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 pancreatic β-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 p mand 10 n mfor CGRP and amylin, respectively. The selective antagonist at CGRP 1-receptors, CGRP 8–37(100 n m), 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 μ m), reduced significantly the incorporation of l-[ 14C] phenylalanine into cellular protein in response to phenylephrine (1 μ m), employed as a positive control, but did not inhibit the response to insulin (1 unit/ml), employed as a negative control. BIM (5 μ m) reduced significantly the responses to FCS (10% v/v), amylin (10 n m) and CGRP (10 p m), but did not inhibit the response to CGRP (100 p m). 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 m), employed as a positive control, and CGRP (10 p m) was significantly greater than in membranes prepared from cardiomyocytes not subjected to agonist stimulation. Phenylephrine (1 μ m) 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 mand 100 n mCGRP 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 novoprotein synthesis since total content of cellular RNA and incorporation of l-[ 14C] phenylalanine into cellula