Holger H. Sigusch

Nitric oxide donors inhibit the coxsackievirus B3 proteinases 2A and 3C in vitro, virus production in cells, and signs of myocarditis in virus-infected mice

The antiviral effect of nitric oxide (NO)-releasing compounds was investigated. Using bacterially expressed and purified proteinases 2A and 3C of coxsackievirus B3, in vitro assays demonstrated the inhibition of the 2A proteinase activity in the presence of S-nitroso-N-acetyl-penicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), 4-phenyl-3-furoxancarbonitrile (PFC), glyceryl trinitrate (GTN), and isosorbide dinitrate (ISDN). Sodium nitroprusside (SNP), which releases NO after metabolization, had no effect. The 3C proteinase was inactivated by SNAP, GTN, and ISDN. The vasodilators GTN and ISDN, widely used in the treatment of angina pectoris, exhibited antiviral activity in CVB3-infected GMK cells. CVB3-infected NMRI outbred mice showed significantly reduced signs of myocarditis after treatment with GTN or ISDN. Inhibitors of the cellular inducible NO synthase (iNOS) such as NG-nitro-l-arginine methyl ester (L-NAME), NG-nitro-l-arginine (L-NNA), and S-methyl-isothiourea (SMT), had no deleterious effect on CVB3-infected NMRI mice, indicating that endogenous NO synthesis is unlikely to be a major defense mechanism after enterovirus infection of outbred mice.

Low level myocardial parvovirus B19 persistence is a frequent finding in patients with heart disease but unrelated to ongoing myocardial injury

While myocardial parvovirus B19 (B19V), aside from enteroviruses (EV) and adenoviruses (ADV), has recently been found often in patients with myocarditis and idiopathic dilated cardiomyopathy (IDC), the pathogenetic significance of B19V genomes in those patients has not yet been sufficiently elucidated. In the present study, left ventricular endomyocardial biopsies from 24 patients with left ventricular ejection fraction (LVEF) below 55% due to IDC, and tissue from the right atrial appendage of 10 control patients undergoing bypass surgery with normal LVEF (>55%) were investigated for B19V, ADV, and EV genomes by specific nested polymerase chain reaction (PCR), by real time PCR or by reverse‐transcription PCR, respectively. The myocardial tissue samples from the 10 controls were analyzed each in three different virological laboratories for B19V. In the IDC group, the frequency of the myocardial virus genomes found in 54% (13/24) of the patients was as follows: B19V: 50% (12/24), EV: 8% (2/24), including one patient with B19V and EV, and ADV: 0% (0/24). For comparison, the prevalence of B19V genomes was between 30% and 60% in the control group as detected in three different laboratories, but all these control subjects were EV‐ and ADV‐negative. The number of B19V gene copies, however, was very low and similar both in the IDC and control group. In the majority of patients myocardial B19V persistence was associated with a low virus load irrespective of the underlying heart disease so that it may be of no importance in the pathogenesis of IDC. J. Med. Virol. 82:1449–1457, 2010.

Genetic and phenotypic analysis of dilated cardiomyopathy with conduction system disease: Demand for strategies in the management of presymptomatic lamin A/C mutant carriers

One‐third of cases of dilated cardiomyopathy (DCM) is of familial aetiology. Several genes have been reported to cause the autosomal dominant form of DCM.

Identification and validation of selective upregulation of ventricular myosin light chain type 2 mRNA in idiopathic dilated cardiomyopathy

the etiology of idiopathic dilated cardiomyopathy (IDCM) is unknown, methods such as suppression subtractive hybridization (SSH) and DNA microarray technology can help to identify genes which might be involved in the pathogenesis of this disease.

Characterization of coxsackievirus B3-caused apoptosis under in vitro conditions.

