Cardiac edema in zebrafish embryos injected with <i>klf3</i> morpholinos.

<p>(A,C) Enlarged GFP images of embryonic hearts at 72 hpf. GFP expression was driven by the <i>myl7</i> regulatory element (cardiac myosin light chain 2) and marks the myocardium of the ventricle (rostral) and atrium (caudal). (B,D) Light microscopy images of the corresponding zebrafish embryos with the GFP heart image shown in (A,C) superimposed. The GFP heart is in the same orientation as in (A) but has not yet been enlarged. Most embryos injected with <i>klf3</i> morpholino exhibited cardiac abnormalities as shown in (C) and cardiac edema shown by arrows in (D) in comparison to wild type embryos shown in (A,B). Injection of the mismatch control morpholino had no effect on the developing zebrafish heart (not shown).</p

<i>Klf3^H275R</i> mRNA, protein, and impairment in DNA binding.

<p>(A) Similar levels of <i>Klf3</i> mRNA by qRT-PCR (P>0.05) and (B) KLF3 protein by Western blot (WB) in whole H275R homozygotes (Homo) and heterozygotes (Het) versus wild-type (WT) embryos at E12.5. (C) Bacterial expression of GST-zinc finger 1–3 of normal and mutant protein showing that H275R impaired DNA binding to CCACACCCT (canonical β-globin promoter) in electrophoretic mobility shift assay (coomassie blue-stained SDS PAGE gel (below)). (D) Dose-response showing H275R also impaired binding to CCACACCT in electrophoretic mobility shift assays when full length <i>Klf3</i> was expressed in COS cells (western blot with αKlf3 antibody below). Protein identity was validated by eliminating binding with a KLF3 antibody (αKlf3).</p

Morphological evidence for aortic valvular stenosis in <i>Klf3</i>^H275R/+ mice.

<p>(A) Histological sections showing thickened aortic valve leaflets in adult <i>Klf3</i>^H275R/+ (arrow in center and right panels) vs. WT (arrow in left panel). Blebs or hematomas were sometimes observed on aortic valve leaflets of <i>Klf3</i>^H275R/+ mice (e.g. arrow in right panel). (B) Scanning electron microscopy images of adult aortic valves (arrows) from WT (left) and <i>Klf3</i>^H275R/+ (center), with magnified images of valve leaflets (right), that show thickened leaflets in <i>Klf3</i>^H275R/+ mice. (C) Micro-ultrasound images of aortic arch (Ao) in adult <i>Klf3</i>^H275R/+ (below) vs. WT (above) showing aortic dilatation in the left ventricular outflow tract (LVOT) typical of <i>Klf3</i>^H275R/+ mice. (D) Elevated peak blood velocity in the ascending aorta of <i>Klf3</i>^H275R/+ (solid circles) relative to WT controls (open circles). Mean ± SE are shown for each group. The lower dashed line shows the WT mean + 2SD and the upper dashed line the WT mean + 3SD. 20 of 31 <i>Klf3</i>^H275R/+ had peak velocities >3SD above WT.</p

Gene trap insertion sites and effects on <i>Klf3</i> mRNA and protein.

<p>(A) Schematic of <i>Klf3</i> gene showing the DNA insertion site of the gene trap marker in the XS line (XS0187) and in the CH line (CH0516). SD = splice donor site; β-geo = β-galactosidase and neomycin fusion protein; pA = polyadenylation. (B) <i>Klf3</i> mRNA in E12.5 whole embryos from the XS (grey squares) and CH (black circles) gene trap lines measured by qRT-PCR was significantly decreased in homozygotes (homo) but not heterozygotes (het). (C) KLF3 protein in spleens of Klf3^H275R/+ adults was similar to WT (i.e. Klf3+/+, XS WT, CH WT) whereas it was undetectable in XS and CH homozygote spleens by western blot.</p

<i>Klf3</i> gene expression detected by LacZ staining in the developing and adult heart and vasculature.

