Myocardial expression of the anaphylatoxin receptor C3aR is associated with cardiac inflammation and prognosis in patients with non-ischaemic heart failure.

AIM: The aim of this study is to analyse the prognostic value of complement anaphylatoxin receptors in patients with non-ischaemic cardiomyopathy undergoing endomyocardial biopsy. METHODS AND RESULTS: In 102 patients (72.5% male patients, median age 54 years) with non-ischaemic cardiomyopathy, myocardial expression of C3aR was assessed among other parameters. The primary study endpoint was a composite of death, heart transplantation, heart failure-related re-hospitalization, and deterioration of left ventricular ejection fraction within a mean follow-up of 11.9 months. The number of cells, which stained positive for C3aR, was significantly increased in patients with inflammatory compared with non-inflammatory cardiomyopathy (1.75 ± 0.31 cells in inflammatory cardiomyopathy vs. 0.94 ± 0.26 in non-inflammatory cardiomyopathy, P = 0.049). Subsequently, positive expression for C3aR was judged based on a semi-quantitative scoring system. Significantly, more patients with positive MHCII and CD68 expression showed an increased number of C3aR-positive cells. C3aR expression based on this score was more pronounced in patients with human herpesvirus 6 viral genome detection. Kaplan-Meier curves illustrate that the C3aR-negative group reached the primary endpoint significantly more often (mean follow-up 11.9 months, log rank 5.963, P = 0.015). Lack of C3aR expression was a strong independent predictor for the primary endpoint in Cox regression analysis [hazard ratio 0.46 (0.26-0.82, P = 0.009)]. CONCLUSIONS: C3aR-positive cells are found more often in patients with inflammatory cardiomyopathy. The relevance of C3aR-positive cells in patients with non-ischaemic cardiomyopathy should be further evaluated as potential predictors or modulators of adverse cardiac remodelling, the substrate of progressive heart failure.

CMR-Derived Extracellular Volume Fraction as a Marker for Myocardial Fibrosis: The Importance of Coexisting Myocardial Inflammation.

OBJECTIVES: The aim of the present study was to evaluate whether extracellular volume fraction (ECV) can reliably inform on the extent of diffuse fibrosis in the simultaneous presence of myocardial inflammation, which has not been verified to date. BACKGROUND: Diffuse myocardial fibrosis is associated with unfavorable outcome in patients with cardiomyopathy, and is of prognostic relevance. Assessment of ECV bears promise for being a noninvasive surrogate parameter, but it may be altered by other pathologies. METHODS: In this prospective study, 107 consecutive patients with clinical suspicion of inflammatory cardiomyopathy were included. All patients underwent left ventricular (LV) endomyocardial biopsy (EMB) and cardiac magnetic resonance imaging on a 1.5-T scanner. T1 mapping was obtained with the modified Look-Locker inversion recovery sequence, and ECV was calculated. RESULTS: Myocardial inflammation was present in 66 patients. Patients with and without inflammation were of similar age and had comparable LV ejection fraction (37 ± 17% vs. 36 ± 18%; p = 0.9) and symptom duration (median 14 days [interquartile range: 5 to 36 days] vs. median 14 days [interquartile range: 7 to 30 days]; p = 0.73). Although LV collagen volume percentage was comparable between groups (inflammation 12.3 ± 17.8% vs. noninflammation 11.4 ± 7.9%; p = 0.577), ECV was significantly higher in patients with inflammation (0.37 ± 0.06%) than in those without inflammation (0.33 ± 0.08%; p = 0.02). Importantly, ECV adequately estimated the degree of LV fibrosis percentage only in patients without inflammation (r = 0.72; p < 0.0001) and not in those with inflammation (r = 0.24; p = 0.06). CONCLUSIONS: These findings prove the theoretical concept of ECV as an estimate for diffuse myocardial fibrosis, but only in the absence of significant myocardial inflammation. Assuming that various degrees of myocardial inflammation and fibrosis coexist in such a scenario, the measured ECV will reflect a sum of these different pathologies but will not inform solely on the extent of diffuse fibrosis.

α-Galactosidase A Genotype N215S Induces a Specific Cardiac Variant of Fabry Disease.

