Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy.

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy, but the mechanism of this pathology is unknown. Mutations in cytoskeletal proteins such as dystrophin cause hereditary dilated cardiomyopathy, but it is unclear if similar mechanisms underlie acquired forms of heart failure. We demonstrate here that purified Coxsackievirus protease 2A cleaves dystrophin in vitro as predicted by computer analysis. Dystrophin is also cleaved during Coxsackievirus infection of cultured myocytes and in infected mouse hearts, leading to impaired dystrophin function. In vivo, dystrophin and the dystrophin-associated glycoproteins alpha-sarcoglycan and beta-dystroglycan are morphologically disrupted in infected myocytes. We suggest a molecular mechanism through which enteroviral infection contributes to the pathogenesis of acquired forms of dilated cardiomyopathy.

CVB infection and mechanisms of viral cardiomyopathy.

Coxsackievirus infection has been demonstrated to be a cause of acute and fulminant viral myocarditis and has been associated with dilated cardiomyopathy. While considerable attention has focused on the role of the cellular and humoral, antigen-specific immune system in viral myocarditis, the interaction between the virus and the infected host myocyte is also important. Coxsackievirus has a relative tropism for the heart that is in part mediated by relatively high levels of the coxsackievirus and adenovirus receptor (CAR) on the cardiac myocyte. Once within the myocyte, coxsackievirus produces proteases, such as protease 2A, that have an important role in viral replication, but can also affect host cell proteins such as dystrophin. Cleavage of dystrophin may have a role in release of the virus from the myocyte since viral infection is increased in the absence of dystrophin. In addition to the direct effect of viral proteins on cardiac myocytes, there is now evidence that the cardiac myocyte has a potent innate immune defense against coxsackieviral infection. Suppressors of cytokine signaling (SOCS) can inhibit an interferon-independent mechanism within the cardiac myocyte. In summary, the interaction between coxsackievirus and the infected myocyte has a significant role in the pathogenesis of viral myocarditis and the susceptibility to viral infection.

Divergent pathways mediate the induction of ANF transgenes in neonatal and hypertrophic ventricular myocardium.

To determine whether similar or divergent pathways mediate atrial natriuretic factor (ANF) induction in neonatal and hypertrophied adult ventricular myocardium, and to assess whether studies using an in vitro model system of hypertrophy have fidelity to the in vivo context during pressure overload hypertrophy, we generated transgenic mice which harbor either 638 or 3,003 bp of the rat ANF 5' flanking region ligated upstream from a luciferase reporter. Luciferase activity in the ventricles of day 1 transgenic neonates was 8-24-fold higher than the levels expressed in the ventricles of adult mice. Adult mice expressed the luciferase reporter in an appropriate tissue-specific manner. Transverse aortic constriction of adult mice harboring ANF reporter transgenes demonstrated no significant increase in reporter activity in the ventricle. These findings demonstrate that distinct regions of the ANF 5'-flanking region are required for inducible expression of the ANF gene in the hypertrophic adult ventricle compared with those required for atrial-specific and developmentally appropriate expression in the intact neonatal heart. Furthermore, the cis regulatory elements necessary for induction of ANF expression in endothelin-1 or alpha 1-adrenergically stimulated cultured neonatal ventricular myocytes are not sufficient for induction in the in vivo context of pressure overload hypertrophy.

Transgenic expression of replication-restricted enteroviral genomes in heart muscle induces defective excitation-contraction coupling and dilated cardiomyopathy.