<p>LacZ -staining (blue) in homozygous XS mice shows <i>Klf3</i> gene expression is located at discrete but widespread sites in (A) the E10.5 embryo (whole mount) including (B) the embryonic outflow tract (OT), and (C) in histological sections of E12.5 embryos particularly in (D) the outflow tract. In adult XS mice, prominent <i>Klf3</i> gene expression was observed in (E) the atria and ventricles and in (F) blood vessels including those of the skeletal muscle shown here in LacZ stained tissues. Histological images show <i>Klf3</i> expression in the myocardium of the (G) atrium (where blood vessels are also heavily stained (arrows)) and in (H) the ventricle where staining is diffuse and punctate. (I) After removing the atria from the heart, prominent expression was seen in cardiac valves (asterisk indicates aortic valve location) and in the aorta (Ao) and pulmonary artery (PA), and coronary vessels (arrows) of the heart. Histological images show strong staining in (J) the aortic valve leaflets (arrow). <i>Klf3</i> gene expression was prominent in (K) the inferior vena cava (left) and aorta (right) and in (L) histological images of vasculature where staining was found in both the smooth muscle and endothelium as shown here for the aorta. Ao, aorta; h, heart; IVC, inferior vena cava; LA, left atrium; LV, left ventricle; OT, outflow tract; PA, pulmonary artery; RA, right atrium; RV, right ventricle.</p

Abnormal cardiac histology in <i>Klf3^H275R</i> embryos.

<p>In homozygotes, histological images showed thinned and disorganized ventricular myocardium and septum (A) at E12.5 and (B) in a rare homozygous survivor at E14.5 (two section levels from same specimen are shown). At E14.5, ventricular (VSD) and atrial septation defects (ASD) were observed in some homozygote and heterozygote hearts. (B) The interventricular septum of heterozygotes at E14.5 appeared thickened relative to WT and homozygotes (black arrows). (C) Heterozygous <i>Klf3</i>^H275R hearts at E14.5 exhibited thickened myocardial and septal wall thicknesses (arrows), diminished left and right ventricular lumens (*), and (D) abnormal hyperplasia of atrioventricular cushion tissue (arrows) (enlargement from (C)). All homozygotes died prenatally. About half of the heterozygotes died before weaning.</p

Abnormal hearts and aortic valves in <i>Klf3</i>^H275R/+ neonates.

<p>(<b>A</b>) Thoracic images of neonates delivered by cesarean-section at E18.5 (term). The heterozygous <i>Klf3</i>^H275R neonate breathed occasionally for 30 min then stopped breathing and died (right). The wild type (WT) neonate (left) breathed and appeared normal before being euthanized for MRI. The ventricular myocardium (V) and septum (S) were markedly thickened in the heterozygous neonate that died. (<b>B</b>) Aortic valve leaflets (arrows) of a heterozygous <i>Klf3</i>^H275R neonate found dead on postnatal day 0 (P0) were thickened (right) relative to a same age control (left). (C) Optical Projection Tomographic images of aortic valves from apparently healthy neonates on day 1 showing thickened aortic valve leaflets in <i>Klf3</i>^H275R/+ (right) compared to WT (left). (<b>D</b>) Serial measurements of aortic peak blood velocity in <i>Klf3^H275R</i>/+ as neonates on day 1 and at 8 wk as adults (n = 7; solid lines join points). Box plots show WT values for day 1 neonates (n = 14) and 8–14 wk adults (n = 21). At day 1, all 7 <i>Klf3^H275R</i>/+ mice had aortic peak velocities within the normal range whereas 6 of 7 were elevated by 8 wk.</p

Cardiovascular changes in adult homozygous (homo) gene deletion mutants.

