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Figure 2 FRET Detection of Functional Anti-ß1-Abs in Patients
Measuring cAMP by Epac-FRET detects functional anti-ß1-Abs in dilated cardiomyopathy (DCM) patients. (A) None of the IgG prepared from healthy control patients (n = 50) induced a significant cAMP response in living cells (left). The IgG from DCM patients previously judged anti-ß1-Abs-positive (15) (Abs+) elicited marked cAMP responses (49.5 ± 3.8% of maximal Iso signal, right). The IgG from 23 of 38 (60%) previously anti-ß1-Abs-negative judged patients (6) (Abs–) showed a robust but significantly smaller increase in cAMP (31.3 ± 6.8%, p < 0.01 according to the Student t test, center). Representative experiments of at least 3 different cells for each IgG preparation are shown. (B) Histogram with Gaussian distribution curves based on the strength of the FRET signal in healthy control patients versus DCM patients (R2 = 0.98 for a 3-peak Gaussian distribution). The antibody induced FRET signals allow discrimination of 3 groups of activity: no activity (n = 15), low activity (n = 22), high activity (n = 18); grouping was performed as described in the text. (C) Frequency and distribution of FRET signals obtained with IgG preparations from n = 22 previously antibody-typed ischemic cardiomyopathy (ICM) patients. Five of them showed high FRET activity, and the remainder was classified antibody-negative. (D) Concentration-response relationship between high-activator and low-activator IgG showing significantly different activation capacities over a wide range of antibody concentrations (p < 0.05; Student t test). The percent ± SEM of maximal Iso-induced cAMP responses is presented (normalized to maximal changes in FRET induced by high-activator anti-ß1-Abs; representative experiments, n = 4). Abbreviations as in Figure 1.
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