Numerous studies have implicated Coxsackievirus in acute and chronic heart failure. Although enteroviral nucleic acids have been detected in selected patients with dilated cardiomyopathy, the significance of such persistent nucleic acids is unknown. To investigate the mechanisms by which restricted viral replication with low level expression of Coxsackieviral proteins may be able to induce cardiomyopathy, we generated transgenic mice which express a replication-restricted full-length Coxsackievirus B3 (CVB3) cDNA mutant (CVB3DeltaVP0) in the heart driven by the cardiac myocyte-specific myosin light chain-2v (MLC-2v) promoter. CVB3DeltaVP0 was generated by mutating infectious CVB3 cDNA at the VP4/VP2 autocatalytic cleavage site from Asn-Ser to Lys-Ala. Cardiac-specific expression of this cDNA leads to synthesis of positive- and negative-strand viral RNA in the heart without formation of infectious viral progeny. Histopathologic analysis of transgenic hearts revealed typical morphologic features of myocardial interstitial fibrosis and in some cases degeneration of myocytes, thus resembling dilated cardiomyopathy in humans. There was also an increase in ventricular atrial natriuretic factor mRNA levels, demonstrating activation of the embryonic program of gene expression typical of ventricular hypertrophy and failure. Echocardiographic analysis demonstrated the presence of left ventricular dilation and decreased systolic function in the transgenic mice compared with wild-type littermates, evidenced by increased ventricular end-diastolic and end-systolic dimensions and decreased fractional shortening. Analysis of isolated myocytes from transgenic mice demonstrate that there is defective excitation-contraction coupling and a decrease in the magnitude of isolated cell shortening. These data demonstrate that restricted replication of enteroviral genomes in the heart can induce dilated cardiomyopathy with excitation-contraction coupling abnormalities similar to pressure overload models of dilated cardiomyopathy.

Adrenergic control of circulating lymphocyte subpopulations. Effects of congestive heart failure, dynamic exercise, and terbutaline treatment.

The current studies were undertaken to explore the relationship between enhanced sympathetic nervous activity and lymphocyte subset distribution in three settings: congestive heart failure, dynamic exercise, and beta-adrenergic agonist treatment. We compared the number and subset distribution of circulating lymphocytes in 36 patients with congestive heart failure and 31 age-matched control subjects. The number of circulating lymphocytes was lower in heart failure than in control. This was due to a reduction in Tsuppressor/cytotoxic and natural killer cells without significant alteration of Thelper cells. The extent of the alteration was similar in patients with idiopathic and ischemic heart failure, but the reduction was more pronounced in patients with New York Heart Association class III-IV than in class I-II. The plasma catecholamine elevation in heart failure was also independent of etiology but more pronounced in the more severely ill patients. We also assessed lymphocyte subsets after acute stimulation of sympathetic activity by dynamic exercise and after treatment with the beta-adrenergic agonist terbutaline. Dynamic exercise until exhaustion increased the number of circulating lymphocytes in healthy controls and heart failure patients in a subset-selective manner. By contrast, a 7-d treatment with terbutaline caused a reduction in the circulating number of lymphocytes in some subsets that was identical to that seen in heart failure patients. We conclude that prolonged sympathetic activity reduces the number of circulating lymphocytes by a beta-adrenergic mechanism. Such alterations might be involved in the pathophysiology of heart failure and other disease states involving increased activity of the sympathetic nervous system.

Treatment of coxsackievirus-B3-infected BALB/c mice with the soluble coxsackie adenovirus receptor CAR4/7 aggravates cardiac injury.

Coxsackie adenovirus receptor (CAR) is involved in immunological processes, and its soluble isoforms have antiviral effects on coxsackievirus B3 (CVB3) infection in vitro. We explored in this study the impact of CAR4/7, a soluble CAR isoform, on CVB3-induced myocarditis in BALB/c mice. BALB/c mice were treated daily with recombinant CAR4/7, beta-galactosidase (beta-Gal; as control protein) or buffer for 9 days. Half of each group was infected with CVB3 on day 3, and all mice were killed on day 9. Myocardial CVB3 titer, histology, and serology were analyzed. Treatment with CAR4/7 led to a significant reduction of myocardial CVB3 titer, whereas the application of beta-Gal had no detectable effect on the myocardial virus load. CAR4/7 application, however, resulted in increased myocardial inflammation and tissue damage in CVB3-infected hearts, whereas beta-Gal caused a degree of cardiac inflammation and injury similar to that in buffer-treated CVB3-infected control animals. CAR4/7 and beta-Gal treatment induced the production of antibodies against the respective antigens. CAR4/7-, but not beta-Gal-specific, virus-negative sera reacted against myocardial tissue and cellular membranous CAR, and significantly inhibited CVB3 infection in vitro. Thus, CAR4/7 suppressed CVB3 infection in vivo, supporting the concept of receptor analog in antiviral therapy. However, CAR4/7 treatment also leads to an aggravation of myocardial inflammation and injury most likely secondary to an autoimmune process.

