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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-EC_II) 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-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-ß₁-EC_II–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-EC_II–induced receptor activation, which could be achieved, however, by the addition of ß₁-EC_II 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-EC_II–induced receptor activation, which opens new venues for the research on anti-ß₁-Abs and eventual treatment options in heart failure.

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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