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Competency of embryonic cardiomyocytes to undergo Purkinje fiber differentiation is regulated by endothelin receptor expression

Department of Cell Biology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, USA

View larger version (8K): [in a new window]   Fig. 1. Chicken ETA, ETB and ETB2 cDNA clones. Asterisks and double asterisks represent regions used for RNase protection assay and in situ hybridization, respectively. Open box, protein-coding region; line, non-coding region; B, BamHI; E, EcoRI; H, HindIII; K, KpnI; S, SpeI; X, XbaI.

View larger version (127K): [in a new window]   Fig. 2. Whole-mount in situ hybridization of embryonic hearts with antisense riboprobe for VMHC1 (A-D), ETA (E-I), ETB (J-N) and ETB2 (O-T), and with sense probe (insets in F-I). Whole embryos at E3-E3.5 (A,E,J,O) and whole hearts cut frontally into half at E6 (B,F,K,P), E10 (C,G,L,Q), E15 (H,M,S) and E18 (D,I,N) were examined. Purple staining represents positive hybridization signals. (R,T) High power views of the boxed area in Q and S, respectively. Arrows indicate ETB2-signals in valve leaflets. ht, heart tube; at, atrium; rv, right ventricle; lv, left ventricle.

View larger version (56K): [in a new window]   Fig. 3. RNase protection assay of ETA and ETB mRNAs expressed in embryonic chick hearts at various stages during development. (A) Autoradiogram of 32P-labelled probes protected by ETA, VMHC1 and GAPDH mRNAs from whole heart (wh), atrium (at), right ventricle (rv) and left ventricle (lv). VMHC1 and GAPDH served as internal controls to normalize the data of ET receptor mRNAs. Developmental stages are indicated by days of incubation. (B) Quantitation of ETA mRNA levels expressed in ventricular and atrial chambers during heart development. The data from three different experiments were normalized and averaged. (C) Autoradiograms of RNase protection assay for ETA and ETB mRNAs. (D) Quantitation of changes in expression levels of ETA and ETB mRNAs in atrial and ventricular chambers during Purkinje fiber differentiation. The data are presented as the ratio of ETB mRNA to ETA mRNA. (E) Left ventricle-dominant expression of ETB in embryonic hearts. Bars indicate standard deviation.

View larger version (121K): [in a new window]   Fig. 4. Expression pattern of ECE1 and conduction cell markers in embryonic chick hearts (A-F), in monolayer (G,H) and organ culture of myocytes (I-L). (A-C) Whole-mount in situ hybridization of E7 (A) and E13 (B) hearts for ECE1. (C) High power view of the boxed area in B. (D) In situ hybridization on a frozen section of E13 ventricle with antisense ECE1 probe. Purple staining in C,D is positive hybridization signal. Arrows and arrowheads in C,D indicate ECE1-positive arterial and endocardial endothelial cells, respectively. Asterisks in D,E indicate the lumen of ventricular chamber. (E) Immunostaining of a frozen section of E15 ventricle with ALD58 (green signal), a Purkinje fiber marker. (F) Same as E but double immunostaining with ALD58 (green signal) and MF20 (red signal), which detects all sarcomeric myosins. Arrows and arrowheads indicate ALD58-positive periarterial and subendocardial Purkinje fibers, respectively. (G) Monolayer culture of myocytes isolated from E3 heart tube. (H) Same as G but exposed to 10–7 M ET1 for 5 days just after isolation. Blue staining is ALD58-positive signals (arrows). (I) A ventricular segment isolated from E3 heart tube. (J-L) E3 ventricular segment cultured and (J) stained with ALD58, (K) exposed to 10–7 M ET-1 just after isolation and (L) exposed to ET 1 day after isolation. Green signals in J-L are ALD58-positive staining.

View larger version (37K): [in a new window]   Fig. 5. RT-PCR analysis of ETA and ETB mRNAs in heart tubes during organ culture. (A) Amplification of ETA (top) and ETB (bottom) cDNA from heart tubes just after isolation with various cycles of PCR. The number of cycles are indicated. m, molecular weight marker. (B) RT-PCR analysis of ETA and ETB expression during organ culture. The number of days in culture are indicated. GAPDH served as internal control to normalize the data. RT+, RT-PCR with reverse transcriptase. RT-, RT-PCR without reverse transcriptase. (C) Changes in ETA and ETB expression during organ culture. RT-PCR signals of ET receptors (IETR) and GAPDH (IGAPDH) were normalized by an expected degree of PCR amplification, 2m and 2n, respectively. The data are presented as a ratio of ET receptor signal to GAPDH signal from three independent analyses. Bars indicate standard deviation.

View larger version (75K): [in a new window]   Fig. 6. ETA-expression retrovirus and in ovo infection. (A) Proviral structures of control viruses, CXL and CXIZ (Mikawa et al., 1992; Mima et al., 1995) and ETA expression virus, CXIZ-ETA. LTR, long terminal repeat; IRES, internal ribosome entry site; lacZ, ß-galactosidase gene. (B) Northern blot of ETA expression from CXIZ-ETA. Total RNAs (20 µg) from E10 heart (lane a), cells transduced with CXIZ (lane b), and cells transduced with CXIZ-ETA (lanes c and d) were separated on agarose gel, stained with Ethidium Bromide (right panel), hybridized with probe for ETA and autoradiographed (left panel). Endogenous ETA and heterogeneous ETA fused to IRES and lacZ are indicated. 28S, 28S ribosomal RNA; 18S, 18S ribosomal RNA. (C,G) E4 embryos infected with (C) CXIZ and (G) CXIZ-ETA at E3. (D,H) High power views of the boxed areas in C,G. (E,I) E15 hearts infected with (E) CXIZ and (I) CXIZ-ETA. (F,J) Ventricular segments infected with (F) CXIZ and (J) CXIZ-ETA at E3, isolated at E4, and cultured for 5 days. Blue staining in C-J indicates ß-gal positive cells infected with virus.

View larger version (54K): [in a new window]   Fig. 7. Ectopic induction of Purkinje fiber marker by the expression of exogenous ETA. CXIZ- (A-F) or CXIZ-ETA- (G-L) infected heart tubes were removed from embryos, and ventricular segments were isolated and cultured in the absence (A-C,G-I) or the presence (D-F,J-L) of 10–7 M of active ET-1 peptide. Tissues were double immunolabeled in whole mount with anti-ß-gal antibody (red signal) and ALD58 (green signal). Immunolabeled ventricular segments (A,D,G,J) were further processed for frozen sectioning (B,C,E,F,H,I,K,L). (B,E,H,K) ß-gal and (C,F,I,L) ALD58 signals in the same cultures. Arrows indicate cells positive for conduction cell marker ALD58. (M) Ratio of ALD58-positive cells per viral infected cells. In each experimental group, a total of 50-100 cells from five infected hearts were examined and averaged.

View larger version (46K): [in a new window]   Fig. 8. Model for age-dependent differentiation of Purkinje fibers from embryonic myocytes induced by local production of ET ligand and its interaction with ET receptor. In the embryonic chicken heart, there are two types of endothelial cells: ECE-expressing and non-expressing cells. Only the former convert bigET into mature ET. Among ET receptor (ETR)-positive myocytes, only those adjacent to ET production can be induced to differentiate into conduction cells. As embryonic development proceeds, the level of ET receptors in myocytes declines, resulting in the loss of competency to respond to ET ligand and to differentiate into conduction cells.