Systems Analysis Implicates WAVE2 Complex in the Pathogenesis of Developmental Left-Sided Obstructive Heart Defects.

Genetic variants are the primary driver of congenital heart disease (CHD) pathogenesis. However, our ability to identify causative variants is limited. To identify causal CHD genes that are associated with specific molecular functions, the study used prior knowledge to filter de novo variants from 2,881 probands with sporadic severe CHD. This approach enabled the authors to identify an association between left ventricular outflow tract obstruction lesions and genes associated with the WAVE2 complex and regulation of small GTPase-mediated signal transduction. Using CRISPR zebrafish knockdowns, the study confirmed that WAVE2 complex proteins brk1, nckap1, and wasf2 and the regulators of small GTPase signaling cul3a and racgap1 are critical to cardiac development

3-OST-7 regulates BMP-dependent cardiac contraction.

The 3-O-sulfotransferase (3-OST) family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4) expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function

3-OST-7 Regulates BMP-Dependent Cardiac Contraction

<div><p>The 3-O-sulfotransferase (3-OST) family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing <i>tropomyosin4</i> (<i>tpm4</i>) expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin <i>tpm4</i> but not by troponin <i>tnnt2</i>, indicating that <i>tpm4</i> serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of <i>bmp4</i>. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, <i>kcnh2</i>, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, <i>tnnt2</i>, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function.</p></div

3-OST-7 controls region-specific BMP signaling in differentiating heart.

<p>ISH for <i>tbx2b</i> (A and D), <i>notch1B</i> (B and E) showed normal AV-restricted expression, whereas <i>bmp4</i> expression (C and F) showed ectopic expression in ventricular myocardium of 3-OST-7 morphants at 48 hpf (<i>n</i> = 30 for each group). IHC for P-Smad at 48 hpf showed delocalized expression in nuclei of 3-OST-7 morphant ventricle (H) compared to localized AV canal expression in control (G) (<i>n</i> = 10 for each group). (I) Graph depicting increased P-Smad-positive nuclei in the ventricle several unit distances away from the AV in 3-OST-7 morphants compared to P-Smad-positive nuclei localized in the AV for control (error bars, standard deviation). Imaging of live <i>Tg(BRE:d2GFP)</i> fish (J and L) showed GFP expression localized to the AV junction in control (K) and expanded expression in ventricle in morphant (M). V, ventricle; At, atrium; red arrows point to AV; white dashed lines outline the hearts.</p

3-OST-7 regulates cardiac contraction by constraining BMP signaling.

<p>(A) <i>Tg(hs:bmp2b)</i> heterozygotes were crossed to wild-type zebrafish and embryos were either untreated (no hs) or heat-shocked at 12 hpf. Embryos in each group were scored for ventricular contraction, and then genotyped for presence of heat-shock transgene. Graph depicts percentage contraction of embryos with transgene (green) or without (blue) in each treatment group. Induction of BMP signaling led to ventricular noncontraction. (B) <i>bmp4^st72</i> heterozygotes were crossed and embryos were either uninjected or injected with 3-OST-7 MO. Embryos in each group were scored for ventricular noncontraction, and then genotyped for <i>bmp4</i> mutation (RE, digestion with <i>SpeI</i>). Graph depicts percentage contraction of each genotypic class in uninjected embryos (blue) or embryos injected with 3-OST-7 MO (red). Ventricular noncontraction was rescued in 3-OST-7 morphants by <i>bmp4^st72</i> mutation.</p

Model for role of 3-O-sulfation catalyzed by 3-OST-7 in cardiac development.

<p>Under normal conditions, specific 3-OST-7-dependent 3-O-sulfation patterns (pink circles) on endocardial HSPGs constrain <i>bmp4</i> in nonchamber (noncontracting) myocardium (AV junction, red compartment), allowing transcription of <i>tpm4</i> in contracting myocardium (ventricle, green compartment). Tpm4 then stabilizes the sarcomere and ensures proper contraction (Tn, troponin). Knockdown of 3-OST-7 results in loss of 3-O-sulfation, expansion of <i>bmp4</i> and BMP signaling and P-Smad delocalization into ventricular myocardium. High levels of BMP signaling lead to reduced levels of <i>tpm4</i> transcripts and Tpm4 proteins, which then disrupt sarcomere assembly and lead to noncontraction.</p

Noncontraction is correlated with ectopic <i>bmp4</i> expression.

<p>(A) Graph comparing the percentage of normal contraction with 3-OST-7, <i>kcnh2</i>, and <i>tnnt2</i> MO injections. Error bars, SEM (B) Graph comparing patterns of <i>bmp4</i> expression at 48 hpf among control embryos (injected with 3-OST-5 MO), 3-OST-7 morphants, <i>kchn2</i> morphants, and <i>tnnt2</i> morphants. Loss of contraction correlates with ectopic <i>bmp4</i> expression in the ventricle (AV+V) or throughout the entire heart in 3-OST-7, <i>kcnh2</i> and <i>tnnt2</i> morphants.</p

3-OST-7 regulates <i>tpm4</i>-driven myofibrillogenesis, sarcomere assembly and ventricular contraction.

<p>Whole mount IHC was performed on fixed 48(injected with control 3-OST-3Z MO) or 3-OST-7 morphant embryos to detect cardiac sarcomere proteins (<i>n</i> = 30 for each group). The heart was then dissected out of the embryo, mounted on cover slips, and imaged using a confocal microscope (thus, the dorso-ventral orientation of the mounted hearts was random). IHC using anti-Tnnt2 antibody and anti-Tpm antibody revealed levels of these proteins were greatly reduced in 3-OST-7 morphants (B and F, dashed lines outline the hearts) compared to control (A and E) embryos. TEM of control (C) and 3-OST-7 morphant (G) hearts show the presence of organized myofibrils (red arrowheads) in control and absence in morphants. ISH for <i>tpm4</i> showed <i>tpm4</i> transcript levels were decreased in 3-OST-7 morphants (H) compared to control embryos (D) at 48 hpf. (D and H) are ventral views with anterior on top; <i>n</i> = 40 for each group. Overexpression of <i>tpm4</i> rescues the expression of Tnnt2 (I) and Tpm (J) proteins, assembly of myofibrils (K), and the noncontracting ventricle phenotype in 3-OST-7 morphant embryos as assessed by ejection area measurements (L, <i>p</i><0.05, *). The ejection area, a measure of contractility, was obtained by computing the difference between systolic and diastolic area for either atrium or ventricle. At, atrium; V, ventricle; error bars, SEM.</p