Left ventricular morphology and function derived from MRI 8 weeks post myocardial infarction.

<p>Group S are untreated wild-type sham-operated mice; Group MI are untreated wild-type infarcted mice; Group MI+R are wild-type infarcted mice treated with ribose; Group MI+C+R are infarcted creatine transporter overexpressing mice treated with ribose. Infarcted groups were matched for infarct size (A). Left ventricular remodelling and function was measured by cine-MRI (B–F). Data are reported as mean ± SD. *** denotes p<0.001 (1-way ANOVA with Bonferroni’s correction).</p

Morphometry and myocardial biochemistry 8 weeks after myocardial infarction.

<p>All values are mean ± SD. Comparisons were made by one-way ANOVA with Bonferroni’s post-hoc test.</p>*<p>denotes p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001 vs group S and ^#p<0.05,</p>###<p>p<0.001 vs group MI.</p

Oral ribose treatment increases ribose-5-phosphate levels in the heart.

<p>Myocardial ribose-5-phosphate levels following administration of ribose (10% w/v) in drinking water for seven weeks. Control n = 5, ribose n = 4, mean ± SD, ** denotes p<0.01.</p

Left ventricular haemodynamic parameters 8 weeks post myocardial infarction.

<p>Group S are untreated wild-type sham-operated mice; Group MI are untreated wild-type infarcted mice; Group MI+R are wild-type infarcted mice treated with ribose; Group MI+C+R are infarcted creatine transporter overexpressing mice treated with ribose. Heart rate (A), LV end-systolic and end-diastolic pressures (B–C) and maximal and minimal rates of pressure change (D–E) under baseline non-stimulated conditions. Differences analysed by one-way ANOVA with Bonferroni’s correction. ** denotes p<0.01 and *** p<0.001 versus group S. There was no difference between any of the infarcted groups. Panels F–H show heart rate and maximal and minimal rates of pressure change before and after stimulation with dobutamine (16 ng/g BW/min). Effect of genotype and dobutamine assessed by two-way ANOVA with post-hoc Bonferroni’s correction. ^ denotes p<0.05, ^^ p<0.01, ^^^ p<0.001 for dobutamine (black bars) vs baseline (white bars) and * denotes p<0.05, ** p<0.01, *** p<0.001 versus sham at the same dobutamine dose. Data are mean ± SD.</p

Factors influencing ejection fraction by correlation analysis.

<p>Ejection fraction assessed by MRI 8 weeks after myocardial infarction correlated well with infarct size (A), but not with myocardial total adenine nucleotides (B), or myocardial creatine levels (C). Correlation analysis and linear regression is for all groups analysed together. Group MI are untreated wild-type infarcted mice; Group MI+R are wild-type infarcted mice treated with ribose; Group MI+C+R are infarcted creatine transporter overexpressing mice treated with ribose.</p

Functional Significance of SRJ Domain Mutations in <em>CITED2</em>

<div><p>CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ) that is highly conserved in placental mammals. Loss of <em>Cited2</em> in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD) cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T) in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 <em>in vitro</em> at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance <em>in vivo</em>, we generated a T166N mutation of mouse <em>Cited2</em>. We also used PhiC31 integrase-mediated cassette exchange to generate a <em>Cited2</em> knock-in allele replacing the mouse <em>Cited2</em> coding sequence with human <em>CITED2</em> and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using <em>in vivo</em> models and that predictions based on structural conservation and <em>in vitro</em> functional assays, or even <em>in vivo</em> global loss of function models, may be insufficient.</p> </div

Molecular characterization of the <i>Cited2 ^T166N</i>, <i>Cited2 ^MRG1</i> and <i>Cited2 ^HUM</i> alleles.

