The chromogranin A1-373 fragment reveals how a single change in the protein sequence exerts strong cardioregulatory effects by engaging neuropilin-1

Aim: Chromogranin A (CgA), a 439-residue long protein, is an important cardiovascular regulator and a precursor of various bioactive fragments. Under stressful/pathological conditions, CgA cleavage generates the CgA1-373 proangiogenic fragment. The present work investigated the possibility that human CgA1-373 influences the mammalian cardiac performance, evaluating the role of its C-terminal sequence. Methods: Haemodynamic assessment was performed on an ex vivo Langendorff rat heart model, while mechanistic studies were performed using perfused hearts, H9c2 cardiomyocytes and in silico. Results: On the ex vivo heart, CgA1-373 elicited direct dose-dependent negative inotropism and vasodilation, while CgA1-372, a fragment lacking the C-terminal R373 residue, was ineffective. Antibodies against the PGPQLR373 C-terminal sequence abrogated the CgA1-373-dependent cardiac and coronary modulation. Ex vivo studies showed that CgA1-373-dependent effects were mediated by endothelium, neuropilin-1 (NRP1) receptor, Akt/NO/Erk1,2 pathways, nitric oxide (NO) production and S-nitrosylation. In vitro experiments on H9c2 cardiomyocytes indicated that CgA1-373 also induced eNOS activation directly on the cardiomyocyte component by NRP1 targeting and NO involvement and provided beneficial action against isoproterenol-induced hypertrophy, by reducing the increase in cell surface area and brain natriuretic peptide (BNP) release. Molecular docking and all-atom molecular dynamics simulations strongly supported the hypothesis that the C-terminal R373 residue of CgA1-373 directly interacts with NRP1. Conclusion: These results suggest that CgA1-373 is a new cardioregulatory hormone and that the removal of R373 represents a critical switch for turning “off” its cardioregulatory activity.