Dissociation of sarcoglycans and the dystrophin carboxyl terminus from the sarcolemma in enteroviral cardiomyopathy.

Enteroviral infection can cause an acquired form of dilated cardiomyopathy. We recently reported that dystrophin is cleaved, functionally impaired, and morphologically disrupted in vitro as well as in vivo during infection with coxsackievirus B3. Genetic dystrophin truncations lead to a marked decrease in dystrophin-associated glycoproteins, whereas expression of only the naturally occurring dystrophin carboxyl terminus, Dp-71, restores the sarcolemmal association of the dystrophin-associated glycoproteins. We sought to determine whether acute cleavage of dystrophin leads to a dissociation of the carboxyl-terminal dystrophin fragment and of the sarcoglycans from the sarcolemma during coxsackievirus B3 infection. We found that in cultured cardiac myocytes and murine hearts infected with coxsackievirus B3, the sarcolemmal localization of the dystrophin carboxyl terminus is lost. The dystrophin-associated glycoproteins alpha-, beta-, gamma-, and delta-sarcoglycan and beta-dystroglycan were markedly decreased in the membrane fraction of infected cells in culture, and the typical sarcolemmal localization for each of these proteins was lost in coxsackievirus-B3-infected cardiomyocytes in vivo. Furthermore, sucrose gradient ultracentrifugation demonstrated that delta-sarcoglycan was physically dissociated from dystrophin within the membrane fraction. In vivo, the sarcolemmal integrity was functionally impaired with Evans blue dye uptake even though there was no generalized disruption of the sarcolemma of infected myocytes evidenced by intact wheat germ agglutinin staining. In analogy to hereditary sarcoglycanopathies, this disintegration of the sarcoglycan complex may, in addition to the dystrophin cleavage, play an important role in the pathogenesis of enterovirus-induced cardiomyopathy. These results imply a potential role for disruption of the sarcoglycans in an acquired form of heart failure.

Unsolved medical issues and new targets for further research in viral myocarditis and dilated cardiomyopathy.

Meaningful advances have been made in understanding the mechanisms that contribute to dilated cardiomyopathy and myocarditis. Our data confirmed the hypothesis that there is an interaction of genetic predisposition and acquired factors, in that both can affect the dystrophin-glycoprotein complex. We could show that dystrophin deficiency increases susceptibility to viral infection. Our experiments addressed the role of coxsackievirus in the pathogenesis of cardiomyopathy, while other viruses may be involved, such as adenovirus, parvovirus, influenza virus, etc. Furthermore, we could demonstrate that cardiac myocyte-specific transgenic expression of SOCS1 inhibited coxsackievirus-induced signaling of Janus kinase (JAK) and signal transducer and activator of transcription (STAT), with accompanying increases in viral replication, cardiomyopathy, and mortality in infected mice. Future treatment strategies may include the development of coxsackie-adenovirus receptor (CAR) inhibitors and enteroviral protease 2A inhibitors. Additional studies are ongoing to determine the effectiveness of these inhibitors on viral infection in culture and in the intact heart.

Cardioselective infection with coxsackievirus B3 requires intact type I interferon signaling: implications for mortality and early viral replication.

BACKGROUND: Interferons (IFNs) play an important role in antiviral defense and have therapeutic potential in coxsackievirus heart disease. However, little is known about the relative contributions of type I and type II IFN signaling in coxsackievirus B3 (CVB3) infection or their role in the cardioselective nature of CVB3 infection. METHODS AND RESULTS: Wild-type mice and mice deficient for either the type I or the type II IFN receptor (IFNR) were infected with CVB3. Infection of the type I IFNR-deficient mice with >10(3) plaque-forming units (pfu) of CVB3 resulted in 100% mortality within 2 to 4 days after infection. Death was rare in wild-type and type II IFNR-deficient mice after inoculation with as much as 10(8) pfu of CVB3. Surprisingly, the early mortality in the type I IFNR-deficient mice was not accompanied by higher virus titers in the heart. Unexpectedly, a dramatic increase of viral RNA in the liver was found to correlate with early mortality in type I IFNR-deficient mice. CONCLUSIONS: Type I but not type II IFN signaling is essential for the prevention of early death due to CVB3 infection. Interestingly, neither type I or type II IFN signaling has a dramatic effect on early viral replication in the heart. However, lethal viral replication in the liver is controlled by type I IFNs. These results demonstrate that the IFN system is capable of modulating both viral pathogenicity and tissue tropism.

