FcγReceptors IIa on Cardiomyocytes and Their Potential Functional Relevance in Dilated Cardiomyopathy

ObjectivesThe purpose of this study was to investigate how cardiac autoantibodies might contribute to cardiac dysfunction in patients suffering from dilated cardiomyopathy (DCM).BackgroundIn the majority of DCM patients, it is possible to detect antibodies with negative inotropic effect on cardiomyocytes. The manner in which these antibodies impair cardiac function is poorly understood.MethodsImmunoglobulin (Ig)G was prepared from plasma of 11 DCM patients containing antibodies that induced a negative inotropic effect on cardiomyocytes. We analyzed the effects of antibodies/IgG fragments on calcium transients and on systolic cell shortening of adult rat cardiomyocytes and investigated the dependency of these effects on potential cardiomyocyte Fc receptors.ResultsIn contrast to control subjects, intact IgG from DCM patients reduced calcium transients and cell shortening of cardiomyocytes. The F(ab′)2fragments of these antibodies did not induce these effects but inhibited the functional effects of DCM-IgG of the respective patients’ IgG. These effects were also inhibited by Fc fragments of normal IgG. Reconstitution of the Fc part by incubation of cardiomyocytes with DCM-F(ab′)2fragments followed by goat-anti-human-F(ab′)-IgG again induced reduction of cell shortening and of calcium transients. In rat and human ventricular cardiomyocytes, Fcγreceptors IIa (CD32) were demonstrated by immunofluorescence.ConclusionsOur findings indicate that DCM-IgG-F(ab′)2bind to their cardiac antigen(s), but the Fc part might trigger the negative inotropic effects via the newly detected Fcγreceptor on cardiomyocytes. These results point to a novel potential mechanism for antibody-induced impairment of cardiac function in DCM patients