BACKGROUND: Hypertrophic cardiomyopathy is the most common type of cardiomyopathy, but many patients lack sarcomeric/myofilament mutations. We studied whether cardio-specific α-galactosidase A gene variants are misinterpreted as hypertrophic cardiomyopathy because of the lack of extracardiac organ involvement. METHODS AND RESULTS: All subjects who tested positive for the N215S genotype (n=26, 13 females, mean age 49±17 [range, 14-74] years) were characterized in this prospective monocentric longitudinal cohort study to determine genotype-specific clinical characteristics of the N215S (c.644A>G [p.Asn215Ser]) α-galactosidase A gene variant. All subjects were initially referred with suspicion of genetically determined hypertrophic cardiomyopathy. Cardiac hypertrophy (interventricular septum, 12±4 [7-23] mm; left ventricular posterior wall, 11±4 [7-21] mm; left ventricular mass, 86±41 [46-195] g/m2) was progressive, systolic function mainly preserved (cardiac index 2.8±0.6 [1.9-3.9] L/min per m2), and diastolic function mildly abnormal. Cardiac magnetic resonance imaging revealed replacement fibrosis in loco typico (18/26, 69%), particularly in subjects >50 years. Elderly subjects had advanced heart failure, and 6 (23%) were suggested for implantable cardioverter-defibrillator therapy. Leukocyte α-galactosidase A enzyme activity was mildly reduced in 19 subjects and lyso-globotriaosylceramide slightly elevated (median, 4.9; interquartile range, 1.3-9.1 ng/mL). Neurological and renal impairments (serum creatinine, 0.87±0.20; median, 0.80; interquartile range, 0.70-1.01 mg/dL; glomerular filtration rate, 102±23; median, 106; interquartile range, 84-113 mL/min) were discreet. Only 2 subjects developed clinically relevant proteinuria. CONCLUSIONS: α-Galactosidase A genotype N215S does not lead to the development of a classical Fabry phenotype but induces a specific cardiac variant of Fabry disease mimicking nonobstructive hypertrophic cardiomyopathy. The lack of prominent noncardiac impairment leads to a significant delay in diagnosis and Fabry-specific therapy.

Clinical Characteristics, Histopathological Features, and Clinical Outcome of Methamphetamine-Associated Cardiomyopathy.

OBJECTIVES: This study aimed to assess characteristics including endomyocardial biopsy and outcome of patients with methamphetamine (MA)-associated cardiomyopathy in a series of patients treated in Germany. BACKGROUND: MA abuse is an increasing problem worldwide. METHODS: The cases of 30 consecutive MA-abusing patients with a left ventricular (LV) ejection fraction of <40% and endomyocardial biopsy performed at initial diagnosis were analyzed. Baseline characteristics were collected retrospectively, whereas follow-up was prospective. The primary endpoint was a composite of death, nonfatal stroke, and rehospitalization for heart failure. RESULTS: Patients were 30.3 ± 1.9 years of age, predominantly male (93.3%), and highly symptomatic; 83.3% had New York Heart Association functional class III or IV dyspnea. Echocardiography revealed marked LV dilatation (mean LV end-diastolic diameter 67.1 ± 7.4 mm) and impaired LV ejection fraction (mean 19 ± 6%). One-third of the patients had intraventricular thrombi. Endomyocardial biopsy revealed markers of inflammation and fibrosis; the fibrosis correlated with the duration of MA abuse. At follow-up, discontinuation of MA abuse together with medical therapy partially improved cardiac function (LV ejection fraction, 19 ± 6 vs. 43 ± 13; p < 0.001) and symptoms (p = 0.056), whereas patients with continued abuse did not show any improvement. The improvement in cardiac function was independently associated with the extent of fibrosis. The primary endpoint occurred more often in patients with continued MA abuse (57.1% vs. 13.0%; p = 0.037). CONCLUSIONS: MA-associated cardiomyopathy is characterized by severe heart failure and depressed cardiac function. The extent of myocardial fibrosis seems to predict the recoverability of LV function. Cessation of MA abuse is associated with improvement in cardiac function and symptoms, whereas continued MA abuse leads to ongoing heart failure and worse outcome.

Parvovirus B19-induced vascular damage in the heart is associated with elevated circulating endothelial microparticles.

