Martin Landsberger

Removal of cardiodepressant antibodies in dilated cardiomyopathy by immunoadsorption.

OBJECTIVES The objective of this study was to investigate whether immunoadsorption (IA) removes cardiodepressant antibodies from the plasma of patients with dilated cardiomyopathy (DCM), as well as to describe their effects on isolated rat cardiomyocytes. BACKGROUND Immunoadsorption induces early hemodynamic improvement in patients with DCM. The mechanisms for this improvement remain to be elucidated. METHODS Patients with DCM (n = 11; left ventricular ejection fraction < 30%, cardiac index [CI] < 2.5 l/min per m(2)) were treated with IA on three consecutive days, with one IA session daily, by application of specific antibody columns directed against human immunoglobulin (Ig). Immunoadsorption was also conducted on 500 ml of blood taken from nine healthy donors (control subjects). After passage of plasma, the IA columns were regenerated. Column eluent (CE) was collected and dialyzed (100 kD). Confocal laser scanning microscopy was used to analyze the effects of CE on cell contraction and on Ca(2+)-dependent fluorescence in isolated, field-stimulated adult rat cardiomyocytes loaded with cell-permeable Fluo-3. Immunoprecipitation with different preparations of myocardial protein fractions was used for characterization of cardiotropic antibodies. RESULTS During IA, the IgG plasma level decreased from 10.7 +/- 0.6 to 2.4 +/- 0.1 g/l (mean +/- SEM), and the CI increased from 2.2 +/- 0.1 to 2.7 +/- 0.2 l/min per m(2) (p < 0.01). The CE obtained from control subjects did not influence Ca(2+) transients or cell shortening of cardiomyocytes. In contrast, in patients with DCM, the CE collected during the first regeneration cycle of the first IA session caused an immediate and dose-related decrease of Ca(2+) transients (dilution 1:5; -22.7 +/- 5.5%; p < 0.01) and cell shortening (dilution 1:5; -29.9 +/- 6.0%, p < 0.01). Early hemodynamic improvement among the patients correlated with the cardiodepressant effect of CE on the isolated cardiomyocytes. Purification of CE by protein A adsorption indicated that the cardiodepressant substances are antibodies. Immunoprecipitation revealed that the eliminated antibodies are capable of binding to various myocardial proteins. CONCLUSIONS Cardiac autoantibodies play a functional role in DCM, and their removal may induce early hemodynamic improvement.

Activation of Akt is essential for acetylcholine to trigger generation of oxygen free radicals

OBJECTIVES Acetylcholine (ACh) receptor activation in the heart causes mitochondrial production of reactive oxygen species (ROS) that is dependent on mitochondrial K(ATP) channel opening. Recent data show that Akt (also known as protein kinase B) is phosphorylated at its activation site following exposure to ACh. However, since no reliable Akt inhibitor is available, it has not been possible to determine whether Akt activation is an actual step in the protective pathway. METHODS Cultured rat vascular smooth muscle cells (A7r5) were transiently transfected with a dominant negative Akt (Akt-AAA), thus inhibiting the ability of ACh in these cells to phosphorylate Akt. Transfected cells were identified by co-transfection of enhanced green fluorescent protein (EGFP). ROS production was determined by incubating the cells for 15 min with 1 mM reduced MitoTracker Red which becomes fluorescent only after reacting with ROS. Cells were then triple-washed to remove any voltage-dependent pool of dye and single cell fluorescence was measured. RESULTS ACh exposure (2 mM) led to a 1.64+/-0.15-fold increase in the average fluorescence over that seen in untreated cells (P=0.002). A similar increase in ROS production occurred after treatment with either the K(ATP) channel opener diazoxide (DIAZ) or the potassium ionophore valinomycin (VAL). Akt-AAA transfection abolished ACh-induced ROS production, but not increased ROS production after treatment with either DIAZ or VAL. CONCLUSION Thus, at least in the smooth muscle cell model, Akt phosphorylation is an important step in the signal transduction pathway leading from ACh receptor activation to the generation of ROS. The experiments reveal that Akt is positioned between the receptor and the K(ATP) channel in this model.

