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Am J Physiol Heart Circ Physiol 289: H1153-H1160, 2005. First published April 29, 2005; doi:10.1152/ajpheart.00048.2005
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Dilated cardiomyopathy in Erb-b4-deficient ventricular muscle

Hernán García-Rivello,1 Julián Taranda,1 Matilde Said,2 Patricia Cabeza-Meckert,3 Martin Vila-Petroff,2 Jorge Scaglione,1 Sergio Ghio,1 Ju Chen,4 Cary Lai,5 Ruben P. Laguens,3 Kent C. Lloyd,6 and Cecilia M. Hertig1

1Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Buenos Aires; 2Centro de Investigaciones Cardiovasculares, Universidad de La Plata, La Plata; and 3División de Patología, Universidad Favaloro, Buenos Aires, Argentina; 4Medical School, University of California San Diego, and 5The Scripps Research Institute, La Jolla; and 6Center for Comparative Medicine, University of California Davis, Davis, California

Submitted 18 January 2005 ; accepted in final form 24 April 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The neuregulin receptor tyrosine kinase Erb-b4, initially linked to early cardiac development, is shown here to play a critical role in adult cardiac function. In wild-type mice, Erb-b4 protein localized to Z lines and to intercalated disks, suggesting a role in subcellular and intercellular communications of cardiomyocytes. Conditional inactivation of erb-b4 in ventricular muscle cells led to a severe dilated cardiomyopathy, characterized by thinned ventricular walls with eccentric hypertrophy, reduced contractility, and delayed conduction. This cardiac dysfunction may account for premature death in adult erb-b4-knockout mice. This study establishes a critical role for Erb-b4 in the maintenance of normal postnatal cardiac structure and function.

erb-b2; neuregulin; conditional knockout; mouse; heart

DILATED CARDIOMYOPATHY, a leading cause of heart failure, is believed to result from an abnormal remodeling of cardiac tissue in response to a variety of stressors (for reviews see Refs. 8, 31). Although the majority of dilated cardiomyopathies are of unrecognized etiology, an increasing number of genes have been implicated in this process (31). We present evidence suggesting that the receptor protein tyrosine kinase Erb-b4 is required for the maintenance of normal heart structure and function in adult animals. Erb-b4 is a molecular component of the neuregulin signaling pathway, structurally related to the epidermal growth factor (EGF) receptor family (5, 36). Neuregulin signaling is required for cardiac development, inasmuch as mice that lack the expression of neuregulin-1 (NRG-1) or the presence of the related receptors Erb-b2 and Erb-b4 exhibited an impaired trabeculation of the ventricular wall (11, 21, 25). Biochemical and genetic evidence has demonstrated that NRG-1 acts through Erb-b2/Erb-b4 heterodimers and perhaps through Erb-b4 homodimers in a paracrine manner in cardiac muscle development (14, 22).

To investigate the role of neuregulin signaling in the heart, we determined the consequences of the loss of Erb-b4 function in ventricular muscle cells in vivo. Because of the early demise of erb-b4–/– embryos (11), we generated a conditional erb-b4-knockout (KO) mouse through the cardiomyocyte-specific deletion of an essential exon using Cre-loxP methodologies (6, 23). Mice that are homozygous for deletion of the erb-b4 allele [subsequently referred to as erb-b4 cardiac-KO (erb-b4-KO) mice] survive postnatally. Despite an apparently normal cardiac morphology at birth, erb-b4-KO mice develop a severe dilated cardiomyopathy and an abnormal conduction, leading to premature death. Accordingly, this murine model provides in vivo evidence for the critical role of Erb-b4 signaling in postnatal remodeling of cardiomyocytes and maintenance of the contractile function of the myocardium.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Generation of erb-b4F/F mice. The targeting construct was made from a 15-kb genomic DNA including the entire coding sequence of exon 2 and flanking intronic sequence (obtained from M. Gassmann, MRC Labs, London) cloned into the targeting vector pFLRT. In the pFLRT-erb-b4 targeting construct, two loxP sequences flanked exon 2, while downstream was the positive neomycin (neo) selection marker flanked by two frt sites. After positive/negative selection of pFLRT-erb-b4-transfected R1 embryonic stem cells (27) with G418 and 1-[2-deoxy-2-fluoro-{beta}-D-arabinofuranosyl]-5-iodouracil and screening for homologous recombination by Southern blot analysis, 20–30 R1 cells were microinjected into each of 12–16 C57Bl/6 blastocysts, which were subsequently transferred into the uterus of timed-pseudopregnant CD1 females (18). Chimeric mice were bred with C57Bl/6 females to establish germ-line transmission of the targeted allele.

