The identification of adenylyl-cyclase modulators as potential receptors for 6-nitrodopamine in human induced pluripotent stem cell (hiPSCs)-derived cardiomyocytes and their relevance in the heart inotropism

Irene Cipollone¹Vittoria Monaco¹José Britto-Júnior^2*Antonio Tiago Lima²EDSON ANTUNES²Andre Sampaio Pupo³Ilaria Iacobucci¹Flora Cozzolino¹MARIA MONTI¹Silvia Parisi⁴Giuseppina Divisato⁴Emanuela Cascone⁴Alfonso De Simone⁵Angela Corvino⁵Ferdinando Fiorino⁵Francesco Frecentese⁵Vincenzo Santagada⁵Beatrice Severino⁵Rosa Sparaco⁵Pierfrancesco Cinque⁵Stefania Vertuccio⁵Giuseppe Caliendo⁵Gilberto De Nucci²

• ¹Department of Chemical Sciences, University of Naples Federico II, Naples, Italy
• ²State University of Campinas, Campinas, Brazil
• ³Department of Biophysics and Pharmacology, Institute of Biosciences of Botucatu, Botucatu, Brazil
• ⁴Department of Molecular Medicine and Medical Biotechnology, School of Medicine and Surgery, University of Naples Federico II, Naples, Campania, Italy
• ⁵Department of Pharmacy, School of Medicine and Surgery, University of Naples Federico II, Naples, Campania, Italy

6-Nitrodopamine (6-ND) has potent positive chronotropic and inotropic effects. At very low dose i.e. 10fM causes potentiation of the positive chronotropic effects induced by the catecholamines in the rat atria, indicating a distinct mechanism of action. Cyclase-associated proteins (CAP-1 and CAP-2) were identified as potential receptors for 6-ND in human cardiomyocytes. Since cAMP plays a fundamental role in the positive inotropic effect of classical catecholamines, it was further investigated whether 6-ND potentiates the positive inotropic effects induced by the classical catecholamines in the rat isolated perfused heart. Human induced pluripotent stem cell (hiPSCs)-derived cardiomyocytes were harvested and lysed, and following appropriate separation procedures, membrane proteins were incubated with the 6-ND derivatized agarose, centrifuged and the proteins retained in the agarose eluted with 6-ND 1mM). The proteins deriving from chemical pull-down were fractionated by SDS-PAGE, the bands were cut and hydrolyzed in situ by trypsin, separated and sequenced. A list of 817 proteins was generated and following screening using UniProt “Retrieve/ID Mapping” function and Gene Ontology cellular component category, 124 proteins were identified as membrane proteins. The experiments above identified a group of 3 proteins that modulate adenylyl-cyclase activity (CAP-1, CAP-2 and STIM1), which are compatible with the pharmacological findings reported for 6-ND in the rat heart. As expected, 6-ND strongly potentiates the inotropic effect induced by noradrenaline in Langendorff’s preparation. In conclusion, 6-ND induced potentiation of the catecholamine-induced chronotropic and inotropic effects is due to modulation of adenylyl cyclase activity probably via direct interactions with CAP-1 and CAP-2.