Most of the above mechanisms are implicated in

Most of the above mechanisms are implicated in attenuation of myocardial I/R injury. However, the role of GalR2 receptor activation in cardioprotection remains unclear because of the lack of receptor subtype selectivity of peptides G2 and G3. It has been shown that Ala5-galanin (2–11) (G4) has a high preference for GalR2 receptor compared to both GalR1 and GalR3 subtypes. This ligand was tested in 125I-galanin competitive binding studies for GalR1-3 receptors and its signaling properties were examined using different cell lines [15]. Although peptide G4 is the shortest GalR2 specific agonist, its use in physiological experiments is difficult due to poor solubility in water. Given the need to test pharmacological agonists on models of myocardial I/R injury, we synthesized a novel ligand of galanin receptors. Its design was based on a galanin fragment (2–15) but with a substitution of two amino acids, His14 and Ala15 to βAla14 and His15 (G5). Such the amino tandospirone sequence provides high solubility in aqueous media and should respond to a greater resistance to degradation by carboxypeptidases. It was found in preliminary studies that peptide G5 is able to improve functional recovery of isolated rat heart during reperfusion after global ischemia.
Materials and methods
Results
Discussion Acute myocardial infarct size is a predictor of long-term disturbances of LV function and adverse clinical outcome in patients. Clinical investigations suggest that there is a direct relationship between mortality and the extent of infarct size, because the myocardium does not have the ability to regenerate [17]. In the present study, we used a rat model of acute myocardial infarction that mimics human ischemic heart disease and serves a tool to elucidate the signaling mechanisms involved in cardiac remodeling and heart failure [18]. The obtained results show for the first time that the full-length galanin G1 and its modified fragments (2-11) G4 and (2-15) G5 have protective effects in myocardial I/R injury in rats in vivo. This finding complements previously obtained data on the ability of natural galanin fragments G2 and G3 to reduce infarct size in a similar model of myocardial damage [5], [6]. Furthermore, the reduction in the infarction area (MI/AAR,%) under the action of the peptides G1-5 was accompanied by a significant decrease in CK-MB and LDH release into the blood during reperfusion. The decrease in the plasma activity of these necrosis markers might be related to maintenance of the membrane integrity of myocardial cells. We found that the overall protective effect of a modified galanin fragment G5 was the highest among the studied peptides. Treatment with G5 limited infarct size and decreased the plasma CK-MB and LDH activity in almost the same extent as the full-length galanin G1 and significantly less influenced SAP and HR. In contrast to G5, the GalR2-specific agonist G4 exhibited the lowest cardioprotective efficacy. This fact implies that triggering GalR2-mediated signaling pathways is insufficient for beneficial outcomes in myocardial I/R injury in vivo and/or agonist G4 has lower binding affinity on plasma membranes of rat myocardial cells. The potency of G5 as the ligand for galanin receptor subtypes GalR1-3 has not been studied yet. It is known that the N-terminal fragments of galanin are of critical importance for its biological activity including cardiovascular regulation, and the first 15 amino acid residues are conserved in most species, while the C-terminal region (residues 17–29) varies among species and lacks receptor affinity [19], [20]. Docking of galanin into the GalR1 receptor revealed that galanin (1–13) interacts with this receptor subtype via binding of Gly1, Trp2, Asn5 and Tyr9 to the receptor [21]. The deletion of the Gly1 residue results in loss of affinity for GalR1 [22]. The Trp2 and Tyr9 residues, together with Asn5, Gly8, Leu10 and Gly12, were identified as the critical pharmacophores for selective galanin (1–16) binding to the GalR2 receptor [23]. Galanin (1–16) displays a low affinity and efficacy for GalR3 receptor [24]. Thus, most likely, the peptide G5synthesized by conjugation of galanin (2–13) with the dipeptide carnosine might act as a chimeric ligand having a preferential binding towards GalR2. To delineate the contribution of galaninergic system in protective effects of peptide G5 is difficult due to: a lack of ligands with receptor subtype specificity [15], [22], the complexity of intracellular signaling cascades [25], switching between different conformational states of G protein-coupled receptors in vivo [21], the existence of Gal1-3 heteroreceptor complexes [15], [21] and antioxidant properties of G5 [5], [6]. Despite this uncertainty, galanin receptor subtypes can be considered as putative drug targets in various cardiac pathology.