Fig. 3. I_SA 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.