Unmasking features of the auto-epitope essential for ß1 -adrenoceptor activation by autoantibodies in chronic heart failure.

AIMS: Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac ß1 -adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human ß1 -adrenoceptor (ß1 ECII ) that is targeted by stimulating ß1 -receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. METHODS AND RESULTS: Non-conserved amino acids within the ß1 ECII loop (compared with the amino acids constituting the ECII loop of the ß2 -adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine-serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking ß1 ECII  ± the above replacements, and (ii) by (auto)antibody stimulation of human ß1 -adrenoceptors bearing corresponding point mutations. With the use of stimulating ß1 -receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the ß1 ECII loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK211-214 motif and (ii) the intra-loop disulfide bond C209 ↔C215 . Of note, aberrant intra-loop disulfide bond C209 ↔C216 almost fully disrupted the functional auto-epitope in cyclopeptides. CONCLUSIONS: The conformational auto-epitope targeted by cardio-pathogenic ß1 -receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the ß1 ECII loop bearing the NDPK211-214 motif and the C209 ↔C215 bridge while lacking cysteine C216 . Such molecules provide promising tools for novel diagnostic and therapeutic approaches in ß1 -autoantibody-positive CHF.

11C-Methionine PET of Myocardial Inflammation in a Rat Model of Experimental Autoimmune Myocarditis.

Myocarditis represents a major cause of dilated cardiomyopathy and sudden cardiac death in younger adults. Currently, definitive diagnosis of myocarditis requires endomyocardial biopsy, which is highly invasive and has the drawback of variable sensitivity due to inherent sampling error. Therefore, reliable noninvasive methods to detect and monitor cardiac inflammation are clinically relevant. In this study, we explored the potential of radiolabeled methionine to assess myocardial inflammatory activity in a rat model of experimental autoimmune myocarditis (EAM). METHODS: Autoimmune myocarditis was induced by immunizing Lewis rats twice with porcine cardiac myosin and Freund complete adjuvant. Control animals were treated with adjuvant alone. Dual-tracer autoradiography was performed to assess 14C-methionine uptake and to compare the distributions of 14C-methionine versus 18F-FDG. Hematoxylin and eosin staining and anti-CD68 macrophage staining were performed for histologic analysis. Additionally, cardiac 11C-methionine PET was performed to evaluate the feasibility of in vivo imaging. 18F-FDG PET was also conducted to compare the in vivo uptake of 11C-methionine and 18F-FDG. RESULTS: Multiple focal cardiac inflammatory lesions were histologically identified in myosin-immunized rats, whereas no cardiac lesions were observed in the controls. Autoradiographic images clearly showed a high-density accumulation of 14C-methionine in inflammatory lesions of EAM rats, whereas no significant uptake was observed in the control animals. 14C-methionine uptake was significantly higher in inflammatory lesions than in remote noninflammatory areas and control rat hearts. The distribution of 14C-methionine correlated well with that of 18F-FDG and with macrophage density. The contrast between inflammatory and noninflammatory areas was higher for 18F-FDG than for 14C-methionine (3.45 ± 0.68 vs. 2.07 ± 0.21, respectively; P < 0.05). In the PET imaging study, the regional 11C-methionine uptake (percentage injected dose per cubic centimeter) observed in EAM rats was significantly higher than the values obtained for control animals (0.64 ± 0.09 vs. 0.28 ± 0.02, respectively; P < 0.001). A good positive correlation between 11C-methionine and 18F-FDG uptake was found. CONCLUSION: In a rat model of autoimmune myocarditis, we demonstrated the colocalization of radiolabeled methionine accumulation with 18F-FDG uptake in histologically proven inflammatory lesions. These data suggest that 11C-methionine might represent a promising candidate for the noninvasive detection and monitoring of myocarditis.