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. 2009 Nov;7(11):e1000246.
doi: 10.1371/journal.pbio.1000246. Epub 2009 Nov 17.

Reversing blood flows act through klf2a to ensure normal valvulogenesis in the developing heart

Julien Vermot  1 , Arian S Forouhar, Michael Liebling, David Wu, Diane Plummer, Morteza Gharib, Scott E Fraser
Affiliations

Reversing blood flows act through klf2a to ensure normal valvulogenesis in the developing heart

Julien Vermot et al. PLoS Biol. 2009 Nov.

Abstract

Heart valve anomalies are some of the most common congenital heart defects, yet neither the genetic nor the epigenetic forces guiding heart valve development are well understood. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop, there is considerable regurgitation, resulting in reversing (or oscillatory) flows between the atrium and ventricle. As reversing flows are particularly strong stimuli to endothelial cells in culture, an attractive hypothesis is that heart valves form as a developmental response to retrograde blood flows through the maturing heart. Here, we exploit the relationship between oscillatory flow and heart rate to manipulate the amount of retrograde flow in the atrioventricular (AV) canal before and during valvulogenesis, and find that this leads to arrested valve growth. Using this manipulation, we determined that klf2a is normally expressed in the valve precursors in response to reversing flows, and is dramatically reduced by treatments that decrease such flows. Experimentally knocking down the expression of this shear-responsive gene with morpholine antisense oligonucleotides (MOs) results in dysfunctional valves. Thus, klf2a expression appears to be necessary for normal valve formation. This, together with its dependence on intracardiac hemodynamic forces, makes klf2a expression an early and reliable indicator of proper valve development. Together, these results demonstrate a critical role for reversing flows during valvulogenesis and show how relatively subtle perturbations of normal hemodynamic patterns can lead to both major alterations in gene expression and severe valve dysgenesis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Transvalvular oscillatory flow patterns change during heart valve morphogenesis and gene expression in the AV canal.
(A–C) Average transvalvular flow direction as a function of time for wild-type hearts as seen in the AV canal (light magenta box) or atrium (light blue box) between 36, 48, and 58 hpf in the area highlighted in the heart drawings (ventral view, anterior to the top). Anterograde flow from the atrium to ventricle is shown in black, retrograde flow from the ventricle to the atrium in red, and no flow between the chambers is shown in white. The sequence of time segments with retrograde, anterograde, and no-flow fractional periods are depicted in red, black, and white, respectively. The retrograde flow fraction (RFF) is the fraction of the cardiac cycle that is red. (D–F) cmlc2 expression reveals changes in heart morphology. cmlc2 is expressed in the heart tube in the anterior region at 36 hpf (D), and is expressed strongly in the ventricle and weakly in the atrium at 48 and 54 hpf (E and F). (G–O) Expression of notch1b, klf2a, and bmp4 progressively becomes localized to the AV canal during valve specification. mRNA distribution of notch1b (G–I), klf2a (J–L), bmp4 (M–O) at 36 hpf (D, G, J, and M), 48 hpf (E, H, K, and N), and 56 hpf (F, I, L, and O). notch1b is found in the anterior part of the heart tube at 36 hpf (G), and becomes stronger in the AV canal and in the ventricle at 48 hpf (H). At 54 hpf, notch1b expression becomes restricted in the AV canal and the outflow tract (I). klf2a expression is found throughout the heart tube at 36 hpf (J) and becomes stronger in the AV canal and in the atrium at 48 hpf (K). At 56 hpf, klf2a is exclusively expressed in the AV canal and the outflow tract, displaying an expression pattern very similar to notch1b (L). bmp4 expression is found in the anterior part of the heart tube at 36 hpf (M) and becomes progressively concentrated at the level of the AV canal from 48 to 54 hpf (N and O). Anterior to the top, white arrows point to the AV canal, black arrows to the outflow tract. Scale bar indicates 50 µm.
Figure 2
Figure 2. Decreased retrograde flow via lowered blood viscosity affects valve morphogenesis.
(A–D) Flow pattern at 48 hpf in (A) control and after (B) gata1, (C) gata2, and (D) gata1/2 knock down. gata2 inactivation leads to a dramatic decrease in the RFF, whereas gata1 and gata1/2 knock downs exhibit increased RFF compared to the control. (E–H) Confocal sections of the valve-forming region in (E) control, (F) gata1, (G) gata2, and (H) gata1/2 morphants. Only gata2 morphants at 96 hpf show valve dysgenesis. Scale bar indicates 50 µm. (I–K) Quantitative RT-PCR showing the expression level of several flow-responsive genes after gata1, gata2, or gata1/2 knock down. **p<0.01, ANOVA. (L) Percentage of embryos displaying valve malformation at 96 hpf (red bar), hematocrit level (yellow bar), and RFF (blue bar) observed in morphants and controls at 48 hpf. The proportions were significantly different at a 10% level of significance (α = 0.1). (M) Outline summarizing the experimental outcome of manipulating oscillatory flow by decreasing circulating blood cells. The color code for gene expression is the same as in (I).
Figure 3
Figure 3. Decreased oscillatory flow decreases klf2a expression.