Among several mechanisms of pathogenesis of the frequent and sometimes serious infections with coxsackievirus B3 (CVB3), one detail is apoptosis. Recently, a new apoptotic mechanism involving the specific interaction between the capsid protein VP2 of the highly virulent variant CVB3H3 and the proapoptotic host protein Siva was identified. The relevance of this observation for virus pathogenicity was shown in a BALB/c mouse model using CVB3H3 and the interaction-deficient mutant virus CVB3H310A1. In this study these results were verified and extended under in vitro conditions. The different apoptotic capability of CVB3H3 versus CVB3H310A1 was demonstrated by apoptotic nuclear condensation, DNA fragmentation, expression of Siva mRNA, and caspase-3 activation. The virus-specific differences were caused by the VP2 capsid proteins, which was shown by overexpression of the single VP2H3 and VP2H310A1 protein. Furthermore, the involvement of apoptosis in virus progeny production and the associated appearance of the cytopathic effect was demonstrated by application of the pan-caspase inhibitor Z-VAD-FMK. These in vitro results indicate that the induction of apoptosis during CVB3H3 infection is based on the interaction between the capsid protein VP2 and the proapoptotic protein Siva, independently from the complex situation in vivo.

Coxsackievirus B3-induced myocarditis: differences in the immune response of C57BL/6 and Balb/c mice

Coxsackievirus B3 (CVB3) infections are the most frequent causes of human myocarditis, often resulting in chronic stages characterized by fibrosis and loss of function. This disease is called dilated cardiomyopathy (DCM). Persistent virus in the myocardium may lead to chronic activation of fibroblasts, and subsequently, to fibrosis of the myocardium. Studies with immunodeficient mice have shown that certain defects of the immune system retard the rate at which virus is eliminated from the heart, thus leading to viral persistence. Therefore, we followed the immune response of two immunocompetent mouse strains (C57BL/6 and Balb/c) to CVB3 infection. These two strains have been reported to develop different immune responses to infections and we expected a similar reaction to viral infections as well. The two mouse strains recovered completely from CVB3 infection and expressed identical levels of cytokine mRNA in the heart. However, the virus in heart tissue decreased more slowly in Balb/c than in C57BL/6 mice. This was accompanied by a strong virus-specific IgG and weak IgM response in the C57BL/6 mice, in comparison to the Balb/c mice. We conclude, therefore, that viral-specific IgG is of importance for CVB3 elimination from infected hearts.

Impact of an exercise-induced increase in cardiac troponin I in chronic heart failure secondary to ischemic or idiopathic dilated cardiomyopathy

In summary, in patients with stable chronic heart failure, an exercise-induced increase in cardiac troponin I was correlated with levels of 3 circulating inflammatory cytokines: tumor necrosis factor-a, interleukin-1β, and interleukin-6. Patients with postexercise troponin levels above the the cutoff for minor myocardial damage are believed to have a poorer outcome. These data are first indicators for a pathologic role of exercise-induced kinetics of cardiac troponin I in heart failure.

Immunopathogenesis of dilated cardiomyopathy. Evidence for the role of TH2-type CD4+T lymphocytes and association with myocardial HLA-DR expression.

Background: An immunological pathogenesis underlying dilated cardiomyopathy and myocarditis has been suggested on the basis of the subtype of lymphocyte infiltrates and the degree of HLA expression in cardiac tissue. In the present study, we investigated the relation between the peripheral CD4+T-cell subset and the degree of HLA expression in the heart. Methods: Fifty-four patients with heart insufficiency included in the study were biopsied after coronary heart disease had been excluded. Immunohistological staining of the left ventricular tissue were performed employing anti-CD3, -CD4, -CD8, -CD14, and HLA-DR monoclonal antibodies. Intracellular expression of IL-2, IL-4, IL-5, IFN-γ, and TNF-α in peripheral CD4+T lymphocytes was determined using flow cytometry. The severity of heart insufficiency was determined by measurement of brain natriuretic peptide (BNP) and the NYHA class. On the basis of HLA expression in the heart, the patients were divided into three groups: Group I (mild-to-none), Group II (moderate), and Group III (strong-to-very strong). Results: Of the 54 patients included in this study, 33 (61%) patients were diagnosed as having idiopathic dilated cardiomyopathy and 10 (18.5%) borderline or healing myocarditis according to the Dallas criteria. Both patient groups were found in all three HLA-DR groups. There was no difference in BNP level or NYHA class between the three groups. However, a significant difference in the proportion of CD4+T lymphocytes producing IL-2 (39.2 versus 21.8%), IFN-γ (19.5 versus 7.8%), and TNF-α (35.8 versus 16.1%) between Groups I and III could be detected, whereas the distribution of IL-4 and IL-5 producing CD4+T lymphocytes was similar. The myocardium of Group III patients exhibited a significant higher number of CD3+T cells (11.4 versus 4.3 per mm2) and CD4+T cells (4.7 versus 0.8 per mm2) compared to Group I patients, while no difference existed with respect to CD8+T cells. Conclusion: High myocardial expression of the HLA-DR antigen is associated with an increase of peripheral-blood CD4+T lymphocytes expressing cytokines of the TH2 subset. The degree of HLA-DR expression is not associated with the degree of heart insufficiency or underlying diagnosis, but correlates with an increase of activated T cells in the myocardium. The data suggest that CD4+T lymphocytes infiltrating cardiac tissue may play a pathogenic role in dilated cardiomyopathy.

