Hypertrophic Effects of Calcitonin Gene-related Peptide (CGRP) and Amylin on Adult Mammalian Ventricular Cardiomyocytes

Calcitonin gene-related peptide (CGRP), a neuropeptide localized in the cardiac autonomic nervous supply, shares 46% similarity in sequence of amino acids with amylin, a peptide synthesized in pancreaticβ-cells. In the present study, the question was addressed whether these peptides could exert hypertrophic effects in cardiomyocytes isolated from the ventricles of adult rats and maintained in short-term, serum-free primary culture. FCS (10% v/v), employed as a positive control, increased the incorporation ofl-[14C] phenylalanine into cellular protein, total content of cellular RNA and total mass of cellular protein significantly. CGRP and amylin also increased each of these parameters significantly and in a concentration-dependent manner; maximum responses occurred at 100 p and 10 n for CGRP and amylin, respectively. The selective antagonist at CGRP1-receptors, CGRP8–37(100 n ), inhibited significantly the incorporation ofl-[14C] phenylalanine into cellular protein in response to CGRP and amylin. The selective inhibitor of protein kinase C (PKC), bisindolylmalemide (BIM) (5μ ), reduced significantly the incorporation ofl-[14C] phenylalanine into cellular protein in response to phenylephrine (1μ ), employed as a positive control, but did not inhibit the response to insulin (1 unit/ml), employed as a negative control. BIM (5μ ) reduced significantly the responses to FCS (10% v/v), amylin (10 n ) and CGRP (10 p ), but did not inhibit the response to CGRP (100 p ). The activity of protein kinase C in membranes prepared from intact cardiomyocytes pre-treated for 10 min with the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (100 n ), employed as a positive control, and CGRP (10 p ) was significantly greater than in membranes prepared from cardiomyocytes not subjected to agonist stimulation. Phenylephrine (1μ ) increased significantly the specific activity of creatine kinase but not of lactate dehydrogenase in day 1 cultures of freshly isolated cardiomyocytes. Significant induction of creatine kinase, but not lactate dehydrogenase, was also stimulated by CGRP and amylin; the maximum responses occurred at 100 p and 100 n CGRP and amylin, respectively. In conclusion, CGRP and amylin exert hypertrophic effects directly on ventricular cardiomyocytes from the hearts of adult ratsin vitro. These effects are: (1) due tode novoprotein synthesis since total content of cellular RNA and incorporation ofl-[14C] phenylalanine into cellular protein were also increased; (2) mediated by a common population of CGRP1-preferring receptors at which amylin binds with lower potency; (3) mediated, at least partly, by the activation of PKC; (4) may be associated with a fetal shift in gene expression, characterized by selective induction of creatine kinase.