Abstract 772: Calcitonin-gene Related Peptide Receptor Regulation In Human Heart Failure
Human alpha-Calcitonin gene-related peptide (CGRP) is a 37 amino-acid neuropeptide that is a potent vasodilator found widely in the nervous and cardiovascular systems. Plasma CGRP levels are significantly upregulated in conditions of cardiac-pathophysiology. CGRP binds to the calcitonin receptor-like receptor (CRLR). The CRLR acts as a CGRP receptor only when associated with receptor activity modifying protein-1 (RAMP1). In contrast, association of CRLR with receptor activity modifying protein-3 (RAMP3) causes CRLR to act as a receptor for adrenomedullin. Since the presence of CRLR in human hearts has not been previously confirmed nor its regulation elucidated, we tested for the CGRP receptor complex expression in human hearts. Cardiac plasma membrane fractions were isolated from non-failing unmatched organ donors without cardiac disease (n=4) and human heart failure samples diagnosed with dilated cardiomyopathy (n=6). The plasma membrane fractions were subjected to CGRP radio-ligand binding assay in the absence and presence of synthetic human alpha-CGRP (VasoGenix Pharmaceuticals Inc., Lenexa, KS). Radio-ligand binding showed a significant increase (38%) in the CGRP receptors (CRLR/RAMP1 complex) in dilated cardiomyopathy compared to the controls (15.25 ± 1.34 vs. 11.05 ± 1.48 fmol/mg protein, p< 0.02). To test whether the increase in receptors is due to augmentation in the CRLR or change in the RAMP composition, RT-PCR analysis was carried out for CRLR, RAMP1 and RAMP3. RT-PCR analysis for CRLR showed no difference between heart failure and control samples. Importantly, RAMP1 was upregulated in heart failure compared to unmatched controls. In contrast, RAMP3 was significantly downregulated in heart failure. Western immunoblotting on the plasma membrane fractions from human heart failure and controls supports our RT-PCR data. RAMP1 was significantly upregulated and contrastingly RAMP3 was downregulated in heart failure compared to the unmatched controls. Taken together these data demonstrate that CGRP receptors are upregulated in human heart failure and the cause for upregulation of CGRP receptors is due to dynamic changes in the RAMP protein that alters the membrane composition to CGRP receptors.