Heparin-binding EGF-like growth factor and ErbB signaling is essential for heart function

Abstract

The heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR) and the related receptor tyrosine kinase, ErbB4. HB-EGF-null mice (HB(del/del)) were generated to examine the role of HB-EGF in vivo. More than half of the HB(del/del) mice died in the first postnatal week. The survivors developed severe heart failure with grossly enlarged ventricular chambers. Echocardiographic examination showed that the ventricular chambers were dilated and that cardiac function was diminished. Moreover, HB(del/del) mice developed grossly enlarged cardiac valves. The cardiac valve and the ventricular chamber phenotypes resembled those displayed by mice lacking EGFR, a receptor for HB-EGF, and by mice conditionally lacking ErbB2, respectively. HB-EGF-ErbB interactions in the heart were examined in vivo by administering HB-EGF to WT mice. HB-EGF induced tyrosine phosphorylation of ErbB2 and ErbB4, and to a lesser degree, of EGFR in cardiac myocytes. In addition, constitutive tyrosine phosphorylation of both ErbB2 and ErbB4 was significantly reduced in HB(del/del) hearts. It was concluded that HB-EGF activation of receptor tyrosine kinases is essential for normal heart function.

Figure 1

Generation of HB-EGF-null mice. (A) Gene-targeting strategy. Mouse HB-EGF cDNA containing the polyadenylation (pA) sequence flanked by loxP sequences was fused with the first exon of the mouse HB-EGF gene. The lacZ gene was inserted downstream of the HB-EGF cDNA. The targeting vector also contains the neomycin-resistance gene (neo), driven by the phosphoglycerate kinase (PGK) promoter, and the diphtheria toxin A-fragment (DT-A) gene. Cre-mediated recombination results in the deletion of HB-EGF cDNA and in the expression of the lacZ gene. Exon sequences are indicated as black boxes. cDNAs encoding HB-EGF and LacZ are indicated as red and blue boxes, respectively. The loxP sites are indicated by yellow-hatched triangles. E, EcoRI; H, HindIII; K, KpnI; S, SacII; V, EcoRV; X, XhoI. (B) Southern blot analysis of the recombination of the HB^lox allele. Genomic DNA from tails of WT (+/+), HB^lox/+ (lox/+), HB^lox/lox (lox/lox), and HB^del/del (del/del) adult mice was digested with KpnI and hybridized with a lox probe. This probe yields a 9.0-kb fragment from the WT allele, a 2.0-kb fragment from the HB^lox allele, and a 5.3-kb fragment from the HB^del allele. (C) RT-PCR analysis of the expression of HB-EGF in WT (+/+) and HB^del/del (del/del) mice. Transcripts encoding HB-EGF were observed in the heart and kidney, but not in the liver, of WT mice. No HB-EGF transcripts were found in HB^del/del mice. GAPDH (G3PDH) mRNA was measured as a control for RNA preparation. (D) HB-EGF LacZ staining of a 12-wk-old HB^del/+ heart section. (Bar = 50 μm.)

Figure 2

HB-EGF-null mice have heart defects. (A) Representative embryonic day 19.5 (E19.5) WT (+/+) and HB^del/del (del/del) hearts. (B) Representative 6-wk-old WT (+/+) and HB^del/del (del/del) hearts. (C–H) Hematoxylin/eosin staining of transverse sections of hearts at the papillary muscle level. (C and D) WT and HB^del/del hearts, respectively, from an E19.5 embryo. (E and F) WT and HB^del/del hearts, respectively, from 12-wk-old mice. In D blood clots are indicated by asterisks. (G and H) High-magnification pictures of E and F, respectively. (Bars = 2 mm in A and B, 300 μm in C and D, 3 mm in E and F, and 20 μm in G and H.)

Figure 3

Echocardiography. (A) representative records of 2D echocardiograms of WT (+/+) and HB^del/del (del/del) mice. Arrows indicate the end-diastolic (d) and end-systolic (s) dimensions, respectively. (B) Physiological parameters of 8- to 12-wk-old mice. BW, body weight; HR, heart rate; sBP and dBP, systolic and diastolic blood pressure, respectively; FS, fractional shortening calculated as [(LVDd − LVDs)/LVDd] × 100; n.s., not significant. All values are ±SEM.

Figure 4

Cardiac valve defects in HB^del/del mice. (A) Schematic illustration of the heart depicting positions of the valves. (B–E) LacZ staining of longitudinal sections through the hearts of E19.5 HB^del/+ (corresponds to WT) embryos. (B) HB-EGF is expressed at the margins of the pulmonic valve (asterisks) and in tricuspid and mitral valves (arrows). pa, pulmonary artery. (C–E) Higher magnification of areas indicated by the boxes in B. Pulmonic valve (C, asterisks), tricuspid valve (D, arrows), and mitral valve (E, arrow) are shown. (F–K) Histological analysis of cardiac valves. Shown are hematoxylin/eosin-stained longitudinal sections of hearts of E19.5 embryos (F–I) and of 12-wk-old mice (J and K). Mice are WT (F, H, and J) and HB^del/del (G, I, and K). E19.5 aortic valves (F and G, asterisks), E19.5 mitral valves (H and I, arrows), and 12-wk-old aortic (J and K, asterisks) and mitral (J and K, arrows) valves are shown. Pulmonic and tricuspid valves are also enlarged (not shown). (Bars = 150 μm.)

Figure 5

Tyrosine phosphorylation of the ErbB receptor family. (A) DMEM (−) or 5 μg/ml recombinant soluble HB-EGF (HB) was perfused directly into the WT heart. Each ErbB receptor was immunoprecipitated from lysates with its corresponding Ab. The immunoprecipitates (IP) were analyzed by Western blotting (WB) using anti-phosphotyrosine Ab (PY) or each of the four corresponding anti-ErbB Abs. (B) Comparison of the constitutive phosphorylation and protein levels of ErbB receptors in WT (+/+) and HB^del/del (del/del) hearts. Hearts perfused with DMEM were homogenized and analyzed by immunoprecipitation and Western blotting as in A.