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Wnt11 patterns a myocardial electrical gradient through regulation of the L-type Ca(2+) channel.

Panáková D, Werdich AA, Macrae CA - Nature (2010)

Bottom Line: Although the traditional planar cell polarity pathway is not involved, we obtained evidence that Wnt11 acts to set up this gradient of electrical coupling through effects on transmembrane Ca(2+) conductance mediated by the L-type calcium channel.These data reveal a previously unrecognized role for Wnt/Ca(2+) signalling in establishing an electrical gradient in the plane of the developing cardiac epithelium through modulation of ion-channel function.The regulation of cellular coupling through such mechanisms may be a general property of non-canonical Wnt signals.

View Article: PubMed Central - PubMed

Affiliation: Brigham and Women's Hospital/Harvard Medical School, Cardiovascular Division, 75 Francis Street, Thorn 11, Boston, Massachusetts 02115, USA.

ABSTRACT
Electrical gradients are critical for many biological processes, including the normal function of excitable tissues, left-right patterning, organogenesis and wound healing. The fundamental mechanisms that regulate the establishment and maintenance of such electrical polarities are poorly understood. Here we identify a gradient of electrical coupling across the developing ventricular myocardium using high-speed optical mapping of transmembrane potentials and calcium concentrations in the zebrafish heart. We excluded a role for differences in cellular excitability, connexin localization, tissue geometry and mechanical inputs, but in contrast we were able to demonstrate that non-canonical Wnt11 signals are required for the genesis of this myocardial electrical gradient. Although the traditional planar cell polarity pathway is not involved, we obtained evidence that Wnt11 acts to set up this gradient of electrical coupling through effects on transmembrane Ca(2+) conductance mediated by the L-type calcium channel. These data reveal a previously unrecognized role for Wnt/Ca(2+) signalling in establishing an electrical gradient in the plane of the developing cardiac epithelium through modulation of ion-channel function. The regulation of cellular coupling through such mechanisms may be a general property of non-canonical Wnt signals.

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Formation of a myocardial electrical gradient in the developing zebrafish ventriclea, b, Isochronal maps of wildtype hearts at 24hpf (a) and 72hpf (b). Each line represents the action potential wavefront position at 5 ms intervals. The colour code depicts the timing of electrical activation (blue areas activated before red areas). Squares indicate regions of interest (ROI) for conduction velocity estimation. The red arrows represent the average velocity vectors: an integral of the mean direction and speed of electrical impulse propagation, in the corresponding ROI. OC=outer curvature, IC=inner curvature.c, Mean estimated conduction velocities from ROIs in a and b. Student's t-test, *p<0.05.d, Averaged action potentials (n=5) from OC and IC ROIs at 72hpf.e, Upstrokes (grey) and derived upstroke velocities (black) of action potentials in d.f, Z projection of 2µm confocal section from a wild-type heart stained with anti-Cx43. The arrowhead points to outflow tract cardiomyocytes with slightly elevated Cx43 levels.g, Average fluorescence intensities of respective ROIs from Cx43-stained hearts (n=10). Student's t-test, ns:p=0.13.h, i, Z projection of 1µm confocal section from a wild-type heart (h) and silent heart (i) stained with anti-β-catenin.j, Mean ventricular cardiomyocyte circularity index and cell area from wildtype (n=4) and silent heart embryos (n=4). Student's t-test, *p<0.05 (wt circularity), ns:p=0.13 (sih circularity), *p<0.05 (wt area), ns:p=0.85 (sih area).f, h, i, 72hpf hearts, atrium at top. Scale bar = 10 µm. c, g, j. Error bars depict SEM.
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Figure 1: Formation of a myocardial electrical gradient in the developing zebrafish ventriclea, b, Isochronal maps of wildtype hearts at 24hpf (a) and 72hpf (b). Each line represents the action potential wavefront position at 5 ms intervals. The colour code depicts the timing of electrical activation (blue areas activated before red areas). Squares indicate regions of interest (ROI) for conduction velocity estimation. The red arrows represent the average velocity vectors: an integral of the mean direction and speed of electrical impulse propagation, in the corresponding ROI. OC=outer curvature, IC=inner curvature.c, Mean estimated conduction velocities from ROIs in a and b. Student's t-test, *p<0.05.d, Averaged action potentials (n=5) from OC and IC ROIs at 72hpf.e, Upstrokes (grey) and derived upstroke velocities (black) of action potentials in d.f, Z projection of 2µm confocal section from a wild-type heart stained with anti-Cx43. The arrowhead points to outflow tract cardiomyocytes with slightly elevated Cx43 levels.g, Average fluorescence intensities of respective ROIs from Cx43-stained hearts (n=10). Student's t-test, ns:p=0.13.h, i, Z projection of 1µm confocal section from a wild-type heart (h) and silent heart (i) stained with anti-β-catenin.j, Mean ventricular cardiomyocyte circularity index and cell area from wildtype (n=4) and silent heart embryos (n=4). Student's t-test, *p<0.05 (wt circularity), ns:p=0.13 (sih circularity), *p<0.05 (wt area), ns:p=0.85 (sih area).f, h, i, 72hpf hearts, atrium at top. Scale bar = 10 µm. c, g, j. Error bars depict SEM.