Funktionelle Effekte kardiotroper Antikörper bei Dilatativer Kardiomyopathie

Die Dilatative Kardiomyopathie (DCM) ist eine häufige Herzmuskelerkrankung und ist durch eine eingeschränkte systolische Pumpfunktion mit Erweiterung des linken, des rechten oder beider Ventrikel gekennzeichnet. Es sind eine Vielzahl von Ursachen bekannt, u. a. genetische, infektiöse und toxische. Des Weiteren sind eine Vielzahl von kardiotropen Antikörpern nachgewiesen worden, die bei der Pathogenese der DCM eine Rolle spielen. In dieser Dissertationsschrift sollten die funktionellen Effekte kardiotroper Antikörper bei Patienten mit DCM untersucht werden und ein eventuell bestehender Zusammenhang zwischen der Häufigkeit der Antikörper mit einer begleitenden Virusinfektion, Inflammation oder dem Stadium der Krankheit hergestellt werden. Die Untersuchung des Bindungsmechanismus der Antikörper an kardiale Antigene, bzw. eventuell beteiligte Rezeptoren auf den Kardiomyozyten war ein weiteres Anliegen der vorliegenden Arbeit. Die therapeutischen Erfolge einer Entfernung von Antikörpern aus dem Plasma von DCM-Patienten (Immunadsorption) sollten mit den gewonnenen Ergebnissen korreliert werden. Die Wirkung der aufgereinigten IgG-Fraktion aus Plasma von Patienten mit DCM auf Kontraktilität und Ca2+-Ratio während eines Kontraktionszyklus wurde auf isolierten Rattenkardiomyozyten getestet. Dabei konnte festgestellt werden, dass das Plasma von 57 % der DCM-Patienten eine negativ inotrope Wirkung auf die isolierten Rattenkardiomyozyten hat. Die Subgruppenanalysen der untersuchten Patienten ergaben keine Hinweise auf eine Korrelation der negativen Inotropie mit einer bestehenden Virusinfektion oder Myokardinflammation. Allerdings konnte bei männlichen Patienten, bei Patienten mit nur leicht reduzierter Pumpfunktion und kürzerer Krankheitsdauer ein erhöhtes Auftreten negativ inotroper Antikörper festgestellt werden. Durch Pepsin-Spaltung negativ inotrop wirksamer Antikörper und der Vorinkubation von isolierten Rattenkardiomyozyten mit F(ab)2-Fragmenten und Fc-Fragmenten konnte gezeigt werden, dass für die negativ inotrope Wirkung sowohl die antigenbindenden Strukturen als auch der Fc-Teil des Antikörpers verantwortlich sind. In diesem Zusammenhang wurde auf Kardiomyozyten erstmals ein Fc-Rezeptor (Fc-Rezeptor IIa; CD32A) mittels Immunfluoreszenz, Westernblot und Elektronenmikroskopie detektiert. Die Therapieerfolge der Entfernung der Antikörper aus dem Plasma von Patienten mittels der Immunadsorptionstherapie konnten mit den in-vitro Untersuchungen korreliert werden. Patienten mit negativ inotrop wirksamen Antikörpern profitieren stärker von der Therapie. Auch der Genotyp des exprimierten Fc-Rezeptors beeinflusst die Verbesserung der kardialen Parameter der Patienten durch die Therapie. Patienten homozygot für Arginin an der Aminosäure 131 des Proteins besitzen den niedrigaffinen Rezeptor und profitieren stärker von einer Entfernung der Antikörper als Patienten mit dem hochaffinen Histidin-Genotyp. Die Ergebnisse dieser Arbeit zeigen, dass bei einem Teil der DCM-Patienten kardiodepressive Antikörper im Plasma vorhanden sind, welche sowohl über den antigenbindenden F(ab)-Teil als auch über den Fc-Teil die Funktion der Kardiomyozyten beeinflussen. Die Entfernung dieser Antikörper führt zu einer Besserung der kardialen Funktion. Die unterschiedliche Affinität der adsorbierten Antikörper zum Fc-Rezeptor IIa, welche beeinflusst wird durch den H/R-131 Genpolymorphismus, bestimmt hierbei das Ansprechen der Patienten auf die Therapie. Mit der vorliegenden Arbeit ist die aktive Rolle kardiotroper Antikörpern in der Pathogenese der Dilatativen Kardiomyopathie unterstrichen worden und mit der Entdeckung des Fc-Rezeptors IIa ein Mechanismus zum Verständnis der physiologischen Wirkungsweise der Antikörper mit verschiedensten Zielepitopen aufgezeigt worden.Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and is characterized by ventricular chamber enlargement and systolic dysfunction. The cause of DCM remains uncertain but there are different factors that are important, e.g. a genetic disorder, infection and toxic substances. Besides in DCM various antibodies against different cardiac cell proteins have been identified. In this thesis the functional effects of cardiotropic antibodies from patients with DCM and a probably correlation of the prevalence of cardiotropic antibodies and a virus infection, inflammation or state of the disease should be investigated. Besides the mechanism of the binding of the antibodies to cardiac epitopes and associated receptors were studied. The therapeutical effect of the removal of the antibodies by immunoadsorption therapy (IA) should be correlated with the experimental results. The effect of purificated IgG from patients suffering from DCM on the contractility and Ca2+-ratio of isolated rat cardiomyocytes was tested and a negative inotropic reaction was observed for 57% of the patients. Subgroup analysis of the tested patients showed no correlation between a myocardial inflammation, virus infection and the reaction of the rat cardiomyocytes. However, a higher prevalence of negative inotropic IgG was found for male patients, patients with shorter disease duration and less impaired ejection fraction. The negative inotropic reaction of the cardiomyocytes was triggered by binding of the antibodies with their antigen binding domain as well as with the Fc-fragment. This was discovered by using pepsin digestion of the negative inotropic IgG and preincubation of the isolated rat cardiomyocytes with F(ab)2- and Fc-fragments. Thereby the cardiac Fcgamma receptor IIa was identified on the surface of rat and human cardiomyocytes by immunofluorescence, electron microscopy and western blotting. The therapeutical outcome of the removal of antibodies by immunoadsorption therapy could be correlated with the in-vitro experiments. Patients where negative inotropic antibodies could be detected improved more in left ventricular function. Besides an influence of the expressed genotype of the Fcgamma receptor IIa regarding the benefit of the therapy was disclosed. A homozygote expression of the low affinity receptor type with argentine at amino acid position 131 resulted in a higher increase of left ventricular ejection fraction than for patients expressing the high-affinity receptor isoform Fcã receptor IIa-H131 (Histidin). The results of this thesis could show that for a subgroup of DCM-patients cardio depressive antibodies are detectable which impact the function of the cardiomyocytes via their F(ab)2- and their Fc-part. The removal of the antibodies could improve cardiopulmonary function. The different affinity of the adsorbed antibodies towards the Fcgamma receptor IIa which is controlled by the H/R-131 gene polymorphism has an influence on the patients’ response to immunoadsorption therapy. This thesis points out the active role of cardiotropic antibodies in the pathogenesis of dilated cardiomyopathy. A new mechanism for understanding the physiological effect of antibodies with different cardiac epitopes is hypothesized by detecting the Fcgamma receptor IIa

