Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein–coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal–regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt.These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. In here we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.

Our present data demonstrate that in dilated cardiomyopathy patients with severe left ventricular dysfunction basal IGF-I levels are reduced whereas the IGF-I response to low rhGH doses is preserved. These findings suggest a normal peripheral GH sensitivity in dilated cardiomyopathy. On the other hand, though nocturnal mean GH concentration in dilated cardiomyopathy patients is similar to that in normal subjects, the somatotroph responsiveness to GHRH, but not that to hexarelin, is reduced. Thus, subtle alterations in the activity of GH/IGF-I axis are present in dilated cardiomyopathy.

Growth hormone secretagogues (GHSs) are synthetic peptidyl and nonpeptidyl molecules that possess strong growth hormone-releasing activity acting on specific pituitary and hypothalamic receptor subtypes. Differently from nonpeptidyl GHSs, peptidyl molecules such as hexarelin, a hexapeptide, possess specific high-affinity binding sites in animal and human heart and, after prolonged treatment, protect rats in vivo from ischemia-induced myocardial damage. To verify the hypothesis that peptidyl GHSs protect heart cells from cell death, we have investigated the cellular effects of hexarelin on H9c2 cardiomyocytes, a fetal cardiomyocyte-derived cell line, and on Hend, an endothelial cell line derived from transformed murine heart endothelium. We show that (i)H9c2 cardiomyocytes show specific binding for 125I-Tyr-Ala-hexarelin, which is inhibited by peptidyl GHSs such as Tyr-Ala-hexarelin and hexarelin but not by the nonpeptidyl GHS MK-0677, (ii) hexarelin promotes survival of H9c2 cardiomyocytes induced to die by doxorubicin, and (iii) that hexarelin inhibits apoptosis, as measured by DNA fragmentation, induced in both H9c2 myocytes and endothelial cells. In conclusion, our findings show that peptidyl GHSs such as hexarelin act as survival factors for cardiomyocytes and endothelium-derived cells in culture. These findings suggest that the inhibitory activity of hexarelin on cardiomyocytes and endothelial cell death could explain, at least partially, its cardioprotective effect against ischemia recorded in rats in vivo.

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