Mentions: Zebrafish cardiomyocytes begin to spontaneously depolarize in the bilateral cardiac primordia before they assemble into the linear heart tube and by 24 hours post fertilization (hpf), synchronous contractions start. Over the next 24 hours the heart loops and atrium and ventricle form. During morphogenesis, intercellular coupling between differentiating cardiomyocytes is established and refined. Studies of higher vertebrates suggest that transmural and apex-to-base electrical gradients, which emerge during development, are crucial for the electrical stability and mechanical efficiency of the adult heart5. Using high-speed optical mapping of transmembrane potentials we characterized electrical conduction in the embryonic zebrafish heart at cellular resolution (Methods and Supplementary Fig. 1). At 24hpf, action potentials propagate slowly and homogenously throughout the linear heart tube (Fig. 1a, c, Supplementary Movie 1). From 48hpf there is substantial acceleration of impulse propagation in both chambers with the concomitant emergence of zones of slow conduction at sinoatrial, atrioventricular and ventriculo-aortic boundaries (Fig. 1b, Supplementary Movie 2), as previously revealed using lower resolution techniques6,7. We observed a gradient of impulse propagation velocities across the ventricle (Fig. 1b); outer curvature (OC) myocardium, that becomes the ventricular apex, conducts action potentials three times faster than the inner curvature (IC), the future ventricular base (Fig. 1c). This electrical gradient emerges at 48hpf and persists into larval stages (Fig. 2h). These data demonstrate functional heterogeneity across the plane of the cardiac epithelium even in the two-chambered zebrafish heart.


Wnt11 patterns a myocardial electrical gradient through regulation of the L-type Ca(2+) channel.

Panáková D, Werdich AA, Macrae CA - Nature (2010)