Collecting duct principal cell transport processes and their regulation.

The principal cell of the kidney collecting duct is one of the most highly regulated epithelial cell types in vertebrates. The effects of hormonal, autocrine, and paracrine factors to regulate principal cell transport processes are central to the maintenance of fluid and electrolyte balance in the face of wide variations in food and water intake. In marked contrast with the epithelial cells lining the proximal tubule, the collecting duct is electrically tight, and ion and osmotic gradients can be very high. The central role of principal cells in salt and water transport is reflected by their defining transporters-the epithelial Na(+) channel (ENaC), the renal outer medullary K(+) channel, and the aquaporin 2 (AQP2) water channel. The coordinated regulation of ENaC by aldosterone, and AQP2 by arginine vasopressin (AVP) in principal cells is essential for the control of plasma Na(+) and K(+) concentrations, extracellular fluid volume, and BP. In addition to these essential hormones, additional neuronal, physical, and chemical factors influence Na(+), K(+), and water homeostasis. Notably, a variety of secreted paracrine and autocrine agents such as bradykinin, ATP, endothelin, nitric oxide, and prostaglandin E2 counterbalance and limit the natriferic effects of aldosterone and the water-retaining effects of AVP. Considerable recent progress has improved our understanding of the transporters, receptors, second messengers, and signaling events that mediate principal cell responses to changing environments in health and disease. This review primarily addresses the structure and function of the key transporters and the complex interplay of regulatory factors that modulate principal cell ion and water transport

A Protein Kinase A-Independent Pathway Controlling Aquaporin 2 Trafficking as a Possible Cause for the Syndrome of Inappropriate Antidiuresis Associated with Polycystic Kidney Disease 1 Haploinsufficiency

Renal water reabsorption is controlled by arginine vasopressin (AVP), which binds to V2 receptors, resulting in protein kinase A (PKA) activation, phosphorylation of aquaporin 2 (AQP2) at serine 256, and translocation of AQP2 to the plasma membrane. However, AVP also causes dephosphorylation of AQP2 at S261. Recent studies showed that cyclin-dependent kinases (cdks) can phosphorylate AQP2 peptides at S261 in vitro. We investigated the possible role of cdks in the phosphorylation of AQP2 and identified a new PKA-independent pathway regulating AQP2 trafficking. In ex vivo kidney slices and MDCK-AQP2 cells, R-roscovitine, a specific inhibitor of cdks, increased pS256 levels and decreased pS261 levels. The changes in AQP2 phosphorylation status were paralleled by increases in cell surface expression of AQP2 and osmotic water permeability in the absence of forskolin stimulation. R-Roscovitine did not alter cAMP-dependent PKA activity but specifically reduced protein phosphatase 2A (PP2A) expression and activity in MDCK cells. Notably, we found reduced PP2A expression and activity and reduced pS261 levels in Pkd1(+/-) mice displaying a syndrome of inappropriate antidiuresis with high levels of pS256, despite unchanged AVP and cAMP. Similar to previous findings in Pkd1(+/-) mice, R-roscovitine treatment caused a significant decrease in intracellular calcium in MDCK cells. Our data indicate that reduced activity of PP2A, secondary to reduced intracellular Ca(2+) levels, promotes AQP2 trafficking independent of the AVP-PKA axis. This pathway may be relevant for explaining pathologic states characterized by inappropriate AVP secretion and positive water balance

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 ≤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 β1-adrenergic receptor (second extracellular loop, cardiac β1-adrenergic receptor [SEL-β1-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-β1-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-β1-AR, and cTnI appear to be associated with DCM. Antibodies against KChIP2 may enhance cell death of rat cardiomyocytes

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