No influence of hemochromatosis-related gene mutations on restenosis rate in a retrospective study of 137 patients after coronary stent implantation

BACKGROUND: Recent publications have shown an increased risk of coronary artery disease and myocardial infarction in patients with alteration of the hemochromatosis-related gene (HFE gene). The HFE gene mutation is associated with elevated iron uptake and serum iron overloading. Iron plays an important role in promoting the oxidation of LDL cholesterol. The iron deposition in the endothelium and in the media is closely associated with the progression of atherosclerosis. However, it is unclear whether the mutation of the HFE gene also influences the rate of restenosis after coronary stent implantation. METHODS: In a retrospective analysis, 137 patients (pts.) who underwent elective coronary stent implantation were angiographically reevaluated after six months. All patients were part of the OPTICUS-study population which investigated optimized stent implantation guided by intravascular ultrasound. Computerized quantitative analysis was performed in all procedures in a double-blinded fashion. At six-month follow-up, DNA fragments containing the substitution of tyrosine for cytosine at codon 282 were amplified by PCR. The results were analyzed by polyacrylamide gel electrophoresis. Statistical analysis was performed by multivariate linear regression. RESULTS: According to the HFE gene polymorphism we formed two subgroups: 129 pts. (94%) did not show changes in HFE gene (NH), 8 pts. (6%) were heterozygous for HFE Cys282Tyr (H). The groups did not differ in age, gender, extent of coronary artery disease, initial degree and length of stenosis and all patients underwent re-angiography. At six-month follow-up the average luminal narrowing in the stented vessel was 36.2 - 20.3% in the NH group compared with 27.8 - 20.0% in the H group which was statistically not significant (n. s.). The minimal luminal diameter was 1.9 - 0.71 mm in the NH group and 2.2 - 0.66 mm in the H group respectively (n. s.). 33 pts (26%) in the NH group versus 2 pts (25%) in the H group had S 50% diameter narrowing at follow-up (n. s.). The odds ratio of stent restenosis in H patients was 0.932. CONCLUSIONS: The authors did not find any association between restenosis rate and HFE gene alteration and therefore, we conclude that the polymorphism of the HFE gene is not a risk factor for restenosis after coronary stent implantation.