Breeding and analysis of erb-b4 gene-targeted mice. All experiments were performed in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (US DHHS Publication No. 85-23, Revised 1996, Protocol No. 1-159). The erb-b4F/F mice were crossed into erb-b4+/+:MLC2vCreKI+/– mice and maintained in colonies. The genotype of individual animals was determined by PCR on tail DNA. Southern and Northern analyses were performed as previously described (15). RT-PCR was performed in 0.5 µg of total RNA isolated from ventricles using reverse transcriptase (Life Technologies, Rockville, MD) and followed by PCR amplification using primers corresponding to exons 1 and 4 of erb-b4 cDNA (a gift from M. Gassmann). Ventricles from a mouse of the same genotype were pooled and homogenized or treated for cardiomyocyte isolation following established protocols (13). Western blot analysis for Erb-b4 and Erb-b2 detection was performed in immunoprecipitates using antibodies against Erb-b4 (rabbit polyclonal 0615 and 0618, obtained from C. Lai) or Erb-b2 (Santa Cruz Biotechnology, Santa Cruz, CA), as described elsewhere (14). Briefly, equivalent amounts of proteins from ventricles or isolated cardiomyocytes were lysed in 0.4 ml of buffer containing 1% Triton X-100 and protease inhibitors. The membranes were blotted with primary antibodies, washed, incubated with peroxidase-coupled goat anti-rabbit antibody (Amersham), and developed using enhanced chemiluminescence (ECL, Amersham). Relative amounts of Erb-b4 protein per sample represent the ratio of Erb-b4 to Erb-b2 band intensity. Animals of the following genotypes were used as controls because of their similar Erb-b4 protein level: erb-b4+/+:MLC2vCreKI+/–, erb-b4F/+:MLC2v+/+, erb-b4F/F:MLC2v+/+, and erb-b4F/+:MLC2vCreKI+/–.

Histochemical and morphological analyses. Dissected hearts from female and male control and KO mice were washed in saline phosphate buffer and then treated as follows. For electron microscopy, pieces of ventricular tissue were fixed in 2.5% glutaraldehyde and then in 1% osmium tetraoxide and embedded in epoxy resins. For optical microscopy, hearts were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded 5-µm-thick serial tissue sections were stained with hematoxylin and eosin. The number of myocyte nuclei and the morphometric analyses of free ventricular walls and interventricular septum diameter were determined with an image analyzer on 40 consecutive fields in heart sections stained with hematoxylin and eosin. The morphometric analysis of individual cardiomyocytes was performed in adult myocytes isolated using an alkaline dissociation method (33). The intercalated disks (IDs) were counted in 20 consecutive fields of 28,547 µm2 per sample in tissue sections immunostained with antibodies against ID-related proteins (catenin and connexin isotypes). The cardiomyocyte ploidy index was determined in Feulgen-stained tissue sections on 300 myocytes per sample, which contained longitudinally oriented nuclei and were compared with diploid cells, and was analyzed in digital images (VIDAS Kontron, Zeiss) (35). Cardiomyocyte synthesis of DNA and apoptosis were analyzed by bromodeoxyuridine incorporation (14) (Zymed, San Francisco, CA) and TdT-mediated dUTP nick end-labeling reaction (15) (Intergen), respectively. Immunohistochemical analyses were performed in paraffin-embedded tissue sections by indirect immunostaining using antibodies against Erb-b4 (Labvision, Fremont, CA), Erb-b2 and ZO-1 (Santa Cruz Biotechnology), and sarcomeric {alpha}-actinin and vinculin (Sigma, St. Louis, MO). The primary antibodies were detected by fluorescent labeling with biotin-coupled antibodies and streptavidin-FITC (Amersham), as previously described (13, 14). Immunostained sections were analyzed by confocal microscopy.

Statistical significance between groups was analyzed by ANOVA and Student's t-test.