Nitric oxide inhibits dystrophin proteolysis by coxsackieviral protease 2A through S-nitrosylation: A protective mechanism against enteroviral cardiomyopathy.

BACKGROUND: Infection with enteroviruses like coxsackievirus B3 (CVB3) as well as genetic dystrophin deficiency can cause dilated cardiomyopathy. We recently identified cleavage and functional impairment of dystrophin by the viral protease 2A during CVB3-infection as a molecular mechanism that may contribute to the pathogenesis of enterovirus-induced cardiomyopathy. Nitric oxide (NO) is elevated in human dilated cardiomyopathy, but the relevance of this finding is unknown. In mice, NO inhibits CVB3 myocarditis. Therefore, we investigated the effects of NO on the coxsackieviral protease 2A. METHODS AND RESULTS: In vitro, NO donors like PAPA-NONOate inhibited the cleavage of human and mouse dystrophin by recombinant coxsackievirus B protease 2A in a dose-dependent manner (IC(50), 51 micromol/L). In CVB3-infected HeLa cells, addition of the NO donor SNAP inhibited protease 2A catalytic activity on dystrophin. Because this inhibitory effect was reversed by the thiol-protecting agent DTT, we investigated whether NO S:-nitrosylates the protease 2A. In vitro, NO nitrosylated the active-site cysteine (C110) of the coxsackieviral protease 2A, as demonstrated by site-directed mutagenesis. Within living COS-7 cells, SNAP-induced S:-nitrosylation of this site was confirmed with electron spin resonance spectroscopy. CONCLUSIONS: These data demonstrate inactivation of a coxsackieviral protease 2A by NO through active-cysteine S:-nitrosylation in vitro and intracellularly. Given that the enteroviral protease 2A cleaves mouse and human dystrophin, NO may be protective in human heart failure with an underlying enteroviral pathogenesis through inhibition of dystrophin proteolysis.

Alpha 1-adrenergic stimulation inhibits 3,5,3'-triiodothyronine-induced expression of the rat heart sarcoplasmic reticulum Ca2+ adenosine triphosphatase gene.

The interactions between the beta-adrenergic system and thyroid hormone (T3) on cardiac function have been investigated in detail. In addition to beta-adrenoceptors, alpha 1-adrenergic receptors are present in the mammalian heart. The interactions between T3 and the alpha 1-adrenergic system remain, however, poorly understood. T3 stimulates the expression and transcription of the sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA2) gene, a protein vital in the control of cardiac calcium transients and contractility. We show that in rat cardiac myocytes, the stimulatory effect of T3 on SERCA2 messenger RNA expression and gene transcription is inhibited by an alpha 1-adrenergic agonist. We demonstrate that direct activation of the alpha 1-adrenergic signaling pathway, using a mutant constitutively active G protein (Gq) similarly down-regulated the T3 effect on SERCA2 transcription. The combined effect of thyroid hormone receptor and retinoid X receptors on T3-stimulated SERCA2 gene transcription was also markedly attenuated by alpha 1-adrenergic stimulation. These results suggested that activation of the alpha 1-adrenergic signaling pathway has an inhibitor effect on T3-dependent SERCA2 gene transcription. As this inhibitory effect of alpha 1-adrenergic stimulation occurs when only one thyroid hormone response element (TRE) drives reporter expression, it is most likely mediated by an alteration of the nuclear factors binding to the TRE or by influencing the interaction of the TRE complex with the basal transcriptional machinery.

Enteroviral cardiomyopathy: bad news for the dystrophin-glycoprotein complex.