<p>(A) Western blot of total protein lysates from mouse embryonic fibroblasts (MEFs), probed with anti-CITED2 antibody. CITED2 and CITED2-MRG1 are indicated, as is a non-specific band (N.S.) that migrates at 25 kDa. (B) RT-PCR showing RNA products expressed by embryos of various genotypes. PCR primers were designed to differentiate between the endogenous mouse <i>Cited2</i> transcript and the <i>Cited2 ^MRG1</i> transcript by their size difference. Wild type mouse <i>Cited2</i>, containing the SRJ domain produces the larger 725 bp band. (C) Southern blots of <i>Cited2 ^T166N</i> allele. Top, Southern blot of <i>EcoRI</i> digested genomic DNA probed with a 5′-probe. Middle, Southern blot of <i>BglII</i> digested genomic DNA, probed with a 3′-probe. Probe positions are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046256#pone-0046256-g003" target="_blank">Fig. 3</a>. Bottom, Southern blot of <i>SpeI</i> digested genomic DNA hybridized with an internal (Neomycin) probe, to confirm single copy integration. (D) Southern blots of <i>Cited2 ^MRG1</i> and <i>Cited2 ^HUM</i> alleles. Top, Southern blot of <i>EcoRI/SacII</i> digested genomic DNA, probed with a 5′-probe. Middle, Southern blot of <i>BglII</i> digested genomic DNA, probed with a 3′-probe. The position of the probes is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046256#pone-0046256-g003" target="_blank">Fig. 3</a>. Bottom, Southern blot of <i>EcoRI</i> digested genomic DNA hybridized with an internal (Puromycin) probe to confirm single copy integration.</p

Synonymous and non-synonymous mutations identified through direct sequencing of 1126 cases with CHD and 1227 controls.

<p>5 unique non-synonymous variants are observed in the cases and 2 in the control group. Abbreviations: PS, pulmonic stenosis; TGA, transposition of great arteries; ASD, atrial septal defect; TOF, Tetralogy of Fallot; AVSD, atrioventricular septal defect; VSD, ventricular septal defect.</p

Structure of CITED2.

<p>(A) Schematic diagram of human CITED2 showing conserved regions (CR) 1–3 and the serine-glycine rich junction (SRJ). Also indicated are the locations of CITED2 variants. All unique non-synonymous variants found in this study are shown above the figure with variants found in the SRJ highlighted. Unique non-synonymous variants found by Sperling et al., 2005 (*) and Yang et al., 2010 (⁺) are shown below. SRJ comprises AA 161 to 199. (B) The CITED2 peptide sequence is shown for <i>Homo sapiens</i> (Human, HSCITED2, AF129290), CITED2-MRG1 (Human, HSCITED2 MRG1 isoform lacking the entire SRJ), <i>Pan troglodytes</i> (Chimapanzee, PTCITED2), <i>Nomascus leucogenys</i> (Gibbon, NLCITED2), <i>Loxodonta africana</i> (Elephant, LACITED2), <i>Sus scrofa</i> (Pig, SSCITED2), <i>Bos taurus</i>, (Cow, BTCITED2), <i>Canis familiaris</i> (Dog, CFCITED2), <i>Mus musculus</i> (Mouse, MMCITED2), <i>Rattus norvegicus</i> (Rat, RNCITED2), <i>Monodelphis domestica</i> (Opossum, MDCITED2), <i>Gallus gallus</i> (Chicken, GGCITED2), <i>Anolis carolinensis</i> (Anole lizard, ACCITED2), <i>Xenopus laevis</i> (African clawed frog, XLCITED2), <i>Xenopus tropicalis</i> (Western clawed frog, XTCITED2), and <i>Danio rerio</i> (Zebrafish, DRCITED2). Sequence alignments taken from the latest genome builds from EBI Ensembl. SRJ domain spans region marked with grey bar above corresponding to AA161 to 199 in the human protein sequence.</p

Phenotypic analysis of mouse embryos expressing CITED2 variants.

<p>MRI analysis of embryos 15.5 days post coitum (dpc). Genotypes are indicated as shown. Sagittal sections through the left kidney (A–E) are shown to indicate the left adrenal gland where present (arrows), and absent (arrowhead). Transverse sections through the thorax (F–J) and 3D reconstructions (K–O) are shown to demonstrate cardiac anatomy. Loss of <i>Cited2</i> leads to adrenal agenesis (B, arrowhead), right atrial isomerism, ventricular septal defect (VSD) and common atrium (G), and abnormal ventricular topology (L). Embryos expressing only the T166N variant, the MRG1 isoform, or full length human CITED2 have normal adrenal glands and hearts. RA, Right Atria; RV, Right Ventricle; LV, Left Ventricle; IVS, Interventricular Septum; IAS, Intra-atrial Septum; AAo, Aorta; AoA, Aortic Arch; Tr, Trachea; DAo, Dorsal Aorta; LSVS and RSVS, Left and Right Systemic Venous Sinus. Axis: A, Anterior; P, Posterior; V, Ventral; D, Dorsal; L, Left; R, Right. Scale bars: 0.5 mm.</p