Contractility determination. Isolated hearts from 3-mo-old female and male wild-type (WT, n = 5) and erb-b4-KO (n = 5) mice were perfused according to the Langendorff technique at constant temperature (37°C), flow (4 ml/min), and heart rate (360 beats/min), as previously described (30). The basal mechanical data obtained in erb-b4-KO mice were age and gender matched to control, WT or heterozygous, mice. The mechanical activity was assessed through an intracardiac water-filled latex balloon connected to a pressure transducer (Perceptor disposable transducer, Namic), achieving a left ventricular end-diastolic pressure of ~10 mmHg. Left ventricular contractile performance was evaluated from the developed pressure at the maximal rise time (+dP/dt) and from the half-relaxation time (t). The response to isoproterenol (300 nM) was determined as percentage of the basal +dP/dt in each sample.

Indo 1 fluorescence and cell-shortening measurements. Myocytes were isolated from adult mouse hearts by a collagenase-based enzymatic digestion, as previously described (34). Rod-shaped myocytes with distinct striations and marked shortening and relaxation on stimulation were loaded with cell-permeant indo 1-AM (17 µM), as previously described (34). Cells were placed on an inverted microscope (Diaphot 200, Nikon) adapted for epifluorescence. Myocytes were superfused with Krebs-Henseleit buffer (pH 7.4) at a constant flow and field stimulated via two platinum electrodes on either side of the bath (square waves, 2-ms duration, and 20% above threshold) at 0.5 Hz. Excitation light was 350 nm, and emission was at 410 and 490 nm to obtain a fluorescence ratio after subtraction of background. The ratio of the indo 1 emission was taken as an indicator of intracellular Ca2+. Cells were illuminated with red light (640–750 nm) through the bright-field illumination optics to allow simultaneous measurements of fluorescence and shortening. Resting cell length and cell shortening were measured by a video-based motion detector (Crescent Electronics) and stored (PowerLab/400 ADInstruments) for offline analysis.

ECG recording. WT and KO mice of both genders and from the same litter were anesthetized with avertin (0.25 mg/g body wt) at 7–10 wk of age. An in vivo electrophysiological monitoring system was used in these studies. The electrodes were placed in the limbs to obtain the six frontal leads or in the chest to obtain the precordial leads. Intracardiac electrophysiological tests were performed with a 2-Fr catheter of eight bipolar pairs (Numed, Hopkinton, NY) (2) placed inside the heart to transmit stimuli and record electrophysiological data. The catheter was connected to an electrical stimulator, which delivered 0.5-mA pulses for 0.2 ms. Additional override pacing was achieved with an SlSl pulse for 200 ms and two extrastimuli for 90 and 70 ms. Bipolar electrograms were acquired with an EXXER multichannel recorder.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Generation of ventricular muscle-specific erb-b4-KO mice. We generated mice containing a "floxed" erb-b4 allele, in which a 3-kb region, including exon 2 of the erb-b4 gene, was flanked by loxP sites. The Cre-recombinase-mediated excision of exon 2 introduced a shift in the reading frame, resulting in the early termination of translation (Fig. 1A). To induce the specific ablation of the erb-b4 floxed alleles, we employed a strain of mice carrying Cre-recombinase coding sequences in the locus of the ventricular myosin light chain MLC2v-Cre knock-in (KI) (6). Coincident with the early expression of MLC2v in ventricular muscle, the MLC2v-Cre-driven recombination events occurred no later than embryonic day 8.5 (7). Mice heterozygous for the MLC2v-CreKI allele display normal cardiac development and function (26). The erb-b4 floxed allele recombination (F*) was observed in the ventricles of heterozygous and homozygous erb-b4-KO mice, but not in other tissues analyzed (Fig. 1, B and C). Genotypic analyses of the offspring showed that erb-b4-KO mice were present at the expected Mendelian ratios, indicating that the loss of this receptor in the ventricles did not result in embryonic lethality.