<p>(A) Elevated peak blood velocity in the ascending aorta of XS and CH gene trap mutants (solid circles) relative to WT controls (open circles). Group means are indicated by horizontal lines. The lower dashed line shows the mean + 2SD and the upper dashed line the mean + 3SD for WT point mutant controls. 5 of 6 XS, and 4 of 5 CH mice had elevated peak velocities (>2SD relative to WT point mutants). (B) Scanning electron microscopy images of the aortic valve of a WT littermate (left) and a XS homozygote with aortic valvular stenosis (right). Note marked thickening of the valve leaflets of the mutant. (C) Arterial blood pressure by tail cuff plethysmography in XS and CH lines. *** P<0.001. N is shown in the bar. Mean ± SE. Note that significant hypotension was observed in the CH line only. (D) Cross-sectional view of the heart of a WT and CH homozygous mutant. Note the enlarged right ventricle (RV) in the mutant. Prominent RV enlargement was observed in 2 of 7 XS and 6 of 8 CH homozygotes but no WT controls. (E) Doppler velocity waveform in the proximal pulmonary artery in a WT and CH homozygous deletion mutant. Reversed diastolic blood velocities (arrows) indicate pulmonary valve regurgitation (observed in 5 of 7 XS and 8 of 8 CH homozygotes but no WT).</p

Adult cardiac parameters measured by micro-ultrasound echocardiography.

<p>Mean ± SE, N is shown in parentheses in column heading or in cell if different, missing N was due to fusion of E and A waves (indicated by #) or for other technical reasons.</p>***<p>P<0.001,</p>**<p>P<0.01,</p>*<p>P<0.05 vs. WT; results were compared with age-matched WT mice with same background (usually littermates).</p><p>WT, wild type; Het, heterozygous; Homo, homozygous.</p><p>BW, body weight; HR, heart rate; PV-Ao, peak velocity in ascending aorta; CO, cardiac output; LV, left ventricle; ESV, end-systolic volume; EDV, end-diastolic volume; EF, ejection fraction; LVWAd, left ventricular wall anterior dimension; LVWPd, left ventricular wall posterior dimension; E-wave, early ventricular filling velocity; A-wave, ventricular filling velocity during atrial contraction; E/A, E-wave to A-wave ratio; ICT, isovolumetric contraction time; IRT, isovolumetric relaxation time; ET, ejection time; Tei Index, myocardial performance index = (ICT + IRT)/ET; LA area, left atrial area; PV-PA, peak velocity pulmonary artery; RV, right ventricle; RA area, right atrial area.</p

Hemodynamic evidence for aortic valvular stenosis in <i>Klf3^H275R</i>/+ mice.

<p>Adult <i>Klf3^H275R</i>/+ mice with high aortic blood velocities and their cage-mate controls were studied. (<b>A</b>) Doppler ultrasound was used to measure transvalvular blood velocity in <i>Klf3^H275R/</i>+ mutants (dark bars) relative to WT (light bars). Blood velocity in the left ventricular outflow tract (LVOT) was similar in mutants and WT but became elevated at the level of the aortic valve and remained elevated in the proximal ascending aorta (24–33 wk adult mice, n = 5 per group, mean ± SE). (<b>B</b>) The transvalvular pressure gradient was measured by pulling a catheter from the left ventricle (LV) to the aorta. The gradient in systolic blood pressure from the LV to the aorta was greater in <i>Klf3^H275R</i>/+ mutant (right) than WT mice (left). (<b>C</b>) Left ventricular systolic blood pressures (LV-SBP) were similar but aortic systolic (Ao-SBP) and aortic diastolic (Ao-DBP) blood pressures were lower in <i>Klf3^H275R</i>/+ mutants (dark bars on right) than WT mice (light bars on left). The LV-SBP to Ao-SBP gradient (mean ± SE above bars) was significantly greater in mutants suggesting aortic stenosis (18–25 wk adult mice, n = 10 WT, n = 9 mutants, mean ± SE). *** P<0.001 <i>Klf3^H275R</i>/+ vs. WT.</p