Formation of a myocardial electrical gradient in the developing zebrafish ventriclea, b, Isochronal maps of wildtype hearts at 24hpf (a) and 72hpf (b). Each line represents the action potential wavefront position at 5 ms intervals. The colour code depicts the timing of electrical activation (blue areas activated before red areas). Squares indicate regions of interest (ROI) for conduction velocity estimation. The red arrows represent the average velocity vectors: an integral of the mean direction and speed of electrical impulse propagation, in the corresponding ROI. OC=outer curvature, IC=inner curvature.c, Mean estimated conduction velocities from ROIs in a and b. Student's t-test, *p<0.05.d, Averaged action potentials (n=5) from OC and IC ROIs at 72hpf.e, Upstrokes (grey) and derived upstroke velocities (black) of action potentials in d.f, Z projection of 2µm confocal section from a wild-type heart stained with anti-Cx43. The arrowhead points to outflow tract cardiomyocytes with slightly elevated Cx43 levels.g, Average fluorescence intensities of respective ROIs from Cx43-stained hearts (n=10). Student's t-test, ns:p=0.13.h, i, Z projection of 1µm confocal section from a wild-type heart (h) and silent heart (i) stained with anti-β-catenin.j, Mean ventricular cardiomyocyte circularity index and cell area from wildtype (n=4) and silent heart embryos (n=4). Student's t-test, *p<0.05 (wt circularity), ns:p=0.13 (sih circularity), *p<0.05 (wt area), ns:p=0.85 (sih area).f, h, i, 72hpf hearts, atrium at top. Scale bar = 10 µm. c, g, j. Error bars depict SEM.
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Figure 1: Formation of a myocardial electrical gradient in the developing zebrafish ventriclea, b, Isochronal maps of wildtype hearts at 24hpf (a) and 72hpf (b). Each line represents the action potential wavefront position at 5 ms intervals. The colour code depicts the timing of electrical activation (blue areas activated before red areas). Squares indicate regions of interest (ROI) for conduction velocity estimation. The red arrows represent the average velocity vectors: an integral of the mean direction and speed of electrical impulse propagation, in the corresponding ROI. OC=outer curvature, IC=inner curvature.c, Mean estimated conduction velocities from ROIs in a and b. Student's t-test, *p<0.05.d, Averaged action potentials (n=5) from OC and IC ROIs at 72hpf.e, Upstrokes (grey) and derived upstroke velocities (black) of action potentials in d.f, Z projection of 2µm confocal section from a wild-type heart stained with anti-Cx43. The arrowhead points to outflow tract cardiomyocytes with slightly elevated Cx43 levels.g, Average fluorescence intensities of respective ROIs from Cx43-stained hearts (n=10). Student's t-test, ns:p=0.13.h, i, Z projection of 1µm confocal section from a wild-type heart (h) and silent heart (i) stained with anti-β-catenin.j, Mean ventricular cardiomyocyte circularity index and cell area from wildtype (n=4) and silent heart embryos (n=4). Student's t-test, *p<0.05 (wt circularity), ns:p=0.13 (sih circularity), *p<0.05 (wt area), ns:p=0.85 (sih area).f, h, i, 72hpf hearts, atrium at top. Scale bar = 10 µm. c, g, j. Error bars depict SEM.
Mentions: Zebrafish cardiomyocytes begin to spontaneously depolarize in the bilateral cardiac primordia before they assemble into the linear heart tube and by 24 hours post fertilization (hpf), synchronous contractions start. Over the next 24 hours the heart loops and atrium and ventricle form. During morphogenesis, intercellular coupling between differentiating cardiomyocytes is established and refined. Studies of higher vertebrates suggest that transmural and apex-to-base electrical gradients, which emerge during development, are crucial for the electrical stability and mechanical efficiency of the adult heart5. Using high-speed optical mapping of transmembrane potentials we characterized electrical conduction in the embryonic zebrafish heart at cellular resolution (Methods and Supplementary Fig. 1). At 24hpf, action potentials propagate slowly and homogenously throughout the linear heart tube (Fig. 1a, c, Supplementary Movie 1). From 48hpf there is substantial acceleration of impulse propagation in both chambers with the concomitant emergence of zones of slow conduction at sinoatrial, atrioventricular and ventriculo-aortic boundaries (Fig. 1b, Supplementary Movie 2), as previously revealed using lower resolution techniques6,7. We observed a gradient of impulse propagation velocities across the ventricle (Fig. 1b); outer curvature (OC) myocardium, that becomes the ventricular apex, conducts action potentials three times faster than the inner curvature (IC), the future ventricular base (Fig. 1c). This electrical gradient emerges at 48hpf and persists into larval stages (Fig. 2h). These data demonstrate functional heterogeneity across the plane of the cardiac epithelium even in the two-chambered zebrafish heart.

Bottom Line: Although the traditional planar cell polarity pathway is not involved, we obtained evidence that Wnt11 acts to set up this gradient of electrical coupling through effects on transmembrane Ca(2+) conductance mediated by the L-type calcium channel.These data reveal a previously unrecognized role for Wnt/Ca(2+) signalling in establishing an electrical gradient in the plane of the developing cardiac epithelium through modulation of ion-channel function.The regulation of cellular coupling through such mechanisms may be a general property of non-canonical Wnt signals.

View Article: PubMed Central - PubMed

Affiliation: Brigham and Women's Hospital/Harvard Medical School, Cardiovascular Division, 75 Francis Street, Thorn 11, Boston, Massachusetts 02115, USA.

ABSTRACT
Electrical gradients are critical for many biological processes, including the normal function of excitable tissues, left-right patterning, organogenesis and wound healing. The fundamental mechanisms that regulate the establishment and maintenance of such electrical polarities are poorly understood. Here we identify a gradient of electrical coupling across the developing ventricular myocardium using high-speed optical mapping of transmembrane potentials and calcium concentrations in the zebrafish heart. We excluded a role for differences in cellular excitability, connexin localization, tissue geometry and mechanical inputs, but in contrast we were able to demonstrate that non-canonical Wnt11 signals are required for the genesis of this myocardial electrical gradient. Although the traditional planar cell polarity pathway is not involved, we obtained evidence that Wnt11 acts to set up this gradient of electrical coupling through effects on transmembrane Ca(2+) conductance mediated by the L-type calcium channel. These data reveal a previously unrecognized role for Wnt/Ca(2+) signalling in establishing an electrical gradient in the plane of the developing cardiac epithelium through modulation of ion-channel function. The regulation of cellular coupling through such mechanisms may be a general property of non-canonical Wnt signals.

Show MeSH
Related in: MedlinePlus