Conditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy

Abstract

The ErbB2 (Her2) proto-oncogene encodes a receptor tyrosine kinase, which is frequently amplified and overexpressed in human tumors. ErbB2 provides the target for a novel and effective antibody-based therapy (Trastuzumab/Herceptin) used for the treatment of mammary carcinomas. However, cardiomyopathies develop in a proportion of patients treated with Trastuzumab, and the incidence of such complications is increased by combination with standard chemotherapy. Gene ablation studies have previously demonstrated that the ErbB2 receptor, together with its coreceptor ErbB4 and the ligand Neuregulin-1, are essential for normal development of the heart ventricle. We use here Cre-loxP technology to mutate ErbB2 specifically in ventricular cardiomyocytes. Conditional mutant mice develop a severe dilated cardiomyopathy, with signs of cardiac dysfunction generally appearing by the second postnatal month. We infer that signaling from the ErbB2 receptor, which is enriched in T-tubules in cardiomyocytes, is crucial for adult heart function. Conditional ErbB2 mutant mice provide a model of dilated cardiomyopathy. In particular, they will allow a rigorous assessment of the role of ErbB2 in the heart and provide insight into the molecular mechanisms that underlie the adverse effects of anti-ErbB2 antibodies.

Figure 1

ErbB2 and ErbB4 are enriched in the T-tubules of adult mouse ventricular cardiomyocytes. Cryosections were stained with antibodies against ErbB2 (A) or ErbB4 (B) and colabeled with anti-vinculin antibodies (C and D). ErbB2 (A and C, red) and ErbB4 (B and D, red) are localized in a transverse membranous network, which is also labeled by vinculin (C and D, green). T-tubular staining in longitudinal sections appears principally as transverse striations. (Bar = 25 μm.)

Figure 2

Cre-mediated mutation of ErbB2 in ventricular cardiomyocytes. (A) Schematic representation of the ErbB2 locus, indicating restriction enzyme sites (black arrowheads: B, BamH1; RV, EcoRV), loxP sequences (red arrowheads), sizes and positions of fragments obtained by digestion of genomic DNA with EcoRV and BamH1 (red lines), and the position of the probe used for hybridization in (B). Note that the ErbB2⁻ allele is indistinguishable from ErbB2^wt under these conditions. (B Upper) Southern blot analysis of genomic DNA from cardiomyocyte preparations (lanes 1–4) or tails of mice (lanes 5, 6), which was digested with EcoRV and BamH1. The genotypes were MLC2v^cre/+; ErbB2^flox/+ (control mice, lanes 1, 2, 5) or MLC2v^cre/+; ErbB2^flox/− (conditional ErbB2 mutant mice, lanes 3, 4, 6). Conditional mutants had markedly impaired cardiac pumping function at the time of analysis. Similar proportions of cardiomyocytes containing the recombined ErbB2^Δ allele were found in the ventricles of control and conditional mutant mice (lanes 1 and 2, 3 and 4, respectively), demonstrating no overt loss of ErbB2^Δ/− cardiomyocytes. (B Lower) Western blot on immunoprecipitates of ErbB2 and ErbB4 analyzed with antibodies directed against ErbB2 or ErbB4. It should be noted that entire ventricular tissue, containing cardiomyocytes and other cell types, was used for this experiment. In conditional mutants, ErbB2 protein is made by cardiomyocytes with the genotype ErbB2^flox/−, i.e., those that escaped cre-mediated recombination, and by endothelia or other cell types that do not express MLC2v^cre. (C–E) Sections of hearts of embryonic day 10.5 embryos, stained with hematoxylin/eosin. (C) Conditional ErbB2 mutant, and (D) control, showing normal heart histology (arrows). (E) Homozygous mutant ErbB2^Δ/− show the myocardial phenotype seen also in ErbB2^−/− mice, i.e., an absence of ventricular trabeculation. (Bars in C–E = 200 μm.)

Figure 3

Conditional ErbB2 mutants develop marked deficits in heart function. Representative examples of ultrasound images of the left ventricle from control (A) and conditional ErbB2 mutant (B) 3-mo-old mice used to determine left ventricular end-diastolic (LVEDD) and end-systolic diameters (LVESD). Vertical axes in A and B represent the size (Bar = 1 mm), and the horizontal axes represent a time course. (C) LVEDD and (D) fractional shortening (FS) of control animals (clear bars) and conditional ErbB2 mutants (solid bars) of the indicated ages. Conditional mutant mice develop ventricular dilatation [1 mo, not significant (n.s.); 2 mo, P = 0, 0025; 3 mo, P = 0.003] and impaired pumping function, which is reflected in reduced FS (1 mo, n.s.; 2 mo, P = 0.0009; 3 mo, P = 0.0003). Analysis of 3-mo-old conditional mutant and control mice before and 1 week after banding of the abdominal aorta (C and D) revealed a significant difference (P = 0.03) in the change in FS. Mean ± SEM are depicted.

Figure 4

Ventricular dilatation and myofiber hypertrophy in conditional ErbB2 mutant mice. Histological (A–D) and electron microscopical analysis (E and F) of control (A, C, and E) and conditional ErbB2 mutant mice (B, D, and F). (A and B) Hematoxylin/eosin-stained sections of the entire heart of 3-mo-old animals. Note the dilatation of both ventricular chambers (ao, aorta; RV, LV, right and left ventricle, respectively) in conditional mutants. (C and D) High magnifications of toluidine blue-stained semithin sections, with myofiber hypertrophy apparent in conditional mutants. (E and F) A normal myofibrillar ultrastructure was observed by electron microscopy in conditional mutants. Note the increased diameter of some T-tubules in the conditional mutant hearts (arrows). (Bars = A and B, 1 mm; C and D, 25 μm; E and F, 2 μm.)