BACKGROUND: Diagnosis of viral myocarditis is difficult by clinical criteria but facilitated by detection of inflammation and viral genomes in endomyocardial biopsies. Parvovirus B19 (B19V) targets endothelial cells where viral nucleic acid is exclusively detected in the heart. Microparticles (MPs) are released after cell damage or activation of specific cells. We aimed to investigate whether circulating endothelial MPs (EMPs) in human and experimental models of myocarditis are associated with B19V myocarditis. METHODS: MPs were investigated in patients with myocarditis (n = 54), divided into two groups: B19V+ (n = 23) and B19V- (n = 31) and compared with healthy controls (HCTR, n = 25). MPs were also investigated in B19V transgenic mice (B19V-NS1+) and mice infected with coxsackievirus B3 (CVB3). MPs were analyzed with fluorescent activated cell sorting (FACS). RESULTS: In human samples, EMP subpopulation patterns were significantly different in B19V+ compared to B19V- and HCTR (p<0.001), with an increase of apoptotic but not activated EMPs. Other MPs such as platelet- (PMPs) leukocyte-(LMPs) and monocyte-derived MPs (MMPs) showed less specific patterns. Significantly different levels of EMPs were observed in transgenic B19V-NS1+ mice compared with CVB3-infected mice (p<0.001). CONCLUSION: EMP subpopulations are different in B19V+ myocarditis in humans and transgenic B19V mice reflecting vascular damage. EMP profiles might permit differentiation between endothelial-cell-mediated diseases like myocardial B19V infection and other causes of myocarditis.

Histopathological and Immunological Characteristics of Tachycardia-Induced Cardiomyopathy.

BACKGROUND: Tachycardiomyopathy or tachycardia-induced cardiomyopathy (TCM) has been known for decades as a reversible form of nonischemic cardiomyopathy. However, its mechanism and properties remain poorly understood. OBJECTIVES: The current study investigated endomyocardial biopsy samples from patients with TCM and compared them with samples from patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (ICM). METHODS: The study included 189 patients with new-onset heart failure and severely reduced ejection fraction not caused by valvular or ischemic heart disease. Nineteen patients retrospectively fulfilled common criteria of TCM, 79 patients had a diagnosis of DCM, and 91 had a diagnosis of ICM. RESULTS: Patients with TCM, on the basis of clinical criteria, had stronger myocardial expression of major histocompatibility complex class II molecule and enhanced infiltration of CD68+ macrophages compared with patients with DCM. Furthermore, when compared with patients with ICM, the presence of T cells and macrophages was significantly reduced in TCM. Myocardial fibrosis was detected to a significantly lower degree in patients with TCM compared with patients with DCM and ICM. Electron microscopic examination revealed severe structural changes in patients with TCM. A disturbed distribution pattern of mitochondria was predominantly present in TCM. Quantitative assessment of myocyte morphology revealed significantly enhanced myocyte size compared with patients with ICM. Ribonucleic acid expression analysis identified changes in metabolic pathways among the patient groups. CONCLUSIONS: TCM is characterized by changes in cardiomyocyte and mitochondrial morphology accompanied by a macrophage-dominated cardiac inflammation. Thus, further prospective studies are warranted to characterize patients with TCM by endomyocardial biopsy more clearly.

Plasma and Cardiac Galectin-3 in Patients With Heart Failure Reflects Both Inflammation and Fibrosis: Implications for Its Use as a Biomarker.

