CLINICAL RESEARCH: HEART FAILURE
A Novel Fluorescence Method for the Rapid Detection of Functional ß1-Adrenergic Receptor Autoantibodies in Heart Failure
Viacheslav O. Nikolaev, PhD*,1,
Valérie Boivin, PhD*,1,
Stefan Störk, MD, PhD ,
Christiane E. Angermann, MD*,
Georg Ertl, MD,
Martin J. Lohse, MD* and
Roland Jahns, MD*,,*
* Institut für Pharmakologie und Toxikologie, Herz- und Kreislaufzentrum, University of Würzburg, Würzburg, Germany
Medizinische Klinik und Poliklinik I, Herz- und Kreislaufzentrum, University of Würzburg, Würzburg, Germany.
Manuscript received July 21, 2006;
revised manuscript received February 1, 2007,
accepted March 20, 2007.
* Reprint requests and correspondence: Dr. Roland Jahns, Medizinische Klinik und Poliklinik I, Herz- und Kreislaufzentrum, University of Würzburg, Klinikstr. 6-8, 97070 Würzburg, Germany. (Email: jahns_r{at}klinik.uni-wuerzburg.de).
Objectives: This study sought to develop a rapid method for the detection of activating autoantibodies directed against the ß1-adrenoceptor (anti-ß1-Abs) in patients with heart failure.
Background: The anti-ß1-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 ß1-receptor loop (anti-ß1-ECII) in 13% of patients with ischemic cardiomyopathy (ICM) and in 26% with dilated cardiomyopathy (DCM).
Methods: To detect anti-ß1-Abs, we measured ß1-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-ß1-ECII–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-ß1-ECII–negative patients (17 ICM/15 DCM) did not significantly affect cAMP. Surprisingly, our technology detected anti-ß1-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-ß1-Abs were blocked preferentially by peptides corresponding to the first, and "high"-activator anti-ß1-Abs by peptides corresponding to the second ß1-extracellular loop. Beta-blockers alone failed to fully prevent anti-ß1-ECII–induced receptor activation, which could be achieved, however, by the addition of ß1-ECII peptides.
Conclusions: Our novel method of detecting anti-ß1-Abs proved to be fast and highly sensitive. It also revealed an insufficient ability of beta-blockers to prevent anti-ß1-ECII–induced receptor activation, which opens new venues for the research on anti-ß1-Abs and eventual treatment options in heart failure.
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Abbreviations and Acronyms
| | anti-ß1-Abs = activating autoantibodies against the ß1-adrenergic receptor | | ß1-AR = ß1-adrenergic receptor/ß1-adrenoceptor | | ß1-ECI
= the first extracellular loop of the ß1-adrenergic receptor | | ß1-ECII
= the second extracellular loop of the ß1-adrenergic receptor | | cAMP = cyclic adenosine monophosphate | | CFP = cyan fluorescent protein | | DCM = dilated cardiomyopathy | | Epac1-camps = Epac1-based fluorescent cAMP sensor | | FRET = fluorescence resonance energy transfer | | GST = glutathione-S-transferase | | ICM = ischemic cardiomyopathy | | isomax
= maximal signal induced by isoproterenol | | PKA = protein kinase A | | YFP = yellow fluorescent protein |
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