Klinisches Bild und Differentialdiagnose von Kardiomyopathie und Myokarditis

Zusammenfassung□ Die idiopathische dilatative Kardiomyopathie ist gekennzeichnet durch die Dilatation und herabgesetzte Kontraktilität des linken oder beider Ventrikel. Ausgehend davon läßt sich das klinische Bild, das Ausdruck des Vorwärts-und Rückwärtsversagens des linken Ventrikels ist, ableiten. Die Klinik der dilatativen Kardiomyopathie unterscheidet sich jedoch nicht von der jeder anderen sekundären Herzinsuffizienz. Die Symptomatik bei einer Myokarditis kann sowohl durch das Ausmaß der linksventrikulären Funktionseinschränkung als auch durch Angina-pectoris-ähnliche Beschwerden bei Perikardbeteiligung bestimmt sein, so daß eine Abgrenzung zur idiopathischen dilatativen Kardiomyopathie schwierig sein kann.□ Die differentialdiagnostische Abgrenzung der idiopathischen dilatativen Kardiomyopathie zu sekundären Kardiomyopathien gelingt mit nichtinvasiven Verfahren nur unzureichend und führt in einem Drittel der Fälle zu Fehldiagnosen. Die invasive Diagnostik mit Koronarangiographie ist also obligat, um die Abgrenzung zur koronaren Herzkrankheit und anderen selteneren Herzerkrankungen zu erreichen.□ Laborchemische Entzündungsmarker und im peripheren Blut gemessene Zytokinkonzentrationen (zum Beispiel TNF-α) können ebenfalls nicht zur Differenzierung von dilatativer Kardiomyopathie und Myokarditis beitragen, so daß die Endomyokardbiopsie indiziert ist. Die Endomyokardbiopsie wird histologisch, immunhistologisch und hinsichtlich der Persistenz eines entzündlichen Agens untersucht. Unter Beachtung der Ergebnisse der invasiven Untersuchung und der Endomyokardbiopsie kann eine Vierfeldersubklassifikation der idiopathischen linksventrikulären Dysfunktion postuliert werden. Dabei werden die Kriterien Nachweis eines infektiösen Agens und Vorhandensein von entzündlichem Infiltrat im Myokard herangezogen. Dilatative Kardiomyopathie (ca. 70 bis 75%), virusassoziierte dilatative Kardiomyopathie (ca. 20 bis 25%), Myokarditis (ca. 7%) und Autoimmunmyokarditis (ca. 3%) sind die resultierenden vier Formen der idiopathischen links-ventrikulären Dysfunktion.□ Daraus lassen sich potentielle therapeutische Strategien ableiten. Neben der konventionellen medikamentösen Therapie ergeben sich Therapieansätze zum Beispiel mit antiviralen Substanzen (wie etwa Interferon) in der enteroviruspositiven Gruppe und mit Immunsuppressiva bei Patienten mit fehlendem Enterovirusnachweis und dem Nachweis von zellulären Infiltraten (Autoimmunmyokarditis).Summary□ The main feature of idiopathic dilated cardiomyopathy is the dilation and impaired contractility of the left ventricle or both ventricles. The clinical picture with forward and backward failure is based on the pump impairment of the left ventricle. However, the clinical presentation of patients with dilated cardiomyopathy is indistinguishable from any other secondary form of heart failure. The symptoms of myocarditis are also often determined by the degree of left ventricular dysfunction and — apart from perimyocarditis-associated precordial discomfort — therefore also often indistinguishable from dilated cardiomyopathy. The differentiation of dilated cardiomyopathy from other myocardial diseases by noninvasive methods is insufficient. Without invasive tests about 1/3 of the patients will be diagnosed incorrectly. Therefore, invasive diagnostics including coronary angiography are necessary to differentiate dilated cardiomyopathy from other diseases, especially coronary artery disease.□ Standard laboratory findings and cytokine serum concentrations (e. g. TNF-α) are not suitable to differentiate dilated cardiomyopathy and myocarditis and endomyocardial biopsy is indicated. Endomyocardial biopsies have to undergo evaluation by standard histology and immunohistology, and should be tested for the persistence of infectious agents. According to cardiac catheterization and evaluation of the endomyocardial biopsy idiopathic left ventricular dysfunction can be further stratified using the criterion of a myocardial virus persistence and the presence/absence of inflammatory infiltrates. Idiopathic dilated cardiomyopathy (approximately 70 to 75%), virus-associated dilated cardiomyopathy (approximately 20 to 25%), myocarditis (approximately 7%) and autoimmune myocarditis (approximately 3%) are the 4 possible resulting forms of idiopathic left ventricular dysfunction. Beside conventional medical therapy there are new therapeutic concepts e. g. using interferon for enterovirus-positive patients and immunosuppression for autoimmune, virus-negative patients with a cellular infiltrate.