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  		Fig. 1. Gene targeting strategy to mutate erb-b4 in ventricular muscle. A: schematic representation of 15 kb of genomic DNA, including exon 2 of the erb-b4 gene. Targeting construct contains exon 2 (solid rectangle) flanked by loxP sites (solid arrows), followed by the neomycin cassette (PGK-neo). The erb-b4 wild-type (WT, +), floxed (F), and floxed out (F*) alleles are shown. Primers (1, 2, and 3) and probe (A) were used for PCR and Southern blot analyses, respectively. B: PCR using primers indicated in A in heart and tail DNA of erb-b4F/F:MLC2v+/+, erb-b4F/+:MLC2v-CreKI+/–, and erb-b4F/F:MLC2v-CreKI+/– mice. Positions of erb-b4 F and F* allele fragments are indicated. C: representative Southern blot of BamH I-digested DNA from heart and brain, hybridized with probe A. Positions of erb-b4 F and F* allele fragments (in kilobase pairs) are indicated. D: RT-PCR using primers in exons 1 and 4 of erb-b4 mRNA in total RNA from postnatal ventricles at day 10 corresponding to WT heterozygous and knockout (KO) genotypes. In KO hearts, there is significant expression of short transcripts corresponding to the mutated erb-b4. Positions of WT (F, +) and recombined (F*) transcripts (in kilobase pairs) are indicated. E: expression of erb-b4, and not erb-b2, monitored by immunoprecipitation (IP) followed by Western blot, was markedly reduced in protein extracts from enriched KO ventricles or neonatal cardiomyocytes compared with heterozygous or WT cardiomyocytes.

 
There were no detectable differences in the Erb-b4 expression level or the distribution pattern from hearts of WT or erb-b4 floxed allele mice (data not shown). RT-PCR analysis in total RNA prepared from whole ventricles (mice at postnatal day 10) revealed the expression of long transcripts spanning exons 1–4 of the WT or floxed erb-b4 gene and short transcripts corresponding to the excised erb-b4 allele in exon 2 of homozygous erb-b4-KO mice (Fig. 1D). To examine the loss of Erb-b4 in KO compared with WT myocytes, we determined the relative protein level in extracts from ventricles at embryonic day 14.5 (22 ± 5%, n = 4) or isolated adult cardiomyocytes (18 ± 4%, n = 4; Fig. 1E). The reduction of Erb-b4 protein was comparable to the MLC2v-Cre-recombination events when used in conjunction with RXR{alpha} (7), gp130 (15), and VEGF-A floxed allele mice (12).

Subcellular localization of Erb-b4. Inasmuch as the precise sites of receptor expression may provide functionally relevant clues, we analyzed the subcellular distribution of Erb-b4 and Erb-b2 in cardiac ventricular tissue prepared from 3- to 4-wk-old mice. Confocal analysis of immunofluorescent staining revealed detectable levels of Erb-b4 and Erb-b2 expression in myocytes of trabeculae and interventricular septum. Both transmembrane protein receptors localized to Z lines and IDs of cardiomyocytes (Fig. 2, A and B). This clustering represents the continuous invaginations of the sarcolemma into the transverse t tubules, labeled by the staining of transmembrane ZO-1 proteins (Fig. 2C). The t tubules are located in close apposition to the sarcomeric Z lines marked by {alpha}-actinin (Fig. 2D), representing sites of functional protein interactions involved in the electrical-contraction coupling of cardiomyocytes (for review see Refs. 3, 19). In KO mice, there was virtually no staining for Erb-b4 protein in the myocardium (Fig. 2E). Erb-b2 was targeted to Z lines and abnormally accumulated along the cardiomyocyte membrane compared with control mice at 1 mo of age (Fig. 2F). Immunostaining for ZO-1 and {alpha}-actinin showed a regular alignment in Z lines (Fig. 2, G and H). The localization of proteins associated with the IDs in 3- to 4-wk-old erb-b4-KO mice showed a broader pattern in trabeculae and septum as visualized by the immunostaining of vinculin, localized to costameres and to N-cadherin-mediated junctions (Fig. 3, top). This result was verified by electron microscopy, showing less compact interactions among cardiomyocytes in the interventricular septum (Fig. 3, bottom).



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  		Fig. 2. Localization of Erb-b2 and Erb-b4 to Z lines by immunofluorescent staining in ventricular sections of 3- to 4-wk-old mice with antibodies against Erb-b4 (A and E), Erb-b2 (B and F), ZO-1 (C and G), and {alpha}-actinin (D and H). Note localization of Erb-b4 in Z lines, Erb-b2 and ZO-1 in intercalated disks and Z lines, and {alpha}-actinin at the myofibril Z band (green) in WT mice (A–D). Actin filaments were stained with rhodamine-phalloidin (red) in erb-b4-KO ventricular sections (E–H). Erb-b2 is localized to the Z line and accumulated at the lateral side of cardiomyocytes in trabeculae (arrows). Regular localization of ZO-1 and {alpha}-actinin in Z lines (green) of erb-b4-KO mice is shown. Scale bars, 10 µm.