Effects of immunoadsorption and subsequent immunoglobulin G substitution on cardiopulmonary exercise capacity in patients with dilated cardiomyopathy

BACKGROUND Recent data indicate that cardiac antibodies play an active role in the pathogenesis of dilated cardiomyopathy (DCM) and may contribute to cardiac dysfunction in patients with DCM. The present study investigated the influence of immunoadsorption with subsequent immunoglobulin G substitution (IA/IgG) on cardiopulmonary exercise capacity in patients with DCM. METHODS Sixty patients with DCM (New York Heart Association II-IV, left ventricular ejection fraction < or =45%) were included in this single-center university hospital-based case-control study. Patients either were treated with IA/IgG (n = 30) or were followed without IA/IgG (n = 30). At baseline and after 3 months, we compared echocardiographic assessment of left ventricular function and spiroergometric exercise parameters. RESULTS In contrast to controls, left ventricular ejection fraction improved significantly in the IA/IgG group from 33.0% +/- 1.2% to 40.1% +/- 1.5% (P < .001). In the control group, spiroergometric exercise parameters did not change during follow-up. After 3 months, maximum achieved power increased in the treatment group from 114.2 +/- 7.4 to 141.9 +/- 7.9 W (P = .02). Total exercise time increased in the treatment group from 812 +/- 29 to 919 +/- 30 seconds (P < .05). Peak oxygen uptake (Vo(2)) increased from 17.3 +/- 0.9 to 21.8 +/- 1.0 mL min(-1) kg(-1) after IA/IgG (P < .01). Oxygen pulse (peak Vo(2)/maximum heart rate) increased in the treatment group (10.7 +/- 0.7 vs 13.6 +/- 0.7 mL beat(-1) min(-1), P < .01). The Vo(2) at the gas exchange anaerobic threshold increased after 3 months in the treatment group from 10.3 +/- 0.5 to 13.2 +/- 0.5 mL min(-1) kg(-1) (P < .001). The ventilatory response to exercise (V(E)/Vco(2) slope) decreased after IA/IgG therapy from 32.3 +/- 1.5 to 28.7 +/- 0.9 (P = .02). CONCLUSIONS In patients with DCM, IA/IgG therapy may induce improvement in echocardiographic and cardiopulmonary exercise parameters.

Immunoadsorption in dilated cardiomyopathy: long-term reduction of cardiodepressant antibodies

Eur J Clin Invest 2010; 40 (8): 685–691

Potential role of antibodies against cardiac Kv channel-interacting protein 2 in dilated cardiomyopathy

BACKGROUND Growing evidence suggests participation of autoimmune mechanisms in the pathogenesis of dilated cardiomyopathy (DCM). METHODS Patients with heart failure (left ventricular ejection fraction < or =50%) due to DCM (n = 98) or ischemic cardiomyopathy (ICM, n = 49) and controls with normal left ventricular function (n = 98) were included. Immunoglobulin G antibodies were purified from plasma by affinity chromatography and analyzed by surface plasmon resonance analysis. We analyzed the distribution of autoantibodies against Kv channel-interacting protein (KChIP) 2.6, cardiac troponin I (cTnI), and the beta1-adrenergic receptor (second extracellular loop, cardiac beta1-adrenergic receptor [SEL-beta1-AR])-two other known autoantibodies involved in heart failure. Effects of antibodies against KChIP2 on cell death of isolated rat cardiomyocytes were assessed by flow cytometry. RESULTS We detected autoantibodies against KChIP2.6 in 14.3% (P < .015 vs controls, P = .286 vs ICM) of the DCM samples, in 8.2% of the ICM samples (P = .304 vs controls), and in 4.1% of the control samples. Virus persistence was significantly associated with detection of autoantibodies against KChIP2.6 in DCM patients (P = .025). Antibodies against SEL-beta1-AR were more frequent in DCM samples (34.7%, P < .001 vs controls, P = .02 vs ICM) and ICM samples (16.3%, P = .083 vs control) than in control samples (7.1%). Antibodies against cTnI were more frequent in DCM samples (20.4%, P < .001 vs controls, P = .769 vs ICM) and in ICM samples (18.4%, P < .01 vs controls) than in control samples (4.1%). Antibodies against rat KChIP2 enhanced cell death in isolated rat cardiomyocytes. Immunofluorescence indicated cell surface expression of KChIP2. CONCLUSIONS Autoantibodies against KChIP2.6, SEL-beta1-AR, and cTnI appear to be associated with DCM. Antibodies against KChIP2 may enhance cell death of rat cardiomyocytes.

Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 reduces leukocyte adhesion within the intestinal microcirculation in experimental endotoxemia in rats

IntroductionLectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the major endothelial receptor for oxidized low-density lipoprotein, is also involved in leukocyte recruitment. Systemic leukocyte activation in sepsis represents a crucial factor in the impairment of the microcirculation of different tissues, causing multiple organ failure and subsequently death. The aim of our experimental study was to evaluate the effects of LOX-1 inhibition on the endotoxin-induced leukocyte adherence and capillary perfusion within the intestinal microcirculation by using intravital microscopy (IVM).MethodsWe used 40 male Lewis rats for the experiments. Ten placebo-treated animals served as a control. Thirty animals received 5 mg/kg lipopolysaccharide (LPS) intravenously. Ten endotoxemic rats remained untreated. In 10 LPS animals, we administered additionally 10 mg/kg LOX-1 antibodies. Ten further LPS animals received a nonspecific immunoglobulin (rat IgG) intravenously. After 2 hours of observation, intestinal microcirculation was evaluated by using IVM; the plasma levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) were determined; and LOX-1 expression was quantified in intestinal tissue with Western blot and reverse-transcription polymerase chain reaction (PCR).ResultsLOX-1 inhibition significantly reduced LPS-induced leukocyte adhesion in intestinal submucosal venules (P < 0.05). At the protein and mRNA levels, LOX-1 expression was significantly increased in untreated LPS animals (P < 0.05), whereas in animals treated with LOX-1 antibody, expression of LOX-1 was reduced (P < 0.05). MCP-1 plasma level was reduced after LOX-1 antibody administration.ConclusionsInhibition of LOX-1 reduced leukocyte activation in experimental endotoxemia. LOX-1 represents a novel target for the modulation of the inflammatory response within the microcirculation in sepsis.

Prostaglandin receptors mediate effects of substances released from ischaemic rat hearts on non-ischaemic cardiomyocytes

Background  After ischaemia and during reperfusion, rat hearts release cardiodepressive substances that are putatively cyclooxygenase‐2‐dependent. The present study analyses the mechanisms by which these substances mediate their effect downstream of cyclooxygenase‐2.

Molecular changes in the early phase of renin-dependent cardiac hypertrophy in hypertensive cyp1a1ren-2 transgenic rats

An early response to high arterial pressure is the development of cardiac hypertrophy. Functional and transcriptional regulation of ion channels and Ca2+ handling proteins are involved in this process but the relative contribution of each is unclear. In this study, we investigated the expression of genes involved in action potential generation and Ca2+ homeostasis of cardiomyocytes in hypertensive cyp1a1ren-2 transgenic rats. In this model, the transgene prorenin was induced by indole-3-carbinol for 2 weeks allowing the induction of hypertension. Electrophysiological recordings from cardiomyocytes of hypertensive rats revealed a slight increase in membrane capacitance consistent with cellular hypertrophy. L-type calcium current density was reduced by 30%. Left ventricles of hypertensive rats showed a significant increase in transcript and protein levels of the cation channel TRPC6 and FK506-binding protein, whereas levels of SERCA2 and voltage-dependent potassium channels Kv4.2 and Kv4.3 were found to be decreased. Further, a marked nuclear localization of the transcription factors GATA4 and NFATC4 was observed in cardiac tissue of hypertensive rats. The cyp1a1ren-2 transgenic rat thus appears to be a valid model to investigate early changes in cardiac hypertrophy. This study points to roles for TRPC6, FK506BP, SERCA2, Kv4.2, and Kv4.3 in the development of cardiac hypertrophy.

