An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System

Alamandine is a peptide of the Renin Angiotensin System (RAS), either generated from Angiotensin A via the Angiotensin Converting Enzyme 2 (ACE2), or directly from Ang-(1–7). In mammals, it elicits cardioprotection via Mas-related G-protein-coupled receptor D (MrgD), and the NOS/NO system. In teleost fish, RAS is known to modulate heart performance. However, no information is available on the presence of a cardioactive ACE2/Alamandine axis. To fill this gap, we used the cyprinid teleost Carassius auratus (goldfish) for in silico and in vitro analyses. Via the NCBI Blast P suite we found that in cyprinids ace2 is phylogenetically detectable in a subcluster of proteins including ace2-like isoforms, and is correlated with a hypoxia-dependent pathway. By real-time PCR, Western Blotting, and HPLC, ACE2 and Alamandine were identified in goldfish heart and plasma, respectively. Both increased after chronic exposure to low O2 (2.6 mg O2 L1). By using an ex-vivo working goldfish-heart preparation, we observed that in vitro administration of exogenous Alamandine dose-dependently stimulates myocardial contractility starting from 1011 M. The effect that involved Mas-related receptors and PKA occurred via the NOS/NO system. This was shown by exposing the perfused heart to the NOS inhibitor L-NMMA (105 M) that abolished the cardiac effect of Alamandine and was supported by the increased expression of the phosphorylated NOS enzyme in the extract from goldfish heart exposed to 1010 M Alamandine. Our data are the first to show that an ACE2/Alamandine axis is present in the goldfish C. auratus and, to elicit cardiac modulation, requires the obligatory involvement of the NOS/NO system.