BACKGROUND: Galectin (Gal)-3 is a ß-galactoside-binding lectin and currently intensely studied as a biomarker in heart failure. Gal-3 also exerts proinflammatory effects, at least in extracardiac tissues. Objective of this study was to characterize the relationship of plasma and myocardial Gal-3 levels with cardiac fibrosis and inflammation in patients with nonischemic dilated cardiomyopathy and inflammatory cardiomyopathy (iCMP). METHODS AND RESULTS: Endomyocardial biopsies and blood samples were obtained from patients with newly diagnosed cardiomyopathy and clinical suspicion of myocarditis. According to histopathologic findings, patients were classified as having dilated cardiomyopathy (n=40) or iCMP (n=75). Cardiac fibrosis was assessed histologically on endomyocardial biopsy sections. In patients with iCMP, myocardial Gal-3 expression significantly correlated with inflammatory cell count on endomyocardial biopsy (r=0.56; P<0.05). In contrast, an inverse association was observed between myocardial Gal-3 expression and cardiac fibrosis in patients with iCMP (r=-0.59; P<0.05). In patients with dilated cardiomyopathy, myocardial Gal-3 expression correlated with cardiac fibrosis on left ventricular biopsy (P=0.63; P<0.01). Of note, in both groups, plasma Gal-3 levels did not correlate with myocardial Gal-3 levels or left ventricular fibrosis, whereas a positive correlation between plasma Gal-3 levels and inflammatory cell count on endomyocardial biopsy was observed in patients with iCMP. CONCLUSIONS: The present study suggests that myocardial Gal-3 can be considered as a possible marker for both cardiac inflammation and fibrosis, depending on the pathogenesis of heart failure. However, circulating concentrations of Gal-3 do not seem to reflect endomyocardial Gal-3 levels or cardiac fibrosis.

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.

Inhibition of miR-208b improves cardiac function in titin-based dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is the result of maladaptive cardiac remodeling, which involves microRNA regulation. In turn, microRNAs can contribute to the remodeling process by post-transcriptional modulation of gene expression networks. The exact role of microRNAs in the pathogenesis of DCM is largely unknown. Here, we used an inducible DCM mouse model that carries a human truncation mutation in the sarcomeric protein titin to dissect microRNA pathways in DCM development. METHODS AND RESULTS: MicroRNA microarray studies revealed up-regulation of microRNA-208b in the myocardium of DCM mice and DCM patients (p<0.05 compared to controls). In order to investigate the effect of microRNA-208b on cardiac remodeling, loss-of-function and gain-of-function studies were performed by repetitive injections of LNA-modified microRNA-208b mimics and antimiR-208b. MiR-208b overexpression resulted in cardiac hypertrophy, whereas miR-208b antagonisation prevented transition of adaptive to maladaptive remodeling in the DCM mouse model. In vitro studies identified several pro-hypertrophic transcription factors as potential targets of miR-208b, suggesting that microRNA-208b plays an important role in cardiac development and growth. MiR-208b was also upregulated in DCM patients, but not in heart failure patients due to ischemic heart disease or myocarditis. CONCLUSION: Our data suggests that miR-208b is involved in the remodeling process and pathogenesis of DCM by post-transcriptional gene expression modulation. MicroRNA-208b might be a novel therapeutic target for DCM.

Biventricular endomyocardial biopsy in patients with suspected myocarditis: Feasibility, complication rate and additional diagnostic value.

BACKGROUND: Previous retrospective analyses have suggested that biventricular (BV) endomyocardial biopsy (EMB) is superior compared to selective left ventricular (LV) or right ventricular (RV) EMB. This study prospectively assessed the feasibility, safety and diagnostic performance of implementing a routine BV-EMB approach in patients with suspected myocarditis. METHODS: Consecutive patients with clinically suspected myocarditis underwent EMB (n=136). Myocarditis was defined as ≥14 infiltrating leukocytes/mm2 in addition to enhanced human leukocyte antigen class II expression in professional antigen-presenting immune cells. The presence of viral genomes was assessed by nested (reverse transcriptase-) polymerase chain reaction. RESULTS: BV-EMB was attempted in 132 patients (LV thrombus, n=3; complication during RV-EMB, n=1) and resulted in sufficient samples from both ventricles in 127 patients (96.2%). One major complication (pericardial tamponade requiring surgical revision) was observed during the 136 RV-EMB (0.7%). No severe complications occurred during the 132 LV procedures. Of the 127 patients with BV-EMB, myocarditis was diagnosed in 89 patients (70.1%). While 67 patients (75.3%) fulfilled the diagnostic criteria in both ventricles, the diagnosis of myocarditis was based on the results of LV-EMB only in 16 patients (18%) and of RV-EMB only in 6 patients (6.7%). Viral genomes were found in 45 of the 127 patients (35.4%) with evidence of virus genome only in the left ventricle in 10 patients (22.2%) and only in the right ventricle in 3 patients (6.7%). CONCLUSIONS: Implementing a routine BV-EMB approach is feasible and safe. In patients with suspected myocarditis, BV-EMB yields superior diagnostic performance compared to selective RV- or LV-EMB.

