In vivo T2* weighted MRI visualizes cardiac lesions in murine models of acute and chronic viral myocarditis.

OBJECTIVE: Acute and chronic forms of myocarditis are mainly induced by virus infections. As a consequence of myocardial damage and inflammation dilated cardiomyopathy and chronic heart failure may develop. The gold standard for the diagnosis of myocarditis is endomyocardial biopsies which are required to determine the etiopathogenesis of cardiac inflammatory processes. However, new non-invasive MRI techniques hold great potential in visualizing cardiac non-ischemic inflammatory lesions at high spatial resolution, which could improve the investigation of the pathophysiology of viral myocarditis. RESULTS: Here we present the discovery of a novel endogenous T2* MRI contrast of myocardial lesions in murine models of acute and chronic CVB3 myocarditis. The evaluation of infected hearts ex vivo and in vivo by 3D T2w and T2*w MRI allowed direct localization of virus-induced myocardial lesions without any MRI tracer or contrast agent. T2*w weighted MRI is able to detect both small cardiac lesions of acute myocarditis and larger necrotic areas at later stages of chronic myocarditis, which was confirmed by spatial correlation of MRI hypointensity in myocardium with myocardial lesions histologically. Additional in vivo and ex vivo MRI analysis proved that the contrast mechanism was due to a strong paramagnetic tissue alteration in the vicinity of myocardial lesions, effectively pointing towards iron deposits as the primary contributor of contrast. The evaluation of the biological origin of the MR contrast by specific histological staining and transmission electron microscopy revealed that impaired iron metabolism primarily in mitochondria caused iron deposits within necrotic myocytes, which induces strong magnetic susceptibility in myocardial lesions and results in strong T2* contrast. CONCLUSION: This T2*w MRI technique provides a fast and sensitive diagnostic tool to determine the patterns and the severity of acute and chronic enteroviral myocarditis and the precise localization of tissue damage free of MR contrast agents.

A standardised FACS assay based on native, receptor transfected cells for the clinical diagnosis and monitoring of ß1-adrenergic receptor autoantibodies in human heart disease.

BACKGROUND: Autoantibodies against ß1-adrenergic receptors (ß1AR) that stimulate cardiac cAMP-production play a causal role in the pathogenesis of human heart failure. Patients can be subjected to specific therapies, if the presence of potentially cardio-noxious ß1AR-autoantibodies is reliably diagnosed. This requires assessment of IgG-interactions with the native ß1AR because ß1AR-autoantibodies target a conformational epitope inadequately presented by denatured receptors or linear peptides. Here, we report on a standardised diagnostic procedure for the assessment of ß1AR-autoantibodies in heart failure patients, which is based on IgG-binding to native human ß1AR. METHODS: Good laboratory practice (GLP)-conform measurement of ß1AR-autoantibodies was based on flow-cytometric quantification of differential IgG-binding to native HT1080 cells overexpressing biofluorescent human ß1AR or not. Receptor-specific IgG-binding was derived from IgG-related median fluorescence of ß1AR-positive cells corrected for background staining of ß1AR-negative cells admixed to each measurement. The slope of IgG binding at two different concentrations was used as measure for the titre/avidity of ß1AR-autoantibodies. RESULTS: Sensitivity and specificity of the novel procedure for high ß1AR-autoantibody levels in dilated cardiomyopathy patients (n=40, NYHA class III-IV) relative to n=40 matched healthy subjects was >90%. It was similar to functional assays considered the gold standard and vastly superior to existing screening-procedures employing fixed cells or linear receptor-peptides as auto-antigenic targets. Inter-assay scatter was 7%-15% and linear dilution recovery was within ±10% of expected values throughout. CONCLUSIONS: The novel assay possibly provides a tool to determine true prevalence and clinical impact of ß1AR-autoantibodies. Furthermore, it may serve as companion diagnostic for therapies specifically directed at ß1AR-autoantibodies.

Novel receptor-derived cyclopeptides to treat heart failure caused by anti-ß1-adrenoceptor antibodies in a human-analogous rat model.

Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the ß1 adrenergic receptor (ß1EC2). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human ß1EC2 (amino-acids 195-225) every 4 weeks; n = 38/114 rats were control-injected with 0.9% NaCl. Intravenous application of a novel cyclic peptide mimicking ß1EC2 (ß1EC2-CP, 1.0 mg/kg every 4 weeks) or administration of the ß1-blocker bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received ß1EC2-CP/bisoprolol co-treatment. We found that ß1EC2-CP prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, ß1EC2-CP mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free anti-ß1EC2-antibodies and by targeting ß1EC2-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful anti-ß1EC2-antibodies and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to ß1-blockade represents a promising new therapeutic option in immune-mediated heart failure.

