The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that belongs to the family of adhesion molecules. In the postnatal heart, it is localized predominantly at the intercalated disc, where its function is not known. Here, we demonstrate that a first degree or complete block of atrioventricular (AV) conduction developed in the absence of CAR in the adult mouse heart and that prolongation of AV conduction occurred in the embryonic heart of the global CAR-KO mouse. In the cardiac-specific CAR-KO (CAR-cKO) mouse, we observed the loss of connexin 45 localization to the cell-cell junctions of the AV node but preservation of connexin 40 and 43 in contracting myocardial cells and connexin 30.2 in the AV node. There was also a marked decrease in β-catenin and zonula occludens-1 (ZO-1) localization to the intercalated discs of CAR-cKO mouse hearts at 8 weeks before the mice developed cardiomyopathy at 21 weeks of age. We also found that CAR formed a complex with connexin 45 via its PSD-95/DigA/ZO-1-binding (PDZ-binding) motifs. We conclude that CAR expression is required for normal AV-node conduction and cardiac function. Furthermore, localization of connexin 45 at the AV-node cell-cell junction and of β-catenin and ZO-1 at the ventricular intercalated disc are dependent on CAR.

No abstract

Heart failure in children and adults is often the consequence of myocarditis associated with Coxsackievirus (CV) infection. Upon CV infection, enteroviral protease 2A cleaves a small number of host proteins including dystrophin, which links actin filaments to the plasma membrane of muscle fiber cells (sarcolemma). It is unknown whether protease 2A-mediated cleavage of dystrophin and subsequent disruption of the sarcolemma play a role in CV-mediated myocarditis. We generated knockin mice harboring a mutation at the protease 2A cleavage site of the dystrophin gene, which prevents dystrophin cleavage following CV infection. Compared with wild-type mice, we found that mice expressing cleavage-resistant dystrophin had a decrease in sarcolemmal disruption and cardiac virus titer following CV infection. In addition, cleavage-resistant dystrophin inhibited the cardiomyopathy induced by cardiomyocyte-restricted expression of the CV protease 2A transgene. These findings indicate that protease 2A-mediated cleavage of dystrophin is critical for viral propagation, enteroviral-mediated cytopathic effects, and the development of cardiomyopathy. IntroductionCoxsackievirus (CV) is a member of the enteroviral genus of the picornavirus family and is known to be an important cause of myocarditis and heart failure in children and adults. Enteroviral protease 2A cleaves the viral polyprotein and a small number of host cell proteins such as the cytoskeletal protein dystrophin (1) and the eukaryotic translation initiation factors eIF4G1 and eIF4G2 (2-4). Genetic deficiency of dystrophin causes cardiomyopathy in Duchenne muscular dystrophy and increases susceptibility to myocarditis (5, 6). However, the importance of protease 2A-mediated cleavage of dystrophin is not known. We hypothesized that cleavage of dystrophin by protease 2A is important in sarcolemmal membrane disruption, viral propagation, enteroviral-mediated cytopathic effects, and the development of viral myocarditis. In order to address this hypothesis, we knocked in a mutation at the protease 2A cleavage site of the dystrophin gene, thus inhibiting only the cleavage of dystrophin following CVB3 infection. When mice expressing cleavage-resistant dystrophin were infected with CVB3, there was a decrease in the sarcolemmal disruption, cardiac virus titer, and severity of myocarditis compared with control mice expressing cleavable wild-type dystrophin. In addition, the prevention of dystrophin cleavage in protease 2A-expressing transgenic mice (7) markedly inhibited the protease 2A-induced myocytopathic effect and cardiomyopathy. These findings indicate that disruption of the sarcolemma by protease 2A-mediated cleavage of dystrophin can have a critical role in the pathogenesis of viral myocarditis via alterations in viral propagation and enteroviral-mediated cytopathic effects in the intact wild-type heart.