Hypertrophic effects of calcitonin gene-related peptide (CGRP) and amylin on adult mammalian ventricular cardiomyocytes

Abstract

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 pancreatic beta-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 of l-[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 pM and 10 nM for CGRP and amylin, respectively. The selective antagonist at CGRP1-receptors, CGRP8-37(100 nM), inhibited significantly the incorporation of l-[14C] phenylalanine into cellular protein in response to CGRP and amylin. The selective inhibitor of protein kinase C (PKC), bisindolylmalemide (BIM) (5 microM), reduced significantly the incorporation of l-[14C] phenylalanine into cellular protein in response to phenylephrine (1 microM), employed as a positive control, but did not inhibit the response to insulin (1 unit/ml), employed as a negative control. BIM (5 microM) reduced significantly the responses to FCS (10% v/v), amylin (10 nM) and CGRP (10 pM), but did not inhibit the response to CGRP (100 pM). The activity of protein kinase C in membranes prepared from intact myocytes pre-treated for 10 min with the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (100 nM), employed as a positive control, and CGRP (10 pM) was significantly greater than in membranes prepared from cardiomyocytes not subjected to agonist stimulation. Phenylephrine (1 microM) 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 pM and 100 nM CGRP and amylin, respectively. In conclusion, CGRP and amylin exert hypertrophic effects directly on ventricular cardiomyocytes from the hearts of adult rats in vitro. These effects are: (1) due to de novo protein synthesis since total content of cellular RNA and incorporation of l-[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.