Dingding Xiong

The suppressor of cytokine signaling-1 (SOCS1) is a novel therapeutic target for enterovirus-induced cardiac injury.

Enteroviral infections of the heart are among the most commonly identified causes of acute myocarditis in children and adults and have been implicated in dilated cardiomyopathy. Although there is considerable information regarding the cellular immune response in myocarditis, little is known about innate signaling mechanisms within the infected cardiac myocyte that contribute to the host defense against viral infection. Here we show the essential role of Janus kinase (JAK) signaling in cardiac myocyte antiviral defense and a negative role of an intrinsic JAK inhibitor, the suppressor of cytokine signaling (SOCS), in the early disease process. Cardiac myocyte-specific transgenic expression of SOCS1 inhibited enterovirus-induced signaling of JAK and the signal transducers and activators of transcription (STAT), with accompanying increases in viral replication, cardiomyopathy, and mortality in coxsackievirus-infected mice. Furthermore, the inhibition of SOCS in the cardiac myocyte through adeno-associated virus-mediated (AAV-mediated) expression of a dominant-negative SOCS1 increased the myocyte resistance to the acute cardiac injury caused by enteroviral infection. These results indicate that strategies directed at inhibition of SOCS in the heart and perhaps other organs can augment the host-cell antiviral system, thus preventing viral-mediated end-organ damage during the early stages of infection.

Dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy: A genetic predisposition to viral heart disease

Both enteroviral infection of the heart and mutations in the dystrophin gene can cause cardiomyopathy. Little is known, however, about the interaction between genetic and acquired forms of cardiomyopathy. We previously demonstrated that the enteroviral protease 2A cleaves dystrophin; therefore, we hypothesized that dystrophin deficiency would predispose to enterovirus-induced cardiomyopathy. We observed more severe cardiomyopathy, worsening over time, and greater viral replication in dystrophin-deficient mice infected with enterovirus than in infected wild-type mice. This difference appears to be a result of more efficient release of the virus from dystrophin-deficient myocytes. In addition, we found that expression of wild-type dystrophin in cultured cells decreased the cytopathic effect of enteroviral infection and the release of virus from the cell. We also found that expression of a cleavage-resistant mutant dystrophin further inhibited the virally mediated cytopathic effect and viral release. These results indicate that viral infection can influence the severity and penetrance of the cardiomyopathy that occurs in the hearts of dystrophin-deficient individuals.

Coxsackievirus and adenovirus receptor (CAR) mediates atrioventricular-node function and connexin 45 localization in the murine heart

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 beta-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 beta-catenin and ZO-1 at the ventricular intercalated disc are dependent on CAR.

Inducible Cardiac-Restricted Expression of Enteroviral Protease 2A Is Sufficient to Induce Dilated Cardiomyopathy

Background— Enterovirus infection is a cause of cardiomyopathy. We previously demonstrated that enteroviral protease 2A directly cleaves the cytoskeletal protein dystrophin. However, the direct effect of protease 2A in enteroviral cardiomyopathy is less clear because other viral proteins are also expressed with viral infection. Methods and Results— A transgenic mouse with inducible cardiac-restricted expression of enteroviral protease 2A was generated. In the transgenic mouse, a tamoxifen-regulated Cre-loxP system, MerCreMer (MCM), was used to induce genetic recombination in cardiac myocytes, which led to protease 2A expression. Protease 2A and MCM double transgenic (2AxMCM) mice were treated with tamoxifen; the controls included 2AxMCM mice treated with diluents for tamoxifen and tamoxifen-treated MCM littermates. Protease 2A activity was significantly induced after tamoxifen in the 2AxMCM mice compared with controls. Echocardiographic analysis demonstrated an increase in left ventricular end-diastolic and end-systolic chamber size, with decreased fractional shortening in tamoxifen-treated 2AxMCM mice. There was an increase in heart weight-to-body weight ratio in 2AxMCM mice treated with tamoxifen. Only a small increase in interstitial fibrosis and inflammation was found in tamoxifen-treated 2AxMCM mice; however, ultrastructural analysis demonstrated myofibrillar collapse with abnormalities of intercalated discs and sarcolemmal membranes. Evans blue dye–positive myocytes with disruption of dystrophin were present in 2AxMCM mice treated with tamoxifen. Disruption of dystrophin was also found in cultured myocytes isolated from 2AxMCM mice with Cre in the nucleus. Conclusions— Protease 2A has a significant role in enteroviral cardiomyopathy and alone is sufficient to induce dilated cardiomyopathy, which is associated with disruption of the sarcolemmal membrane and cleavage of dystrophin with protease 2A expression.