 


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  		Fig. 3. Localization of vinculin to intercalated disks. Top: immunofluorescent staining in ventricular sections of 3- to 4-wk-old WT and KO mice with antibodies against vinculin (green) and rhodamine-phalloidin (red) to stain actin filaments. Note broader pattern of vinculin at intercalated disks in Erb-b4-deficient myocytes from trabeculae. Scale bar, 10 µm. Bottom: electron micrographs from interventricular septum showing widened intercalated disks in KO compared with WT mice. Scale bar, 0.5 µm.

 
Erb-b4-deficient mice displayed dilated cardiomyopathy and hypertrophy. The erb-b4-KO neonatal mice exhibited normal thickness of right and left ventricular walls and septum (Table 1). However, ventricular chamber dilation was clearly evident by the third mo, as manifested by the significant reduction in wall thickness and the enlargement of the hearts (Fig. 4A, Table 1). Despite relatively normal behavior and activity, erb-b4-KO mice die within the first year of life (Fig. 4B). We investigated a possible reduction in the number of myocytes that could account for the postnatal thinning of the ventricular walls. There were no differences in cell number when nuclei of KO and WT mice were counted at birth (Table 1). There were no changes in cardiomyocyte proliferation or apparent apoptosis monitored by bromodeoxyuridine incorporation into DNA or the TdT-mediated dUTP nick end-labeling reaction, respectively.


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  		Table 1. Phenotypic modifications and cellular hypertrophy

 


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  		Fig. 4. Erb-b4 deficiency results in severe dilation of ventricular chambers. A: transverse ventricular sections of 3-mo-old control WT and KO mice stained with hematoxylin-eosin. Note severe dilation of right and left ventricles (RV and LV) in KO heart. B: survival rate in 139 WT and heterozygous (Het) and 48 KO mice from 23 litters. C: Northern blots of total RNA from adult ventricles of WT, heterozygous, and KO hearts hybridized with skeletal (sk) actin, atrial natriuretic factor (ANF), and brain natriuretic peptide (BNP) cDNA probes. The same blots were stripped and hybridized with GAPDH cDNA probe for sample loading control. D: isolated cardiomyocytes from WT and KO hearts. Myocyte length is increased in KO compared with WT hearts. E: high magnification of ventricular wall from WT (Ct) and KO mice in A. Scale bar, 20 µm. Note enlarged nuclei in KO myocytes compared with Ct. F: electron-microcopic images of ventricles from WT (left) and KO (right) mice. WT myocytes contain myofibrils and mitochondria of normal morphology. Note dilation of tubular membrane system (arrows) in KO myocytes and normally appearing myofibrils and mitochondria. Scale bar, 2.5 µm.

 
The erb-b4-KO mice at 3 mo of age exhibited higher heart-to-body weight ratios than WT, heterozygous (control) mice (8.78 ± 2.55 vs. 5.58 ± 0.80 mg/g) without significant differences in total body weight (30.65 ± 3.2 vs. 30.46 ± 2.9 g), indicative of a hypertrophic growth. The increase in cardiac tissue mass was correlated with the expression of hypertrophy-related genes (29), including skeletal actin and atrial natriuretic factor and brain natriuretic peptide (Fig. 4C). The number of IDs and nuclei per unit area of ventricular muscle (Table 1) was reduced as a result of the myocyte elongation (136.5 ± 34.4 vs. 181.6 ± 50.3 µm, P < 0.05; Fig. 4, D and E). The erb-b4-KO cardiomyocytes showed enlarged nuclei with a significantly higher rate of polyploidy at 3 mo of age (Fig. 4E, Table 1), a parameter also altered in human dilated cardiomyopathy (24). Ultrastructural analysis revealed dilation of the myocyte membrane structures with apparently normal myofibrils and mitochondrial morphology (Fig. 4F).

