Cardiomyocyte-targeted overexpression of the coxsackie–adenovirus receptor causes a cardiomyopathy in association with β-catenin signaling

Abstract

The coxsackie–adenovirus receptor (CAR) is an adhesion molecule found at the intercalated disc of cardiomyocytes in association with other adherens and tight junction proteins. CAR expression is increased at cardiomyocyte junctions in patients with heart failure. It is not known what contribution elevated CAR expression makes to cardiac pathology. We generated a binary transgenic mouse enabling cardiac-restricted doxycycline-regulated expression of Flag-tagged murine CAR (mCAR⁺/αMtTA⁺ mice). Myocardial CAR levels were increased 6-fold in mCAR⁺/αMtTA⁺ mice, localizing to intercalated discs and sarcolemma. Well at birth, mCAR⁺/αMtTA⁺ mice developed a severe cardiomyopathy and died by 4 weeks. Cardiomyocyte hypertrophy was evident at 1 week, with increased heart:body weight ratios by 3 weeks. Disorganization and degeneration of cardiomyocytes were evident with disrupted adherens junctions. Doxycycline administration turned off transgene expression and rescued mice from the development of the cardiomyopathic phenotype. In CAR-overexpressing mCAR⁺/αMtTA⁺ mice, adherens junction proteins were abnormally expressed. N-cadherin protein levels were 83% lower in mCAR⁺/αMtTA⁺ hearts vs controls at 1 week, with levels subsequently increased above controls at 3 weeks. β-catenin expression was 90% and 135% above controls at 1 and 3 weeks, respectively. Nuclear translocation of β-catenin in cardiomyocytes of mCAR⁺/αMtTA⁺ mice was associated with increased c-myc RNA, a target of active β-catenin known to be associated with cardiac hypertrophy. Our study is the first to demonstrate that increased CAR expression can induce a cardiomyopathy and supports a model whereby the pathogenesis is determined by CAR stimulated β-catenin signaling, and/or disruption of the adherens junction.

Introduction

The coxsackie–adenovirus receptor (CAR) is a cell–cell adhesion protein, which has been shown to be a component of the cardiac intercalated disc, the epithelial tight junction [1], and adherens junctions [2], [3], [4]. This 46-kDa integral membrane protein is composed of an extracellular region with two immunoglobulin-like domains, a transmembrane region, and a cytoplasmic domain. CAR mediates attachment and infection by group B coxsackieviruses (CVB) and select adenoviruses, two common causes of viral myocarditis and subsequent dilated cardiomyopathy (DCM) [5], [6].

CAR is ubiquitously expressed and tightly regulated during development. High levels are found in the embryonic heart, and are significantly reduced after birth through adulthood [7], [8], [9]. CAR loss-of-function models have shown that CAR is essential for normal embryonic development [9], [10], [11], [12]. With CAR depletion, altered organization of myofibrils, increased proliferation of cardiomyocytes, and apoptosis within the myocardium have been described [9], [10], [11].

Increased CAR expression is observed in association with myocardial disease and heart failure in humans, such as DCM [2], [13], and in animal models of cardiac injury and inflammation [14], [15]. This suggests a possible role for the protein in the development and progression of cardiomyopathy and heart failure. CAR is found increased at cell–cell contacts in the heart, especially at the intercalated disc [2]. This specialized structure composed of adherens junctions, desmosomes, and gap junctions maintains the structural and the functional integrity of the heart. Conditional knockout of CAR in the adult mouse disrupts intercalated disc proteins and demonstrates that CAR is required for normal atrial-ventricular node function [12], [16]. However, it is not known what the contribution of increased levels of CAR is to the development and progression of heart failure.

To better understand the function of CAR in the heart, we generated a murine CAR (mCAR) transgenic mouse, conditionally overexpressing CAR under the control of a tetracycline/doxycycline (dox)-responsive transcriptional activator (tTA) driven by the α-myosin heavy chain promoter (αMtTA). In this binary transgenic (mCAR⁺/αMtTA⁺) mouse, in the absence of dox, CAR is upregulated during development and in the postnatal mouse. We see a severe cardiomyopathy in mCAR⁺/αMtTA⁺ mice, evolving in association with disruption of the adherens junction and translocation of β-catenin into the nucleus. Subsequently, transcriptional activation and upregulation of c-myc, which is important to developmental and oncogenic growth, as well as implicated in cardiac hypertrophy [17], may contribute to the CAR-induced cardiomyopathic phenotype.