Genetic deficiency of the dystrophin-glycoprotein complex causes hereditary dilated cardiomyopathy. Enteroviruses can also cause cardiomyopathy and we have recently described a potential molecular mechanism for enterovirus-induced dilated cardiomyopathy. The coxsackieviral protease 2A proteolytically cleaves and functionally impairs dystrophin. Additionally, during infection with coxsackievirus B3, the dystrophin-glycoprotein complex becomes disrupted and the sarcolemmal integrity is lost. This review article discusses the importance of the dystrophin cleavage for the development of increased sarcolemmal permeability and potential pathways for mechanisms by which the dystrophin cleavage during coxsackieviral infection may contribute to dilated cardiomyopathy.

Regulated expression of a contractile protein gene correlates with recovery of contractile function after reversible metabolic inhibition in cultured myocytes.

Little is known of the relation between recovery of contraction and the regulation of contractile protein gene expression in ventricular myocytes after severe ATP depletion. We have examined alterations in activation of an MLC-2 luciferase fusion gene in cultured neonatal rat ventricular myocytes produced by exposure to 2 mM Na CN and 20 mM 2-deoxyglucose, and after recovery is serum or serum free medium. The effects of metabolic inhibition followed by recovery on expression on an RSV-luciferase activity were also investigated. Myocytes were co-transfected with a CMV beta-galactosidase fusion gene, and luciferase activities were normalized relative to beta-galactosidase activity to control for transfection efficiency. Two hours of metabolic inhibition produced significant cell injury, as documented by disorganization of myofilaments, and reduction in luciferase and beta-galactosidase activity within transfected cells. Cells allowed to recover for 48 h in serum free hormone supplemented medium showed a further decline in corrected luciferase activity, consistent with a marked reduction in MLC-2 gene transcription. Cells recovered from severe metabolic inhibition in serum free medium also showed failure to redevelop contractile activity, and failure of redevelopment of organized myofibrils. In contrast, myocytes exposed to serum during the 48 h recovery period had a marked increase in luciferase activity, resumed contractile activity and re-established organized myofilaments. There were no significant differences between RSV luciferase activities in cells recovered in serum versus serum free media. In ventricular myocytes in which contraction was inhibited by exposure to 10 microM verapamil, MLC-2 luciferase activity declined by 87%. However, even when contractile activity was inhibited by exposure to verapamil during recovery from metabolic inhibition, exposure to serum containing medium caused a significantly greater increase in MLC-2 luciferase activity than did serum free medium. Thus, the effects of serum on MLC-2 gene expression were not solely due to an effect of serum on recovery of contractile activity. Verapamil had no consistent effect on expression of RSV luciferase. These results suggest that expression of the MLC-2 gene is markedly reduced following recovery from severe metabolic inhibition, an effect largely due to cessation of myocyte contractile activity. Resupply of growth factors present in fetal calf serum reactivate expression of this gene, and this is associated with resumption of contractile activity and redevelopment of organized myofibrils. These results suggest that reactivation of contractile protein gene expression during recovery from metabolic inhibition may be beneficial in allowing cells to recover from this insult.

Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response.

Studies from both in vivo and in vitro model systems have provided an initial skeleton of the potential signaling pathways that might regulate cardiac genes during growth and hypertrophy. One of the first detectable changes in cardiac gene expression is the activation of a program of immediate early gene expression, which is distinct for the hypertrophic response, and is conserved in multiple models of both in vivo and in vitro hypertrophy. Diverse and distinct hormonal stimuli have been documented to activate several features of the hypertrophic response, including several autocrine and paracrine factors. Although the signaling mechanisms that link these factors with the activation of cardiac gene expression are unclear, recent studies suggest that the activation of protein kinase C may represent one of the most proximal common events in this signaling cascade. The activation of cardiac target genes induces a program of embryonic gene expression, including the atrial natriuretic factor (ANF) gene. The cis sequences that mediate cardiac-specific and inducible expression of an embryonic marker gene (ANF) can be segregated by studies in both cultured cell models and in vivo models of hypertrophy in transgenic mice, suggesting that specific sets of regulatory elements may exist for inducible expression of this class of cardiac gene responses. However, the induction of a constitutively expressed contractile protein gene (MLC-2) is mediated by a set of conserved elements that regulate both cardiac-specific and inducible expression. Finally, a subset of cardiac muscle genes appears to be noninducible during in vivo or in vitro hypertrophy in myocardial cells, demonstrating specificity of transcriptional activation during the hypertrophic process. The development of a bona fide in vivo pressure overload model of hypertrophy in a small animal model that can be genetically manipulated, such as the in vivo murine model recently described, should allow a rigorous analysis of the role of these specific signaling mechanisms in the activation of the responses of cardiac genes during the hypertrophic process in vivo.

