A Novel Fluorescence Method for the Rapid Detection of Functional β1-Adrenergic Receptor Autoantibodies in Heart Failure

Objectives: This study sought to develop a rapid method for the detection of activating autoantibodies directed against the β1-adrenoceptor (anti-β1-Abs) in patients with heart failure. Background: The anti-β1-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 β1-receptor loop (anti-β1-ECII) in 13% of patients with ischemic cardiomyopathy (ICM) and in 26% with dilated cardiomyopathy (DCM). Methods: To detect anti-β1-Abs, we measured β1-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-β1-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-β1-ECII–negative patients (17 ICM/15 DCM) did not significantly affect cAMP. Surprisingly, our technology detected anti-β1-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-β1-Abs were blocked preferentially by peptides corresponding to the first, and “high”-activator anti-β1-Abs by peptides corresponding to the second β1-extracellular loop. Beta-blockers alone failed to fully prevent anti-β1-ECII–induced receptor activation, which could be achieved, however, by the addition of β1-ECII peptides. Conclusions: Our novel method of detecting anti-β1-Abs proved to be fast and highly sensitive. It also revealed an insufficient ability of beta-blockers to prevent anti-β1-ECII–induced receptor activation, which opens new venues for the research on anti-β1-Abs and eventual treatment options in heart failure. [Copyright &y& Elsevier]