 µm. AO: Aorta; PA: Pulmonary Artery; LA: Left Atrium; RA: Right Atrium; LV: left ventricle; RV: right ventricle; VSD: Ventricular septum defect; PTA: Persistent truncus arterious.

Figure 3

SOD1 overexpression suppresses maternal diabetes-induced cell apoptosis and caspase activation and normalizes cell proliferation in the developing heart. A and B, representative images of the TUNEL assay showing apoptotic cells (Red signal) and quantified as TUNEL positive cells per section (3 sections from 3 different hearts of each group). Cell nuclei were stained with DAPI (Blue). TUNEL positive cells in the A-V and outflow tract endocardial cushion are shown in A and B, respectively. In A, TUNEL positive cells are also seen in the epicardium lining the ventricle. Bars = 150 µm. C, representative western blot for detection of cleaved caspase 3, 8 and the quantification normalized to β-actin. Human SOD1 (hSOD1), the transgene product, was detected by a specific human SOD1 antibody. Experiments were repeated three times using hearts from embryos of three different dams (n = 3) per group. D, representative images of p-Histone H3 immunostaining, which labels proliferating cells. p-Histone H3 positive cells are labeled by red signal and cell nuclei are stained by DAPI (Blue). In the dot graph, p-Histone H3 positive cells were quantified by the NIH Image J software and expressed as number of cells per section. Three hearts from embryos of three dams (n = 3) per group, and three serial sections per heart were analyzed. Bars = 150 µm. IgG: normal rabbit IgG controls; WT: Wild-type; ND: nondiabetic; DM: diabetic mellitus. * indicates significant difference compared to other groups.

Figure 4

SOD1 overexpression rescues diabetes-increased canonical Wnt antagonists and restores the expression of key canonical Wnt intermediates. A, mRNA abundance of the three canonical Wnt antagonists. B, representative western blots of phosphorylated and non-phosphorylated Dvl2, p-GSK3β and β-catenin. The dot graphs showed the quantification of the immunoblotting. In A and B, three hearts from embryos of three dams (n = 3) per group were analyzed. C, images of β-catenin immunostaining. Rabbit normal IgG served as controls. Red signal was β-catenin and cell nuclei were stained by DAPI (Blue). Three E12.5 hearts from embryos of three dams (n = 3) per group, and three serial sections per heart were analyzed, and similar results were obtained. Bars = 150 µm. * in A, B indicate significant difference compared to other groups. ND: nondiabetic; DM: diabetic mellitus; WT: wild-type hearts; SOD1: SOD1 overexpressing hearts.

Figure 5

Maternal diabetes impairs the non-canonical Wnt pathway and this impairment is abolished by SOD1 overexpression. A, mRNA levels of Wnt5a in E12.5 hearts. B, Wnt5a protein levels in E12.5 hearts. C, images of Wnt5a immunostaining. Rabbit normal IgG served as controls. Red signal was Wnt5a and cell nuclei were stained by DAPI (Blue). Three hearts from embryos of three dams (n = 3) per group, and three serial sections per heart were analyzed, and similar results were obtained. Bars = 150 µm. D, levels of p-NFAT4. E, levels of nuclear NFAT2. F, levels of p-CaMKII. In B, D, E, and F, quantification of immunoblotting was shown in the dot graph. In A, B, D, E, F, experiments were repeated three times using three E12.5 hearts from embryos of three dams (n = 3) per group. * in A, B, D, E, F indicate significant difference compared to other groups. ND: nondiabetic; DM: diabetic mellitus; WT: wild-type hearts; SOD1: SOD1 overexpressing hearts.

Figure 6

SOD1 overexpression rescues maternal diabetes-reduced Wnt target gene expression. A, mRNA levels of canonical Wnt target genes. B, mRNA levels of noncanonical Wnt targe genes. ND: nondiabetic; DM: diabetic mellitus; WT: wild-type hearts; SOD1: SOD1 overexpressing hearts. Five hearts from E12.5 embryos of five dams (n = 5) per group were analyzed. * indicate significant difference compared to other groups.

Figure 7

The SOD1 mimetic, tempol, abolishes high glucose-suppressed Wnt signaling, and both ROS and RNS inhibit Wnt signaling in cultured hearts. A and B, DKK1, β-catenin, Wnt5a and phosphorylated NFAT4 levels in cultured hearts. Experiments were repeated three times. * indicate significant difference compared with other groups.. C. Schematic of oxidative stress-mediated Wnt signaling impairment in the developing heart leading to heart defects under maternal diabetic conditions. Oxidative stress induced by maternal diabetes inhibits both the canonical and noncanonical Wnt signaling pathways through two distinct mechanisms. Maternal diabetes suppresses the canonical Wnt signaling by increasing its antagonist expression and the activity of its negative regulator whereas it inhibits the non-canonical Wnt signaling by downregulating Wnt5a.Major types of heart defects associated with gene deletion of key Wnt intermediates are indicated. p means phosphorylation.

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Superoxide Dismutase 1 In Vivo Ameliorates Maternal Diabetes Mellitus-Induced Apoptosis and Heart Defects Through Restoration of Impaired Wnt Signaling - PubMed (original) (raw)