Detection of anti-ß1-AR autoantibodies in heart failure by a cell-based competition ELISA.

RATIONALE: Autoantibodies directed against the second extracellular loop of the cardiac ß1-adrenergic receptor (ß1-AR) are thought to contribute to the pathogenesis of dilated cardiomyopathy (DCM) and Chagas heart disease. Various approaches have been used to detect such autoantibodies; however, the reported prevalence varies largely, depending on the detection method used. OBJECTIVE: We analyzed sera from 167 DCM patients (ejection fraction<45%) and from 110 age-matched volunteers who did not report any heart disease themselves, with an often used simple peptide-ELISA approach, and compared it with a novel whole cell-based ELISA, using cells expressing the full transgene for the human ß1-AR. Additionally, 35 patients with hypertensive heart disease with preserved ejection fraction were investigated. METHODS AND RESULTS: The novel assay was designed according to the currently most reliable anti-TSH receptor antibody-ELISA used to diagnose Graves disease ("third-generation assay") and also detects the target antibodies by competition with a specific monoclonal anti-ß1-AR antibody (ß1-AR MAb) directed against the functionally relevant ß1-AR epitope. Anti-ß1-AR antibodies were detected in ≈60% of DCM patients and in ≈8% of healthy volunteers using the same cutoff values. The prevalence of these antibodies was 17% in patients with hypertensive heart disease. Anti-ß1-AR antibody titers (defined as inhibition of ß1-AR MAb-binding) were no longer detected after depleting sera from IgG antibodies by protein G adsorption. In contrast, a previously used ELISA conducted with a linear 26-meric peptide derived from the second extracellular ß1-AR loop yielded a high number of false-positive results precluding any specific identification of DCM patients. CONCLUSIONS: We established a simple and efficient screening assay detecting disease-relevant ß1-AR autoantibodies in patient sera yielding a high reproducibility also in high throughput screening. The assay was validated according to "good laboratory practice" and can serve as a companion biodiagnostic assay for the development and evaluation of antibody-directed therapies in antibody-positive heart failure.

Targeting receptor antibodies in immune cardiomyopathy.

Although autoimmunity represents a well-established pathogenetic principle in several endocrine (Graves' disease), rheumatic (systemic lupus erythematosus), and neurological disorders (myasthenia gravis, multiple sclerosis), this mechanism has only recently gained more attention in cardiac diseases. Depending on individual genetic predisposition, heart-directed autoimmune reactions are supposed to emerge as a consequence of cardiomyocyte injury induced by inflammation, ischemia, or exposure to cardiotoxic substances. Myocyte apoptosis or necrosis and subsequent liberation of a "critical amount" of cardiac autoantigens may then induce a self-directed immune response, which in the worst case results in perpetuation of autoantibody-mediated cardiac damage. In particular, functionally active autoantibodies (aabs) directed against the cardiac beta1-adrenergic receptor (beta1-aabs) have been assigned a pivotal role in the pathogenesis of immune cardiomyopathy. Conformational beta1-aabs allosterically activate the sympathetic transmembrane signaling cascade, thereby increasing sarcoplasmatic cyclic adenosine monophosphate (cAMP) and calcium concentrations. Chronic cAMP production and calcium overload are cardiotoxic, leading to myocyte apoptosis, fibrotic repair, subsequent heart muscle dysfunction, and, finally, a dilative cardiomyopathic phenotype. Elimination by (extracorporeal) immunoadsorption or direct neutralization of the harmful receptor autoantibodies in the circulating blood represent promising strategies to protect the heart from beta1-(auto)antibody-induced damage.

Pathological autoantibodies in cardiomyopathy.

Myocardial dilatation and dysfunction in the absence of significant coronary heart disease has been termed "idiopathic" dilated cardiomyopathy (iDCM), which--according to the 1995 task force report on the classification of cardiomyopathies-besides genetic, toxic or infectious causes also includes immune-mediated heart muscle damage in the spectrum of putative DCM etiologies. Incremental research on this topic particularly in the past few years has significantly contributed evidence to the hypothesis that autoimmune reactions against certain myocyte antigens may play a pivotal role in the initiation and/or progression of DCM. Recent transfer experiments in animals (mostly rodents) performed by various groups throughout the world and some preliminary clinical data even indicate that a few of these autoantibodies are indeed "pathogenic", inferring that they can actually cause cardiac dysfunction and heart failure by their own. Dependent on the individual genetic predisposition such harmful autoimmune reactions are supposed to emerge as a consequence of heart muscle damage induced by viral triggers, ischemia or exposure to cardiotoxins leading to myocyte apoptosis (and/or necrosis) and subsequent liberation of a "critical amount" of self-antigens previously hidden to the immune system. The following article will summarize the so far available evidence for an implication of a confined number of harmful autoantibodies directed against specific cardiac antigens in the pathogenesis of DCM.