Comparison of Ventricular Inducibility with Late Gadolinium Enhancement and Myocardial Inflammation in Endomyocardial Biopsy in Patients with Dilated Cardiomyopathy.

BACKGROUND: Risk stratification of patients with non-ischemic dilated cardiomyopathy remains a matter of debate in the era of device implantation. OBJECTIVE: We investigated associations between histopathological findings, contrast-enhanced cardiac MRI and the inducibility of ventricular tachycardia (VT) or fibrillation (VF) in programmed ventricular stimulation. METHODS: 56 patients with impaired left ventricular ejection fraction (LVEF≤50%, mean 36.6±10.5%) due to non-ischemic dilated cardiomyopathy underwent cardiac MRI, programmed ventricular stimulation, and endomyocardial biopsy and were retrospectively investigated. Inducibility was defined as sustained mono- or polymorphic VT or unstable VT/VF requiring cardioversion/defibrillation. Primary study endpoint was defined as the occurrence of hemodynamically relevant VT/VF and/or adequate ICD-therapy during follow-up. RESULTS: Endomyocardial biopsy detected cardiac fibrosis in 18 (32.1%) patients. Cardiac MRI revealed 35 (62.5%) patients with positive late gadolinium enhancement. VT/VF was induced in ten (17.9%) patients during programmed ventricular stimulation. Monomorphic VT was inducible in 70%, while 20% of patients showed polymorphic VT. One patient (10%) presented with VF. Inducibility correlated significantly with the presence of positive late gadolinium enhancement in cardiac MRI (p<0.01). We could not find a significant association between inducibility and the degree of cardiac inflammation and fibrosis in non-site directed routine right ventricular endomyocardial biopsy. During a mean follow-up of 2.6 years, nine (16.1%) patients reached the primary endpoint. Monomorphic VTs were found in 66.7% patients and were terminated by antitachycardia pacing therapy. One patient with polymorphic VT and two patients with VF received adequate therapy by an ICD-shock. However, inducibility did not correlate with the occurrence of endpoints. CONCLUSION: Inducibilty during programmed ventricular stimulation is associated with positive late gadolinium enhancement in cardiac MRI of patients with non-ischemic dilated cardiomyopathy. The presence of myocardial fibrosis or inflammation in undirected endomyocardial biopsy does not seem to be sufficient to predict future ventricular arrhythmias.

Endomyocardial biopsy in patients with cardiomyopathy of unknown origin: does specialized center experience apply to a tertiary care hospital?

BACKGROUND: In patients with cardiomyopathy of unknown origin, endomyocardial biopsy provides the possibility of improved diagnosis and tailored treatment. Specific guidance has been developed based on cardiovascular centre of excellence experience but it is unknown if the benefits also extend into the tertiary care hospital setting. METHODS: Endomyocardial biopsies was performed in patients with cardiomyopathy of unknown origin. The outcomes were mirrored against the current ESC recommendations. RESULTS: A total of 57 patients with cardiomyopathy of unknown origin underwent endomyocardial biopsy with a mean age of 54 years and 28 % being women. In 17 patients (30 %), viruses were detected in the biopsy material, in 6 patients (11 %) cardiac amyloidosis was found of which 3 had also a positive test for viruses. The overall mortality rate was 18 % in the mean follow up period of 30 months, with a rate of 24 % in those with virus detection (mean FU 24 months) and 15 % in those without virus detection (mean FU 31 months. Death rates were 83 % in patients with cardiac amyloidosis (mean FU 10 months). CONCLUSION: We conclude that, limited by uncertainty stemming from the small number of included patients, endomyocardial biopsy may not prove to have a clinical impact on treatment decisions and outcomes in a tertiary care hospital setting. We consider cardiac amyloidosis to be an exception, since the mortality rate with or without concomitant virus load was extremely high.

Cardiovascular magnetic resonance patterns of biopsy proven cardiac involvement in systemic sclerosis.