A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3.

Coxsackievirus B3 (CVB3) infections induce myocarditis in humans and mice. Little is known about the molecular characteristics of CVB3 that activate the cellular immunity responsible for cardiac inflammation. Previous experiments have identified an antibody escape mutant (H310A1) of a myocarditic variant of CVB3 (H3) that attenuates the myocarditic potential of the virus in mice in spite of ongoing viral replication in the heart. We have cloned full-length infectious cDNA copies of the viral genome of both the wild-type myocarditic H3 variant of CVB3 and the antibody escape mutant H310A1. Progeny viruses maintained the myocarditic and attenuated myocarditic potential of the parent viruses, H3 and H310A1. The full sequence of the H3 viral cDNA is reported and compared with those of previously published CVB3 variants. Comparison of the full sequences of H3 and H310A1 viruses identified a single nonconserved mutation (A to G) in the P1 polyprotein region at nucleotide 1442 resulting in an asparagine-to-aspartate mutation in amino acid 165 of VP2. This mutation is in a region that corresponds to the puff region of VP2. Nucleotide 1442 of the H3 and H310A1 cDNA copies of the viral genome was mutated to change amino acid 165 of VP2 to aspartate and asparagine, respectively. The presence of asparagine at amino acid 165 of VP2 is associated with the myocarditic phenotype, while an aspartate at the same site reduces the myocarditic potential of the virus. In addition, high-level production of tumor necrosis factor alpha by infected BALB/c monocytes is associated with asparagine at amino acid 165 of VP2 as has been previously demonstrated for the H3 virus. These findings identify potentially important differences between the H3 variant of CVB3 and other previously published CVB3 variants. In addition, the data demonstrate that a point mutation in the puff region of VP2 can markedly alter the ability of CVB3 to induce myocarditis in mice and tumor necrosis factor alpha secretion from infected BALB/c monocytes.

Low-level expression of a mutant coxsackieviral cDNA induces a myocytopathic effect in culture: an approach to the study of enteroviral persistence in cardiac myocytes.

BACKGROUND: Enteroviral ribonucleic acids have been identified in heart muscle of a subset of patients with myocarditis and dilated cardiomyopathy as well as in a mouse model of persistent coxsackievirus B3 (CVB3) infection, suggesting that persistent viral infection along with activation of an immune response may contribute to the pathogenesis of ongoing cardiac disease and dilated cardiomyopathy in certain patients. It is still not known whether persistence of the viral genome contributes to the pathogenesis of dilated cardiomyopathy. METHODS AND RESULTS: To determine whether low-level enteroviral gene expression similar to that observed with viral persistence can induce myocytopathic effects without formation of infectious virus progeny, the full-length infectious cDNA copy of CVB3 was mutated at the VP0 maturation cleavage site. This prevented formation of infectious virus progeny. In myocytes transfected with this mutated cDNA copy of the viral genome, both positive- and negative-strand viral RNAs were detected, demonstrating that there was replication of the viral genome by the RNA-dependent RNA polymerase. The level of viral protein expression was found to be below limits of detection by conventional methods of protein detection, thus resembling restricted virus replication. Nonetheless, the CVB3 mutant was found to induce a cytopathic effect in transfected myocytes, which was demonstrated by inhibition of cotransfected MLC-2v luciferase reporter activity and an increase in release of lactate dehydrogenase from transfected cells. CONCLUSIONS: This study demonstrates that restricted replication of enteroviral genomes in myocytes in a pattern similar to that observed in hearts with persistent viral infection can induce myocytopathic effects without generation of infectious virus progeny.