A novel fluorescence method for the rapid detection of functional beta1-adrenergic receptor autoantibodies in heart failure.

OBJECTIVES: This study sought to develop a rapid method for the detection of activating autoantibodies directed against the beta1-adrenoceptor (anti-beta1-Abs) in patients with heart failure. BACKGROUND: The anti-beta1-Abs are supposed to play a pathophysiological role in heart failure. However, there is no reliable method for their detection. With a complex screening strategy (enzyme-linked immunosorbent assay, immunofluorescence, cyclic adenosine monophosphate [cAMP]-radioimmunoassay) we have previously identified antibodies targeting the second extracellular beta1-receptor loop (anti-beta1-EC(II)) in 13% of patients with ischemic cardiomyopathy (ICM) and in 26% with dilated cardiomyopathy (DCM). METHODS: To detect anti-beta1-Abs, we measured beta1-receptor-mediated increases in intracellular cAMP by fluorescence resonance energy transfer using a highly sensitive cAMP sensor (Epac1-based fluorescent cAMP sensor). RESULTS: The immunoglobulin G (IgG) prepared from 77 previously antibody-typed patients (22 ICM/55 DCM) and 50 matched control patients was analyzed. The IgG from all 22 previously anti-beta1-EC(II)-positive patients (5 ICM/17 DCM) induced a marked cAMP increase, indicating receptor activation (49.8 +/- 4.2% of maximal isoproterenol-induced signal). The IgG from control patients and 32 previously anti-beta1-EC(II)-negative patients (17 ICM/15 DCM) did not significantly affect cAMP. Surprisingly, our technology detected anti-beta1-Abs in 23 DCM patients formerly judged antibody-negative, but their cAMP signals were generally lower (31.3 +/- 6.8%) than in the previous group. "Low"-activator anti-beta1-Abs were blocked preferentially by peptides corresponding to the first, and "high"-activator anti-beta1-Abs by peptides corresponding to the second beta1-extracellular loop. Beta-blockers alone failed to fully prevent anti-beta1-EC(II)-induced receptor activation, which could be achieved, however, by the addition of beta1-EC(II) peptides. CONCLUSIONS: Our novel method of detecting anti-beta1-Abs proved to be fast and highly sensitive. It also revealed an insufficient ability of beta-blockers to prevent anti-beta1-EC(II)-induced receptor activation, which opens new venues for the research on anti-beta1-Abs and eventual treatment options in heart failure.

Stimulating autoantibodies directed against the cardiac beta1-adrenergic receptor predict increased mortality in idiopathic cardiomyopathy.

BACKGROUND: The aim of this study was to estimate the independent and incremental prognostic value of the presence of stimulating autoantibodies directed against the human beta1-adrenergic receptor (anti-beta1-AR) in patients with chronic heart failure. METHODS: One hundred five antibody-typed chronic heart failure patients with dilated cardiomyopathy (DCM, n = 65) or ischemic cardiomyopathy (ICM, n = 40) were prospectively followed for 10.7 +/- 2.5 years. Information on all-cause and cardiovascular mortality was collected throughout the observation period. RESULTS: Stimulating anti-beta1-AR were prevalent in 26% (17/65) of patients with DCM and 13% (5/40) with ICM. All-cause mortality in antibody-positive patients was 65% in those with DCM and 80% in those with ICM, and in antibody-negative patients 44% and 49%, respectively. In univariate and multivariable Cox regression analysis (P < .05), presence of stimulating anti-beta1-AR was associated with increased all-cause and cardiovascular mortality risk in DCM but not in ICM. Information on antibody status improved the prognostic capacity in models containing already extensive information on clinical profile, Holter electrocardiography, and invasive hemodynamic measurements (area under the receiver operating characteristic curve, 0.91; 95% confidence interval, 0.85-0.97; P < .05 for increase in receiver operating characteristic area). CONCLUSION: The presence of stimulating anti-beta1-AR autoantibodies independently predicts increased all-cause and cardiovascular mortality risk in DCM conferring incremental prognostic value in addition to established risk predictors. Our data indicate a clinical relevance of stimulating anti-beta1-AR in DCM and encourage further research into antibody-directed strategies as a therapeutic principle.

Beta 1-adrenergic receptor-directed autoimmunity as a cause of dilated cardiomyopathy in rats.