Cardiac function was assessed in erb-b4-KO compared with WT hearts. Retrograde perfusion of the left ventricle in isolated erb-b4-KO hearts showed a reduction of the maximal left ventricular developed pressure (49.16 ± 3.05 and 24.58 ± 5.29 mmHg in WT and KO mice, respectively) and +dP/dt (1,241.34 ± 107.94 and 587.02 ± 117.57 mmHg/s in WT and KO mice, respectively), demonstrating depressed myocardial contractility (Fig. 5). Although basal +dP/dt data in WT mice appeared relatively low, these values are comparable to and within the range reported for mice of different genetic backgrounds (17, 30). There were no differences in ventricular relaxation, determined by t1/2, between KO and WT hearts (Fig. 5). The response to {beta}-adrenergic agonists was assayed at a maximal dose of isoproterenol, showing an inotropic response in KO and WT hearts (146.94 ± 20.07 and 150.15 ± 18.98% of basal +dP/dt in WT and KO hearts, respectively). This result suggests that Erb-b4 signaling is not required for this physiological response.



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  		Fig. 5. Contractility assay in KO hearts. Left ventricular developed pressure (LVDP) and developed pressure at maximal rise time (+dP/dt) were reduced in 3-mo-old KO (n = 5) compared with WT (n = 5) mice of both genders, and half-relaxation time (t1/2) was normal in KO and WT mice.

 
The contractile performance in isolated cardiomyocytes appeared normal, inasmuch as there were no detectable differences in Ca2+ transients or fractional shortening in response to increasing Ca2+ concentrations or pacing frequency. (Supplemental data can be found at http://ajpheart.physiology.org/cgi/content/full/00048.2005/DC1.) Taken together, these results suggest that the contractile dysfunction as manifested in the context of the whole Erb-b4-deficient heart may relate to defective intercellular coupling.

In addition to the reduced contractility, the ECG of erb-b4-KO mice at 2–3 mo of age displayed an abnormal conduction profile compared with WT, or heterozygous, mice (Fig. 6). The electrical change was characterized by the significant increase in the width of the QRS complex and a 12.5% prolongation in Q-Tc intervals, as monitored by limb and chest electrical recordings (Table 2, Fig. 6, A and B). Furthermore, programmed intracardiac stimulation showed increased susceptibility to ventricular tachycardia in erb-b4-KO compared with WT mice (Fig. 6C).



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  		Fig. 6. ECGs of WT and KO mice. A: representative ECGs from male KO and WT mice. Comparative limb lead recordings show abnormal QRS complex and Q-T interval in 2- to 3-mo-old WT and KO mice (n = 15). B: chest lead recordings show widened QRS complex in KO (n = 6) compared with WT mice (n = 6). C: intracardiac electrogram profiles of overdrive pacing in WT (n = 5) and KO mice (n = 4) registered on a scale of 2 and 1 mV. Electrical stimulation induced ventricular tachycardia in KO mice.

 

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  		Table 2. ECG measurements

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The primary finding of this study is that, in adult mice, specific lack of erb-b4 expression in ventricular muscle leads to a severe dilated cardiomyopathy, resulting in a shortened lifespan. The cardiac dysfunction was characterized by ventricular dilation, reduced contractility, and delayed conduction. These observations are consistent with the cardiac phenotype mediated by the conditional inactivation of the erb-b2 gene (9, 28). Together, these mutant mice illustrate that the loss of neuregulin signaling through Erb-b2 and Erb-b4 heterodimeric receptors prevents normal postnatal cardiac remodeling and function. These murine models may prove useful in the understanding of progressive cardiac failure. For example, acquired cardiomyopathy has been observed in a subset of breast cancer patients undergoing combined treatments with anthracycline derivatives and the monoclonal anti-Erb-b2 antibody Herceptin (32). The observed dysfunction in erb-b4-KO mice indicates that Herceptin antibody may affect myocardial function not only as a modifier of anthracycline cardiotoxicity but because of impaired neuregulin signaling through Erb-b2 and Erb-b4.