(A) RFF is decreased by alterations in heart rate. The highest RFF is seen at the control heart rate (>30% between 1.5 and 2 Hz) at 48 hpf. Raising fish at lowered or elevated temperatures slows or speeds heart rate and significantly decreases RFF. Lidocaine treatment decreases heart rate and RFF (blue data point). The decreased heart rate and RFF is rescued by elevating the temperature to 34°C (red data point). (B) Decreased RFF from treatment with lidocaine or with high-temperature (34°C) leads to valve defects. The maximal effect is observed when treatment is initiated at 36 h. (C–E) Valve formation in normal and lidocaine treated embryos. (C) Embryos that were raised in control conditions have valve leaflets (white arrows). (D) Embryos in which RFF was decreased by lidocaine treatment from 31 to 55 hpf have endocardial tissue thickening (asterisk) but no valve leaflets are apparent (50%, n = 36). (E) Heart valve dysgenesis in fish exposed to 0.15% lidocaine for 24 h is rescued by incubating it at 34°C to restore normal RFF. Heart valve leaflets are present and function normally (white arrows). All embryos are imaged at 96 hpf. A, atrium; V, ventricle. (F–H) klf2a expression in 46-hpf-old embryos is altered by lidocaine treatment. (F) klf2a expression is localized at the AV boundary in control embryos. (G) klf2a expression decreases after 15-h lidocaine treatment (90%, n = 67). (H) Restoring heart rate and RFF to normal by raising the fish at 34°C restores klf2a expression (90%, n = 45). Anterior to the top. (I–K) nppa expression remains largely unaffected by lidocaine treatment and temperature rescue. (L) Quantitative RT-PCR showing the expression level of several flow-responsive genes after lidocaine treatment. klf2a expression is significantly decreased after 6 and 10 h of treatment and is restored by incubation at 34°C; 100% of expression corresponds to a normal expression level. *p<0.05; **p<0.01, ANOVA. (M) Outline summarizing the experimental outcome of decreasing oscillatory flow by decreasing heart rate. The color code for gene expression is the same as in (L).
Figure 4
Figure 4. Morpholine antisense oligonucleotide treatment decreases expression of the flow-responsive gene klf2a results in valve dysgenesis.
(A–D) Valve leaflets scored at 96 hpf show effects of klf2a MO. (A and C) Sham-injected embryos form normal heart valves. (B and D) klf2a MO-treated embryos display valve dysgenesis, often with a complete absence of valve leaflets. (C and D) Detailed views of valve morphology. (C) Control embryo has clearly distinguishable valve leaflets (arrows). (D) klf2a MO-treated embryo has no valve leaflets forming from the endocardium (arrow) (52%, n = 46). The proportions were significantly different at a level of significance α = 0.01. Scale bars indicate 50 µm. (E and F) Average flow pattern at 48 hpf in controls (E) and klf2a morphants (F) showing that the RFF is unaffected in the mutants but that the heart rate is slightly decreased. (G–L) Expression of three marker genes at 48 hpf in normal and klf2a morphants. (G–J) nppa expression is normal in the klf2a morphants, showing that chamber specification occurs independently of klf2a. (I and J) bmp4 mRNA distribution at 48 hpf showing that expression is decreased in the MO-treated embryo in the AV node region at 48 hpf (n = 23, 40%; compare expression at arrow in panels [I and J]). (K and L) notch1b expression at 48 hpf decreases in the AV boundary of the klf2a morphants (n = 45, 71%). Arrows point to the AV boundary in all panels (G–L). (M) Summary of quantitative RT-PCR showing the expression level of flow-responsive genes in klf2a morphants. Expression of all genes decreases significantly, confirming the down-regulation of bmp4 and notch1b observed by ISH. **p<0.01, ANOVA. (N) Summary diagram of klf2a function during heart valve formation. klf2a acts as a transcriptional relay between the reversing flow generated by the circulating blood cells at the AV canal and several genes activated in the AV endothelial cells (such as notch1b, neuregulin1, and endothelin1). klf2a also affects the expression of bmp4, revealing a possible interaction between myocardium and endothelium essential for valve morphogenesis.
Figure 5
Figure 5. Comparison of the valve phenotype between the different treatments affecting valvulogenesis in transgenic Tg(flk1:EGFP) zebrafish at 72 hpf.
GFP is expressed in the endothelial cell layer and highlights the developing valves. (A, F, and F') control embryo, (B, G, and G') gata1 morphant, (C, H, and H') lidocaine treated, (D, I, and I') gata2 morphant, and (E, J, and J') klf2a morphant. Each treatment lead to an incomplete ingression of the endothelial cells in order to make a functional leaflet except in gata1 morphants. (F'–J') Schematic representation of the panels (F–J) underlining the endothelial cells within valve-forming region (yellow) and the heart lumen (white). A, atrium; V, ventricle. (K–O) Three-dimensional reconstruction of 10 µm depth of the AV area in control (K), gata1 morphants (L), lidocaine-treated embryo (M), and gata2 (N) and klf2a (O) morphants. The white arrows point to the cell that has been reconstructed in three dimensions and which is presented in (P–T). (P–T) Side view (left) and top view (right) of a reconstructed cell of the AV canal. (U) Schematic drawing showing the approach used to define cuboidal versus non-cuboidal cell shape. (V) Graph summarizing the number of cells counted in the AV canal (corresponding to the yellow cells in [F'–J']) (blue bars), and the ratio between cuboidal versus non cuboidal cell shape (red bars). Yellow arrows in (A–E) point to the endocardial ring.
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