BACKGROUND: To determine morphological and functional cardiovascular magnetic resonance (CMR) patterns in histopathologically confirmed myocardial involvement in patients with systemic sclerosis (SSc). METHODS: Twenty patients (6 females; mean age 41 ± 11 years) with histopathologically proven cardiac involvement in SSc in the years 2008-2016 were retrospectively evaluated. Morphological, functional and late gadolinium enhancement (LGE) images were acquired in standard angulations at 1.5 T CMR. Pathologies were categorized: 1) Pericardial effusion; 2) pathologic left (LV) or right ventricular (RV) contractility (hypokinesia, dyssynchrony, and diastolic restriction); 3) reduced left (LV-EF) and right ventricular ejection fraction (RV-EF); 4) fibrosis and/or inflammation (positive LGE); 5) RV dilatation. 95 % confidence intervals (CI) were calculated for appearance of pathologic EF and RV dilatation. RESULTS: Seven patients (35 %) had positive CMR findings in three categories, 9 patients (45 %) in four categories and 4 patients (20 %) in five categories. The distribution of pathologic findings was: minimal pericardial effusion in 7 patients (35 %), moderate pericardial effusion >5 mm in nine patients (45 %); abnormal LV or RV contractility in 19 patients (95 %), reduced LV or RV function in 14 patients (70 %; 95 % CI: 51-88 %), pathologic LGE in all patients, RV dilatation in 6 patients (30 %; 95 % CI: 15-54 %). CONCLUSIONS: CMR diagnosis of myocardial involvement in SSc requires increased attention to subtle findings. Pathologic findings in at least three of five categories indicate myocardial involvement in SSc.

A new monoclonal antibody (Cox mAB 31A2) detects VP1 protein of coxsackievirus B3 with high sensitivity and specificity.

Human enteroviruses, e.g. coxsackieviruses, induce a variety of severe acute and chronic forms of disease, including myocarditis, meningitis and diabetes mellitus type 1. To visualize enterovirus infection with a diagnostic intent, many studies have applied a commercially available antibody (anti-CVB5 VP1, clone 5-D8/1, Dako, Hamburg, Germany) that identifies VP1 of different enteroviral serotypes. Many antibodies, however, have been found to bind non-specifically to proteins of cardiomyocytes and in the interstitial space, resulting in non-specific staining in immunohistochemistry. In this paper we show that the anti-CVB5 VP1 antibody, recognizing VP1 of coxsackieviruses and widely used in diagnostics and research, shows strong cross-reactivity with cellular proteins in the heart (and pancreas) of humans and mice, which calls for a more specific antibody to be used for diagnostic purposes. We observed by Western blot analyses of lysates from human heart tissue samples and HeLa cells two cross-reactive bands when using clone 5-D8/1. Peptide mass fingerprinting (MALDI-TOF) identified these proteins as creatine kinase (B-type) and tubulin, confirming that this mAb detects cellular proteins in addition to viral VP1. In order to overcome the problems of false positive VP1 staining we generated a new highly specific and sensitive monoclonal antibody (Cox mAB 31A2) that recognizes VP1 from CVB3. The new antibody was characterized and was found to function well in immunohistochemistry, immunofluorescence staining, Western blotting, ELISA and FACS analyses.

Changes of myocardial gene expression and protein composition in patients with dilated cardiomyopathy after immunoadsorption with subsequent immunoglobulin substitution.

Immunoadsorption with subsequent immunoglobulin substitution (IA/IgG) represents a therapeutic approach for patients with dilated cardiomyopathy (DCM). Here, we studied which molecular cardiac alterations are initiated after this treatment. Transcription profiling of endomyocardial biopsies with Affymetrix whole genome arrays was performed on 33 paired samples of DCM patients collected before and 6 months after IA/IgG. Therapy-related effects on myocardial protein levels were analysed by label-free proteome profiling for a subset of 23 DCM patients. Data were analysed regarding therapy-associated differences in gene expression and protein levels by comparing responders (defined by improvement of left ventricular ejection fraction ≥20 % relative and ≥5 % absolute) and non-responders. Responders to IA/IgG showed a decrease in serum N-terminal proBNP levels in comparison with baseline which was accompanied by a decreased expression of heart failure markers, such as angiotensin converting enzyme 2 or periostin. However, despite clinical improvement even in responders, IA/IgG did not trigger general inversion of DCM-associated molecular alterations in myocardial tissue. Transcriptome profiling revealed reduced gene expression for connective tissue growth factor, fibronectin, and collagen type I in responders. In contrast, in non-responders after IA/IgG, fibrosis-associated genes and proteins showed elevated levels, whereas values were reduced or maintained in responders. Thus, improvement of LV function after IA/IgG seems to be related to a reduced gene expression of heart failure markers and pro-fibrotic molecules as well as reduced fibrosis progression.