Progressive cardiac dilatation and pump failure of unknown etiology has been termed idiopathic dilated cardiomyopathy (DCM). During recent years a large body of data has accumulated indicating that functionally active antibodies or autoantibodies being able to recognize and to stimulate the cardiac beta(1)-adrenergic receptor (anti-beta(1)-AR) may play an important role in the initiation and/or clinical course of DCM. Recent experiments in rats even point towards a cause-and-effect relation between stimulatory anti-beta(1)-AR antibodies and DCM. Immunization of rats against the second extracellular loop of the human beta(1)-adrenergic receptor (100% sequence-identity between human and rat) resulted in both development of stimulatory anti-beta(1)-AR antibodies and development of progressive cardiac dilatation and dysfunction. Isogenic transfer of stimulatory anti-beta(1)-AR from cardiomyopathic into healthy inbred animals reproduced the disease, hence providing conclusive proof for a beta(1)-receptor-directed autoimmune attack as a possible cause of cardiomyopathy. This kind of cardiomyopathy is now referred to as anti-beta(1)-AR-induced dilated immune-cardiomyopathy (DiCM). The following article reviews recent evidence obtained from experimental animal-models implying a significant role of the cardiac beta(1)-adrenergic receptor as a pathophysiologically and clinically relevant autoantigen also in human DCM.

beta(1)-Adrenergic receptor function, autoimmunity, and pathogenesis of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a heart disease characterized by progressive depression of cardiac function and left ventricular dilatation of unknown etiology in the absence of coronary artery disease. Genetic causes and cardiotoxic substances account for about one third of the DCM cases, but the etiology of the remaining 60% to 70% is still unclear. Over the past two decades, evidence has accumulated continuously that functionally active antibodies or autoantibodies targeting cardiac beta(1)-adrenergic receptors (anti-beta(1)-AR antibodies) may play an important role in the initiation and/or clinical course of DCM. Recent experiments in rats indicate that such antibodies can actually cause DCM. This article reviews current knowledge and recent experimental and clinical findings focusing on the role of the beta(1)-adrenergic receptor as a self-antigen in the pathogenesis of DCM.

False aneurysm formation following cutting balloon angioplasty in the renal artery of a child.

PURPOSE: To report the fortuitous discovery of a false aneurysm 7 years after cutting balloon angioplasty for severe renal artery fibromuscular dysplasia in a child. CASE REPORT: A 3-year-old girl with neurofibromatosis was referred to our institution because of high blood pressure (220/160 mmHg). Computed tomography identified coarctation of the aorta and severe bilateral renal artery stenoses. The coarctation was successfully resected. One month later, bilateral renal artery angioplasty with a 3-mm balloon was unsatisfactory, so a second angioplasty one month later was done with a 3.25-mm cutting balloon. This procedure was complicated by a minimal arterial rupture, which resolved spontaneously after inflation of a regular balloon. Normal blood pressure was restored. The child was lost to follow-up until 7 years later, when recurrent hypertension (200 mmHg systolic) prompted referral again. Arteriography showed a very severe stenosis on the right side and a 30-mm false aneurysm of the left renal artery at the rupture site. Due to her age, the patient underwent surgery, which brought the blood pressure under control. CONCLUSIONS: False aneurysm of the renal arteries is a rare complication of percutaneous angioplasty. In a child, the cutting balloon would appear to be contraindicated for concentric dysplastic stenoses that are resistant to regular balloon angioplasty.

Direct evidence for a beta 1-adrenergic receptor-directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy.

Today, dilated cardiomyopathy (DCM) represents the main cause of severe heart failure and disability in younger adults and thus is a challenge for public health. About 30% of DCM cases are genetic in origin; however, the large majority of cases are sporadic, and a viral or immune pathogenesis is suspected. Following the established postulates for pathogenesis of autoimmune diseases, here we provide direct evidence that an autoimmune attack directed against the cardiac beta(1)-adrenergic receptor may play a causal role in DCM. First, we immunized inbred rats against the second extracellular beta(1)-receptor loop (beta(1)-EC(II); 100% sequence identity between human and rat) every month. All these rats developed first, receptor-stimulating anti-beta(1)-EC(II) Ab's and then, after 9 months, progressive severe left ventricular dilatation and dysfunction. Second, we transferred sera from anti-beta(1)-EC(II)-positive and Ab-negative animals every month to healthy rats of the same strain. Strikingly, all anti-beta(1)-EC(II)-transferred rats also developed a similar cardiomyopathic phenotype within a similar time frame, underlining the pathogenic potential of these receptor Ab's. As a consequence, beta(1)-adrenergic receptor-targeted autoimmune DCM should now be categorized with other known receptor Ab-mediated autoimmune diseases, such as Graves disease or myasthenia gravis. Although carried out in an experimental animal model, our findings should further encourage the development of therapeutic strategies that combat harmful anti-beta(1)-EC(II) in receptor Ab-positive DCM patients.