Viral myocarditis and coagulopathy: increased tissue factor expression and plasma thrombogenicity.

We investigated the effects of viral infection on Tissue Factor (TF) expression and activity in mice within the myocardium to understand increased thrombosis during myocarditis. Mice were infected with coxsackie virus B3 (CVB3) and the hearts were collected at day 4, 8 and 28 post infection (p.i.). Myocardial TF expression and cellular activity as well as plasma activity were analyzed from CVB3 infected mice by Western blot, chromogenic Factor Xa generation assay, in situ staining for active TF and immunohistochemistry. In addition to TF expression, hemodynamic parameters were measured during the time course of infection. Furthermore, we analyzed myocardial tissues from patients with suspected inflammatory cardiomyopathy. TF protein expression was maximally 5-fold elevated 8 days p.i. in mice and remained increased on day 28 p.i. (P<0.001 vs. non-infected controls). Alterations in TF expression were associated with fibrin deposits within the myocardium. The TF pathway inhibitor protein expression in the myocardium was not altered during myocarditis. Active cellular TF co-localized with CD3 positive cells and VCAM-1 positive endothelial cells in the myocardium. The TF expression was positively correlated with the amount of infiltrating CD3 and Mac3 positive cells (Spearman-Rho rho=0.749 P<0.0001 for CD3(+) and rho=0.775 P<0.0001 for Mac3(+); N=35). Increased myocardial TF expression was associated with a 2-fold elevated plasma activity (P<0.05 vs. non-infected controls). In the human hearts, the TF expression correlated positively with an endothelial cell activation marker (rho=0.523 P<0.0001 for CD62E; N=54). Viral myocarditis is a hypercoagulative state which is associated with increased myocardial TF expression and activity. Upregulation of TF contributes to a systemic activation of the coagulation cascade.

Immunoadsorption and subsequent immunoglobulin substitution decreases myocardial gene expression of desmin in dilated cardiomyopathy.

Cardiac autoantibodies play a pathogenic role in dilated cardiomyopathy (DCM). Removal of antibodies by immunoadsorption (IA) induces hemodynamic improvement in DCM patients. The present study investigated the effects of IA on myocardial gene expression of the intermediate cytoskeletal filament desmin, which is upregulated in heart failure. RNA was isolated from five explanted non-failing hearts and five explanted failing hearts of DCM patients, and myocardial gene expression of desmin was estimated by real-time polymerase chain reaction (PCR). In a case-control study in six DCM patients (LVEF < 40%, NYHA II-III), IA and subsequent IgG substitution were performed at monthly intervals until month 3. Endomyocardial biopsies (EMBs) were obtained before and after IA (after 3-6 months). From six DCM patients without IA therapy (controls), EMBs were also obtained over a comparable time interval. Expression of the desmin gene was analyzed in these EMBs by real-time PCR. In failing explanted hearts, expression of desmin was significantly increased (0.88 +/- 0.12 vs 0.45 +/- 0.15 in non-failing hearts, P < 0.05). After IA, myocardial gene expression of desmin was significantly decreased (from 0.26 +/- 0.05 [baseline] to 0.14 +/- 0.04 [P < 0.05] vs baseline and controls). Removal of antibodies by IA modulates myocardial gene expression of desmin in DCM patients.

Autoimmunological features in inflammatory cardiomyopathy.

During recent years, increasing evidence has been obtained that cellular as well as humoral autoimmunity is involved in the pathogenesis of dilated cardiomyopathy (DCM). The immune system is generally activated by viral infections with the objective of virus elimination from the myocardium. However, a relevant number of patients demonstrate viral persistence and/or chronic inflammation in the myocardium. This chronic myocardial inflammation, defined by chronic inflammation, is termed "inflammatory cardiomyopathy" according to the WHO classification of cardiomyopathies. Chronic inflammation is frequently followed by the development of autoimmunity. A breakdown in the control mechanisms protecting against autoimmune reactions by both presentation of normally not accessible self-antigens and bystander- activation, induced by the pathogen, leads to the formation of autoreactive antibodies and T cells. The auto-reactive antibodies interact directly with heart tissue resulting in altered signal transduction or complement activation, whereas the T cell-mediated mechanisms include direct attack by cytotoxic T cells or indirect effects of cytotoxic cytokines released by stimulated T cells or macrophages.

Inhibition of urokinase-type plasminogen activator or matrix metalloproteinases prevents cardiac injury and dysfunction during viral myocarditis.

BACKGROUND: Acute viral myocarditis is an important cause of cardiac failure in young adults for which there is no effective treatment apart from general heart failure therapy. The present study tested the hypothesis that increased expression of the proteinases urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs) is implicated in cardiac inflammation, injury, and subsequent failure during Coxsackievirus-B3 (CVB3)-induced myocarditis. METHODS AND RESULTS: First, we showed increased expression and activity of uPA and MMP-9 in wild-type mice at 7 days of CVB3-induced myocarditis. Targeted deletion of uPA, which resulted in reduced MMP activity and cytokine expression or inhibition of MMPs by adenoviral gene overexpression of tissue inhibitor of metalloproteinases-1, decreased cardiac inflammation and reduced myocardial necrosis at 7 days and decreased cardiac fibrosis at 35 days after CVB3 infection. Importantly, loss of uPA or MMP activity prevented CVB3-induced cardiac dilatation and dysfunction, as determined by serial echocardiography. CONCLUSIONS: Loss of uPA or MMP activity reduces the cardiac inflammatory response after CVB3 infection, thereby protecting against cardiac injury, dilatation, and failure during CVB3-induced myocarditis.

Characterization of cis-elements mediating the stimulation of glucose-6-phosphate transporter promoter activity by glucocorticoids.

The endoplasmatic glucose-6-phosphate transporter is involved in the control of hepatic glucose production and blood glucose homeostasis. In this study, the expression of a luciferase reporter gene under the control of the glucose-6-phosphate transporter gene promoter was examined in transiently transfected hepatoma cells. The promoter activity was stimulated approximately 2.5-fold by dexamethasone. Mutational analyses demonstrated that the regions nucleotide (nt) -215/-209 and nt -197/-183 relative to the translation start site were critical for this regulation. In gel electrophoretic mobility shift assays the transcription factor Fox O1, also called forkhead in rhabdomyosarcoma (FKHR), overexpressed in 293 cells, bound to a probe with the sequence nt -215/-209. The overexpression of Fox O1 stimulated the induction of glucose-6-phosphate transporter promoter activity by dexamethasone via nt -215/-209 in hepatoma cells. Recombinant glucocorticoid receptor DNA binding domain protein bound to a probe with the sequence of nt -197/-183 in gel electrophoretic mobility shift assays and an oligonucleotide with this sequence transferred glucocorticoid responsiveness to a heterologous promoter. The data indicate that the glucose-6-phosphate transporter promoter contains a glucocorticoid response unit consisting of binding sites for Fox O1 and the glucocorticoid receptor.