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First published online 7 January 2004
doi: 10.1242/dev.00947

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Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart

Christopher E. Hall^1,*, Romulo Hurtado^1,*, Kenneth W. Hewett^2,*, Maxim Shulimovich^1,*, Clifton P. Poma^1,*, Maria Reckova², Chip Justus², David J. Pennisi¹, Kimimasa Tobita³, David Sedmera², Robert G. Gourdie² and Takashi Mikawa^1,

¹ Department of Cell and Developmental Biology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, USA
² Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425, USA
³ Department of Pediatrics, Children's Hospital of Pittsburgh, Pittsburgh, PA 15213, USA

View larger version (44K): [in a new window]   Fig. 1. Optical mapping of electrical activation in the embryonic chick heart. (A) Setup consisting of epifluorescence microscope, temperature-controlled oxygenated organ bath and computer-controlled intensified high-speed camera. (B) Brightfield image of E6.5 heart immediately after recording. (C) Epifluorescence signals of di-4-ANEPPS from the same heart captured by the high-speed camera (80x80 pixels). (D) An example of changes in fluorescence intensity over time from a 4x4 pixel region of the ventricle (raw 12 bit data). Drops in fluorescent intensity level correspond to depolarization. (E) A typical action potential recording from a single pixel that was inverted, digitally filtered (white line), and the first derivative (yellow) calculated. Peak of the first derivative, corresponding to maximum upstroke velocity, was determined (orange) to mark the time of activation of individual pixels. x axis scale in milliseconds. (F) An example of two-dimensional array of the optical mapping data. la, left atrium; ra, right atrium; lv, left ventricle; rv, right ventricle.

View larger version (78K): [in a new window]   Fig. 2. Developmental changes of ventricular activation sequence and the expression of Cx40 and ECE1. (A-C) Isochronal maps of action potential propagation recording on the dorsal ventricular surface of looping, and septating stage hearts. The first activation site is indicated in red. Developmental stages and time resolution of each frame are also indicated. (D) Topological shift of ventricular activation sequences from the immature to the mature pattern during embryogenesis is presented as % of hearts exhibiting the apex-to-base pattern. More than 12 hearts examined at each time point. (E-G) Whole-mount in situ hybridization for Cx40 mRNAs (positive signals indicated by white arrows) in hearts cut to half frontally at stages indicated. Insets are controls stained with sense probes. (H) A transverse section of E8.5 heart stained for Cx40. (I) High power view of the boxed area in H. (J-L) As in E-G but for ECE1 mRNAs. (M) As in H but for ECE1. (N) High power view of the boxed area in M. at, atrium; avj, atrioventricular junction; ec, endocardium; ivs, interventricular septum; lv, left ventricle; ot, outflow tract; rv, right ventricle; tb, trabeculum.

View larger version (62K): [in a new window]   Fig. 3. ISA channel antagonist-induced downregulation of ECE1 and Cx40 expression levels in vivo. (A) In ovo image of E3.5 embryo during injection through the anterior or posterior vitelline veins (avv and pvv). (B) High-power view of the microinjection site into the posterior vitelline vein. Arrow indicates direction of blood flow carrying the injected gadolinium (Gd3+)-containing Tyrodes's saline visualized by Trypan Blue. (C) Gel displaying RT-PCR amplicons of GAPDH, ECE1 and Cx40 from control (–) and gadolinium (+)-injected E4 ventricles. (D) Dose response of ECE1 expression to gadolinium injection. The data of three independent experiments were averaged and normalized by those of control. [Gd3+]f indicates the estimated final concentration of injected gadolinium (3 µl of 0 mM, 10 mM and 100 mM), using average wet weight of embryos as the total volume. (E) Time course of ECE1 and Cx40 expression levels following injection of 3 µl of 10 mM gadolinium-containing Tyrode's saline at E7.5. The data are presented as the ratio of expression levels in gadolinium-injected hearts to those in control saline-injected hearts and normalized to GAPDH. (F) Relative expression levels of ECE1 and Cx40 to GAPDH in individual ventricles of E8 hearts injected with control (open circle) and 10 mM gadolinium-containing (filled circle) Tyrode's saline at E7.5. Line corresponds to best fit analysis of data points.

View larger version (55K): [in a new window]   Fig. 4. In situ hybridization for ECE1 (A-C,G-I) and Cx40 (E-F,J-L) in E8-8.5 control (A-F) and gadolinium-injected (G-L) hearts. High power view of boxed areas in transverse sections (B,E,H,K) of the ventricle of whole-mount stained hearts are presented in C,F,I,L, respectively. at, atrium; avj, atrioventricular junction; ec, endocardium; ivs, interventricular septum; lv, left ventricle; ot, outflow tract; rv, right ventricle; tb, trabeculum.

View larger version (56K): [in a new window]   Fig. 5. Delayed patterning of ventricular activation sequences by gadolinium-injection at E7.5. (A) Isochronal map of control E8 heart with the apex-to-base activation pattern. (B) As in A but gadolinium-injected heart still exhibiting an immature lateral activation pattern. (C) Proportion of hearts exhibiting mature (white), intermediate (see text) (gray) and immature (black) activation patterns following no injection (control), buffer and gadolinium injection. Chi Square test between buffer- and gadolinium-injected groups gave a significant P value of 0.00012, while it found an insignificant P value of 0.521 between uninjected (control) and buffer-injected hearts. n, number of hearts examined. at, atrium; avj, atrioventricular junction; ec, endocardium; ivs, interventricular septum; lv, left ventricle; ot, outflow tract; rv, right ventricle; tb, trabeculum.

View larger version (49K): [in a new window]   Fig. 6. Increased levels of ECE1 and Cx40 expression by CTB-induced pressure overloading between E3.5 and E6-6.5. (A) Video-captured image of an E3.5 embryo just after CTB (arrow, suture). (B) High power view of the heart region in A, showing a banding of the outflow tract without constriction at this stage. (C) E6.5 sham-operated heart. (D) E6.5 CTB (arrow) heart. (E) Relative expression levels of ECE1 and Cx40 normalized to GAPDH in individual ventricles of E6-6.5 control (white circle) and CTB (black circle) hearts. Line corresponds to best fit analysis of data points. Inset is an example of RT-PCR amplicons of GAPDH, ECE1 and Cx40 from control and CTB ventricles.

View larger version (57K): [in a new window]   Fig. 7. In situ hybridization for ECE1 (A-C,G-I) and Cx40 (E-F,J-L) in E6-6.5 control (A-F) and CTB (G-L) hearts. High-power view of boxed areas in transverse sections (B,E,H,K) of the ventricle of whole-mount stained hearts are presented in C,F,I,L, respectively. at, atrium; avj, atrioventricular junction; ec, endocardium; ivs, interventricular septum; lv, left ventricle; ot, outflow tract; rv, right ventricle; tb, trabeculum.

View larger version (56K): [in a new window]   Fig. 8. Precocious patterning of ventricular activation sequences by ventricular pressure overload (A) Isochronal map of control E6-6.5 heart with an immature lateral activation pattern across the dorsal surface of the ventricle. (B) As in A but the ventral view. (C) As in A but CTB-heart already exhibiting the apex-to-base activation pattern. (D) As in C but the ventral view with a mature pattern. (E) Proportion of hearts exhibiting immature (nonapex-to-base, open bar) and mature (apex-to-base, filled bar) activation patterns following no operation (control), sham operation and CTB. at, atrium; avj, atrioventricular junction; ec, endocardium; ivs, interventricular septum; lv, left ventricle; ot, outflow tract; rv, right ventricle; tb, trabeculum.