Innate defense mechanism against virus infection within the cardiac myocyte requiring gp130-STAT3 signaling.

Background— Little is known about innate immune mechanisms within the cardiac myocyte that determine susceptibility to enterovirus infection, an important cause of myocarditis and subsequent heart failure. Although interferon (IFN) generally plays a key role in innate immunity, ablation of IFN receptors has little or no effect on acute coxsackievirus B3 infection in the heart. Interestingly, gp130-cytokine–mediated stimulation of neonatal ventricular myocytes has a cytoprotective effect against virus infection in culture that can be inhibited by suppressors of cytokine signaling (SOCS)-3, a physiological inhibitor of gp130 signaling that does not affect IFN signaling. Therefore, we hypothesized that inhibition of gp130 signaling by SOCS3 would change cardiac myocyte susceptibility to virus infection without affecting IFN signaling. Methods and Results— We generated cardiac-specific SOCS3 transgenic mice. Despite an intact IFN-mediated antiviral response in adult transgenic myocytes, there was a marked increase in susceptibility to viral infection in the SOCS3 transgenic mouse hearts. This indicated the presence of IFN-independent innate defense mechanisms within the cardiac myocyte. Subsequently, we demonstrated that cardiac-specific gp130-knockout mice also had increased susceptibility to viral infection. Furthermore, we demonstrated that the gp130-mediated increase in survival of infected myocytes occurred through a signal transducers and activators of transcription-3–dependent mechanism that did not affect viral replication. This was accompanied by a persistent expression of full-length dystrophin after coxsackievirus B3 infection. In addition, we found that both SOCS3 transgenic and gp130-deficient mice had a decrease in α-sarcoglycan. Conclusions— SOCS3-mediated regulation of gp130 signaling can affect susceptibility to viral infection in the heart. Increased cardiac cell survival through gp130–signal transducers and activators of transcription-3 signaling appears to play an important role in preserving nondividing cardiac myocytes until specific immune responses begin to clear the virus.

Inhibition of Coxsackievirus-associated dystrophin cleavage prevents cardiomyopathy

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.

Protection of Cap-dependent protein synthesis in vivo and in vitro with an eIF4G-1 variant highly resistant to cleavage by coxsackievirus 2A protease

The shutoff of host protein synthesis by certain picornaviruses is mediated, at least in part, by proteolytic cleavage of eIF4G-1. Previously, we developed a cleavage site variant of eIF4G-1, termed eIF4G-1SM, that was 100-fold more resistant to in vitro cleavage by Coxsackievirus 2A protease (2APro) than wild-type eIF4G-1 (eIF4G-1WT), but it was still digested at high protease concentrations. Here we identified a secondary cleavage site upstream of the primary site. We changed Gly at the P1′-position of the secondary site to Ala to produce eIF4G-1DM. eIF4G-1DM was 1,000–10,000-fold more resistant to cleavagein vitro than eIF4G-1WT. Full functional activity of eIF4G-1DM was demonstrated in vitroby its ability to restore cap-dependent translation to a 2APro-pretreated rabbit reticulocyte system. An isoform containing the binding site for poly(A)-binding protein, eIF4G-1eDM, was more active in this assay than an isoform lacking it, eIF4G-1aDM, but only with polyadenylated mRNA. Functional activity was also demonstrated in vivowith stably transfected HeLa cells expressing eIF4G-1DMfrom a tetracycline-regulated promoter. Cap-dependent green fluorescent protein synthesis was drastically inhibited by 2APro expression, but synthesis was almost fully restored by induction of either eIF4G-1aDM or eIF4G-1eDM. By contrast, encephalomyocarditis virus internal ribosome entry site-dependent green fluorescent protein synthesis was stimulated by 2APro; stimulation was suppressed by eIF4G-1eDM but not eIF4G-1aDM.

Pathogenesis of Enteroviral Cardiomyopathy: Interaction of Viral Proteins With Infected Myocytes

Dilated cardiomyopathy, one of the leading causes of heart failure in the United States, is a multifactorial disease that includes hereditary and acquired forms.1 Although a hereditary component of dilated cardiomyopathy is recognized in 25% to 35%2 of cases, the majority of cases do not have a clearly defined cause. In addition, children and adults can present with an acute onset of cardiomyopathy that is often called acute myocarditis. This is usually attributed to viral infection, although limitations in our diagnostic abilities prevent precise quantitation of the incidence of viral infection in these cases.