Die Rolle der Zytokine bei der Herzinsuffizienz

Hintergrund: Zytokine haben als wichtige Mediatoren des Immunsystems bei verschiedenen kardialen Erkrankungen eine pathophysiologische Bedeutung. Gegenwärtig sind 18 Zytokine mit dem Namen Interleukin (IL) bekannt, die in pro- und antiinflammatorische Zytokine unterteilt werden können. Sie wirken z. T. negativ inotrop, verursachen einen Myozytenuntergang und eine vermehrte Fibrosierung. Kardial wirksame Zytokine: Das proinflammatorische Zytokin Tumor-Nekrose-Faktor (TNF-)α wirkt kardiodepressiv und apoptotisch. TNF-α, IL-6 sowie löslicher TNF-Rezeptor-1 und -2 stellen unabhängige Prädiktoren für eine erhöhte Mortalität bei Patienten mit Herzinsuffizienz dar. Experimentell und klinisch konnte nachgewiesen werden, dass bei der Herzinsuffizienz die Plasma- und Gewebsspiegel von TNF-α in Bereiche ansteigen, die zumindest z. T. die Symptome der Herzinsuffizienz mit einer Wirkung dieses Zytokins erköären können. Klinische Studien: Ausgehend von erfolgversprechenden Pilotstudien sollte durch zwei Multicenterstudien (RENAISSANCE, RECOVER) geprüft werden, ob der TNF-α-Antagonist (Etanercept) signifikant Morbidität und Mortalität von Patienten mit Herzinsuffizienz beeinflusst. Beide Studien mussten jedoch vorzeitig beendet werden, da kein günstiger Einfluss auf die beiden klinischen Endpunkte nachgewiesen werden konnte. Diese mangelnde Wirksamkeit kann jedoch nicht dahingehend interpretiert werden, dass TNF-α bei Entstehung und Progredienz der Herzinsuffizienz keine pathophysiologisch bedeutsame Rolle spielt.Background: Cytokines are important mediators of the immune system and are of pathophysiological relevance for different cardiac diseases. Currently, 18 cytokines carrying the name interleukin (IL) are known; they can be subdivided into pro- and anti-inflammatory interleukins. Cardiac Cytokines: They partly act in a negative inotropic manner and cause destruction of cardiomyocytes resulting in myocardial fibrosis. The proinflammatory cytokine tumor necrosis factor (TNF-)α induces cardiodepressive effects and causes apoptosis. TNF-α, IL-6, soluble TNF-receptor-1 and -2 are independent predictors of increased mortality of patients with heart failure. Experimental and clinical evidence has shown that plasma and tissue levels of TNF-α were elevated to such extent as to explain at least some of the symptoms of heart failure due to the actions of this cytokine. Clinical Trials: Two multicenter studies (RENAISSANCE, RECOVER), based on promising pilot studies, have disclosed no effect for the TNF-α antagonists (Etanercept) on mortality and morbidity. It is not possible, however, to draw the conclusion from the data of these studies that TNF-α plays no significant pathophysiological role in the etiology and progression of heart failure.

Antibodies against potassium channel interacting protein 2 induce necrosis in isolated rat cardiomyocytes.

Auto‐antibodies against cardiac proteins have been described in patients with dilated cardiomyopathy. Antibodies against the C‐terminal part of KChIP2 (anti‐KChIP2 [C‐12]) enhance cell death of rat cardiomyocytes. The underlying mechanisms are not fully understood. Therefore, we wanted to explore the mechanisms responsible for anti‐KChIP2‐mediated cell death. Rat cardiomyocytes were treated with anti‐KChIP2 (C‐12). KChIP2 RNA and protein expressions, nuclear NF‐κB, mitochondrial membrane potential Δψm, caspase‐3 and ‐9 activities, necrotic and apoptotic cells, total Ca2+ and K+ concentrations, and the effects on L‐type Ca2+ channels were quantified. Anti‐KChIP2 (C‐12) induced nuclear translocation of NF‐κB. Anti‐KChIP2 (C‐12)‐treatment for 2 h significantly reduced KChIP2 mRNA and protein expression. Anti‐KChIP2 (C‐12) induced nuclear translocation of NF‐κB after 1 h. After 6 h, Δψm and caspase‐3 and ‐9 activities were not significantly changed. After 24 h, anti‐KChIP2 (C‐12)‐treated cells were 75 ± 3% necrotic, 2 ± 1% apoptotic, and 13 ± 2% viable. Eighty‐six ± 1% of experimental buffer‐treated cells were viable. Anti‐KChIP2 (C‐12) induced significant increases in total Ca2+ (plus 11 ± 2%) and K+ (plus 18 ± 2%) concentrations after 5 min. Anti‐KChIP2 (C‐12) resulted in an increased Ca2+ influx through L‐type Ca2+ channels. In conclusion, our results suggest that anti‐KChIP2 (C‐12) enhances cell death of rat cardiomyocytes probably due to necrosis. J. Cell. Biochem. 115: 678–689, 2014.