Homomeric Kv7.2 channels are inhibited by tamoxifen.

<p>A, Current traces of Kv7.2 in presence and absence of tamoxifen 1 µM. B, Current-voltage relationship in control (squares, n = 5) and in presence of 1µM tamoxifen (circles, n = 5). C, Kv7.2 activation curves in control (squares, n = 5) and in presence of 1 µM tamoxifen (circles, n = 5). D, Concentration–effect relationship for block of Kv7.2 channels by tamoxifen. IC₅₀ = 0.74 ± 0.16 µM, n_H = 0.4 ± 0.05(n = 5). The line represents the fit of the experimental data by a Hill equation with the values given in the text.</p

Tamoxifen inhibited Kv7.2/Kv7.3 channels expressed in HEK-293 cells.

<p>A and B, ionic currents recorded in control condition (A) and in presence of 1 µM tamoxifen (B) using voltage protocol shown in the inset. C, Current-voltage relationship in control (square, n = 6) and in presence of 1 µM tamoxifen (circles, n = 6). D, Kv7.2/Kv7.3 activation curves in control (squares, n = 5) and in presence of 1 µM tamoxifen (circles, n = 6).</p

The affinity of the heteromeric mutant channels Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 for PIP₂ correlates with the degree of current inhibition by tamoxifen.

<p>A and B, Superimposed representative Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 current traces recorded in control (black) and in presence of 10 µM tamoxifen (gray). Currents were evoked depolarizing the membrane to +40 mV for 3 s and then repolarizing to -60 mV. C, Concentration–effect relationship for the inhibitory effect of tamoxifen on Kv7.2/Kv7.3 WT channels (dashed line) and the mutants Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 (triangles and diamonds respectively). The line represents the fit of the experimental data by a Hill equation with the values given in the text. Each point represents the mean ± SEM from <i>n</i> = 5 to 6 experiments.</p

Tamoxifen inhibition of Kv7.2 channels is related to PIP₂-channel affinity.

<p>A and B, Superimposed representative Kv7.2 R463Q and Kv7.2 R463E current traces recorded in control (black) and in presence of 10 µM tamoxifen (gray). Currents were evoked depolarizing the membrane to +40 mV for 3 s and then repolarizing to -60 mV. C, Concentration–effect relationship for the inhibitory effect of tamoxifen on Kv7.2 WT channels (dashed line) and the mutants Kv7.2 R463Q (triangles) and Kv7.2 R463E (diamonds). The line represents the fit of the experimental data by a Hill equation with the values given in the text. Each point represents the mean ± SEM from <i>n</i> = 5 to 6 experiments. D, Activation curves for Kv7.2 WT (dashed line, n = 5), Kv7.2 R463Q (triangles, n = 6) and Kv7.2 R463E (diamonds, n = 6). E, Deactivation time constants resulting from the monoexponential fit of the tail current decay (τ_deact) at -60 mV after a test pulse to +40 mV, are shown for Kv7.2 WT (n = 5), Kv7.2 R463Q (n = 6) and Kv7.2 R463E (n = 6).</p

Overexpression of PIP5K-2A diminished the effect of tamoxifen on Kv7.2/Kv7.3 and Kv7.2 R463Q channels.

<p>Inhibition of the currents recorded at 40 mV by tamoxifen 1 µM when Kv7.2/Kv7.3 and Kv7.2 R463Q channels were expressed alone compared to the effect of tamoxifen 10 µM when those channels were co-expressed with the kinase.</p

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

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