Erb-b4 is critical for maintenance of ventricular function. By 3 mo of age, erb-b4-KO mice exhibit the hypertrophic growth of myocytes with enlarged polyploid nuclei, as observed after volume overload. Expression of hypertrophy-related genes was detected in young mice, preceding the overt enlargement of the hearts. The absence of significant changes in the relative cell number suggests that the postnatal hypertrophic response may be related to early modifications in the cellular architecture, ultrastructurally visualized by dilation of the tubular membrane system and widening of the IDs. The restricted localization of Erb-b4 and Erb-b2 to the Z line and IDs, critical sites for the electrical-contraction coupling of the ventricular muscle (3, 19), provides clues as to how these receptors may be involved in regulating cardiomyocyte contractility. Functional studies demonstrated that deletion of erb-b4 in ventricular muscle affected the systolic contractile properties of the myocardium without apparent changes in relaxation or in the {beta}-adrenergic response. This dysfunction was also associated with the abnormally widened QRS complex and Q-Tc intervals. A delayed conduction and repolarization may result in the functional substrate for the observed increase in susceptibility to arrhythmias and death in adult erb-b4-KO mice. A shortened lifespan was also manifested in conditional erb-b2 mutants harboring a "null" erb-b2 allele (28) or under increased stress evoked by aortic banding (9). Together with the reported delay in the onset of dilation (9), our findings suggest that conditional erb-b2-KO mice may display a less severe phenotype, probably because of functional Erb-b4 homodimeric receptor complexes in cardiac muscle.

How does Erb-b4 affect cardiomyocyte architecture? In erb-b4-KO myocytes, neuregulin signaling through Erb-b4 and Erb-b2 should be impaired. In this setting, Erb-b2 could potentially permit additional neuregulin-independent signaling events through interactions with other molecules, including the EGF receptor, gp130, and G protein-coupled receptors (1). Therefore, it is reasonable to assume that the defects likely stem from the loss of specific phosphorylation events performed by one or both of these receptors and from the loss of a single target or multiple targets recruited by activated Erb-b receptors. Erb-b4-mediated activation of the phosphatidylinositol 3-kinase/Akt pathway appears to be critical for growth of the developing heart and protection of myocytes from the cardiotoxic effects mediated by anthracycline derivatives or by oxidative stress (14, 10, 20). In this regard, the lack of cardiotoxic signs (e.g., cell apoptosis, relative metabolic changes, or early modifications in the number or structure of mitochondria and vacuoles) suggests that a highly cooperative signaling network might enhance myocyte survival in vivo under the stress conditions displayed in erb-b4-KO mice.

In addition, the loss of Erb-b4 could also disrupt protein-protein and phosphorylation-independent interactions critical for localized signaling of these receptors. Among these, the most notable are the interactions with PDZ domain-containing proteins (4, 5, 16), which may result in the localized targeting of Erb-b receptors to t tubules. The significant loss of Erb-b4 in ventricular muscle affects the characteristic cytoarchitecture by the dilation of the membrane network, also modified in the conditional erb-b2-KO (28). It is noteworthy that in Erb-b4-deficient cardiomyocytes, Erb-b2 appears to lose normal targeting, inasmuch as some cells showed protein accumulation to the membrane, suggesting that the heterodimeric association of Erb-b4 with Erb-b2 may be critical for its proper clustering. This observation is consistent with the abnormal ID structure in myocytes from trabeculae and the interventricular septum, where Erb-b2 and Erb-b4 are expressed at detectable levels.

Overall, our findings suggest that an alteration in the intercellular coupling results in the impaired ability of the myocardium to develop tension without significant changes in Ca2+ handling or myofibril Ca2+ responsiveness in individual myocytes.

An improved understanding of the molecular mechanisms underlying cardiac remodeling may lead to new therapeutic strategies. In this regard, these findings suggest that the development of agents that mediate Erb-b4 signaling may promote cardiac function and prevent progressive cardiac failure.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Fogarty-National Institutes of Health Grants RO3 TW-01152-03 and PICT01-08291 to C. M. Hertig. P. Cabeza-Meckert is a member of the Research Council, Buenos Aires. C. M. Hertig and R. P. Laguens are members of the National Research Council (Consejo National de Investigiones Cientificas y Tecnicas).


   ACKNOWLEDGMENTS
 
We thank Dr. Alicia Mattiazzi for critical reading of the manuscript, Dr. Tomas Santa Coloma for sharing the confocal microscope facility, Micaela Ricca for animal care and catheterization, and Luis Palacios for histology.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. M. Hertig, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, INGEBI, Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina (E-mail: chertig{at}dna.uba.ar)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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