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. 2011 Jun;91(6):468-76.
doi: 10.1002/bdra.20816. Epub 2011 May 11.

Defective sumoylation pathway directs congenital heart disease

Jun Wang  1 , Li Chen, Shu Wen, Huiping Zhu, Wei Yu, Ivan P Moskowitz, Gary M Shaw, Richard H Finnell, Robert J Schwartz
Affiliations

Defective sumoylation pathway directs congenital heart disease

Jun Wang et al. Birth Defects Res A Clin Mol Teratol. 2011 Jun.

Abstract

Congenital heart defects (CHDs) are the most common of all birth defects, yet molecular mechanism(s) underlying highly prevalent atrial septal defects (ASDs) and ventricular septal defects (VSDs) have remained elusive. We demonstrate the indispensability of "balanced" posttranslational small ubiquitin-like modifier (SUMO) conjugation-deconjugation pathway for normal cardiac development. Both hetero- and homozygous SUMO-1 knockout mice exhibited ASDs and VSDs with high mortality rates, which were rescued by cardiac reexpression of the SUMO-1 transgene. Because SUMO-1 was also involved in cleft lip/palate in human patients, the previous findings provided a powerful rationale to question whether SUMO-1 was mutated in infants born with cleft palates and ASDs. Sequence analysis of DNA from newborn screening blood spots revealed a single 16 bp substitution in the SUMO-1 regulatory promoter of a patient displaying both oral-facial clefts and ASDs. Diminished sumoylation activity whether by genetics, environmental toxins, and/or pharmaceuticals may significantly contribute to susceptibility to the induction of congenital heart disease worldwide. Birth Defects Research (Part A) 2011. © 2011 Wiley-Liss, Inc.

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Figures

Figure 1
Figure 1. SUMO-1 transcripts were detected in the developing hearts during mouse embryogenesis
A. In situ hybridization was performed on mouse embryos at E8.5, E9.5, and E10.5 using antisense of SUMO-1 mRNA. Note that SUMO-1 transcripts were detected on the hearts and craniofacial region (arrow and arrowhead, respectively). B. LacZ staining truthfully revealed the endogenous SUMO-1 expression pattern in the heart. LacZ staining was performed on E10.5 of SUMO-1Gal/+ mouse embryo. The arrow indicated the positive staining in the heart.
Figure 2
Figure 2. SUMO-1 mutant mice exhibited abnormal septogenesis
A. P1 frequency of SUMO-1−/− mice was significantly lower than the expected Mendelian rate, and the mortality rate was higher compared with that of wild type (wt) mice. The data were compiled from 18 litters of totally 125 animals. **, p<0.01, Chi-square test was used; ++, p<0.01, Fisher’s exact test was used. B. Both prematurely demised hetero- and homozygous SUMO-1 mice showed ASD and/or VSD. Arrows indicated either an ASD or VSD, respectively. RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; ASD, atrial septal defect; VSD, ventricular septal defect. Bar = 200 µm.
Figure 3
Figure 3. SUMO-1 mutants displayed cardiac dysgenesis
A. SUMO-1Gal/+ mice exhibited ASDs/VSDs. Arrows indicated either an ASD or VSD, respectively. B. Generation of flag-tagged SUMO-1 Tg mice under control of cardiac α-MHC promoter (SUMO-1-Tg). Free flag-SUMO-1 (upper panel) and increased high molecular weight (HMW) conjugates of SUMO-1 (middle panel) were detected in SUMO-1-Tg mice. GAPDH (lower panel) served as a control. Western blots were performed on heart extracts from wild type and SUMO-1-Tg mice and labeled with indicated antibodies. C. Mortality rate of compound SUMO-1-Tg/SUMO-1Gal/+ mice was substantially decreased compared with that of single SUMO-1Gal/+ mice. The data were compiled from 17 litters with a total of 140 animals (wt, 30; SUMO-1-Tg, 32 SUMO-1Gal/+, 32; SUMO-1-Tg/SUMO-1Gal/+, 46). D. Down-regulated genes associated with cell proliferation (upper panel) and up-regulated liver-enriched genes (lower panel) in SUMO-1 mutant mouse hearts. Microarray assay on RNAs purified from SUMO-1 mutant and wild type mouse hearts were performed as described in the section of Materials and Methods. E. Verification of changes in several gene expressions shown in D by quantitative RT-PCR. F. Liver-enriched transcription factor C/EBPβ was suppressed in ES cells during EB formation.
Figure 4
Figure 4. Potential genetic interaction between Nkx2.5 and SUMO-1 genes
A. Compound Nkx2.5+/−/SUMO-1Gal/+ mice exhibited higher mortality rates compared with those of single Nkx2.5+/− and SUMO-1Gal/+ mice. Total number (dead) of animals for analysis was shown. B. Histology of dead compound Nkx2.5+/−/SUMO-1Gal/+ mice revealed severe ASD/VSD. Representative data were shown.
Figure 5
Figure 5. Sequence analysis of DNA from newborn screening blood spots revealed potential SUMO-1 regulatory promoter mutations in infants displaying both oral-facial clefts and ASDs
A. Schematic representation of the locations of mutations relative to the transcription initiation site in the human SUMO-1 gene cis-regulatory region. B. Sequencing graphic revealing mutations in two different human SUMO-1 promoter regions with indicated positive/total number of patients. The observed DNA sequence variants were not detected in a total of 100 control samples. C. Luciferase reporter activity assays revealed ~95% and ~60% decrease in the activity of of mut-16 and A-1239G SUMO-1 promoter respectively, compared with that of wild type promoter. Data were obtained from four independent assays in HL-1 cell line, each carried out in duplicate. Student’s t test was used for statistical significance analysis. *P<0.05; **, P<0.001, compared with wt group.
Figure 6
Figure 6
Model of sumoylation conjugation - deconjugation pathway is highly complex yet under balance to promote normal cardiac development. Reduction of SUMO-1 by half elicited ASD/VSDs as in SUMO-1 haploid-insufficient mice may underscore other genetic defects of the individual SUMO conjugation pathway components and even the cardiac transfactor targets causing similar CHD phenotypes. Environmental toxins, nutrients and drugs may adversely affect the expression of SUMO pathway components.
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