Endomyocardial miR-133a levels correlate with myocardial inflammation, improved left ventricular function, and clinical outcome in patients with inflammatory cardiomyopathy.

AIMS: Inflammatory heart disease represents an important cause of chronic dilated cardiomyopathy (DCM). Predicting the clinical course of patients with inflammatory cardiomyopathy (iCMP) is difficult, and the prognostic value of current biological markers remains controversial. We tested whether expression of selected microRNAs in endomyocardial biopsies (EMBs) is related to LV functional recovery and clinical events in iCMP patients. METHODS AND RESULTS: EMBs were obtained from patients with iCMP (n = 76) and non-inflammatory DCM (n = 22). A set of six microRNAs implicated in inflammation (miR-155 and miR-146b), heart failure (miR-21 and miR-133a), and endothelial cell (miR-126) and skeletal muscle function (miR-206) was pre-defined. Endomyocardial expression of miR-155 and miR-133a, as quantified by reverse transcription-PCR (RT-PCR), was up-regulated in patients with iCMP as compared with patients with DCM. Levels of miR-133a (R = 0.73, P < 0.01) and miR-155 (R = 0.63, P < 0.01) correlated with inflammatory cell count on EMBs from patients with iCMP. Patients with iCMP and preserved LV function at study entry demonstrated higher expression of miR-133a than patients with reduced LV function. Also, increased expression of miR-133a was associated with less fibrosis and myocyte necrosis on EMB, and LV functional recovery during a mean follow-up of 3.1 years. Importantly, patients with iCMP and miR-133a levels in the upper tertile showed longer survival free of death, malignant arrhythmias, and hospitalizations for heart failure. CONCLUSION: The present study demonstrates that miR-133a levels correlate with macrophage infiltration, cardiac injury, improved LV function, and clinical outcome in patients with iCMP. miR-133a may serve as a potential novel biomarker and therapeutic target in human iCMP.

Comprehensive Cardiac Magnetic Resonance Imaging in Patients With Suspected Myocarditis: The MyoRacer-Trial.

BACKGROUND: Data suggest that T1 and T2 mapping have excellent diagnostic accuracy in patients with suspected myocarditis. However, the true diagnostic performance of comprehensive cardiac magnetic resonance (CMR) mapping versus endomyocardial biopsy (EMB) has not been determined. OBJECTIVES: This study assessed the performance of CMR imaging, including T1 and T2 mapping, compared with EMB in an unselected, consecutive patient cohort with suspected myocarditis. It also examined the potential role of CMR field strength by comparing 1.5-T versus 3.0-T imaging. METHODS: Patients underwent biventricular EMB, cardiac catheterization (for exclusion of coronary artery disease), and CMR imaging on 1.5- and 3-T scanners. The CMR protocol included current standard Lake Louise criteria (LLC) for myocarditis as well as native T1, calculation of extracellular volume fraction (ECV), and T2 mapping (only on 1.5-T). Patients were divided into 2 groups according to symptom duration (acute: ≤14 days vs. chronic: >14 days). RESULTS: A total of 129 patients underwent 1.5-T imaging. In patients with acute symptoms, native T1 yielded the best diagnostic performance as defined by the area under the curve (AUC) of receiver-operating curves (0.82) followed by T2 (0.81), ECV (0.75), and LLC (0.56). In patients with chronic symptoms, only T2 mapping yielded an acceptable AUC (0.77). On 3.0-T, AUCs of native T1, ECV, and LLC were comparable to 1.5-T with no significant differences. CONCLUSIONS: In patients with acute symptoms, mapping techniques provide a useful tool for confirming or rejecting the diagnosis of myocarditis and are superior to the LLC. However, only T2 mapping has acceptable diagnostic performance in patients with chronic symptoms. (Magnetic Resonance Imaging in Myocarditis [MyoRacer]; NCT02177630).