Francesca Rusconi1, Paola Ceriotti1, Michele Miragoli, Pierluigi Carullo, Nicolò Salvarani, Marcella Rocchetti, Elisa Di Pasquale, Stefano Rossi, Maddalena Tessari, Silvia Caprari, Magali Cazade, Paolo Kunderfranco, Jean Chemin, Marie-Louise Bang, Fabio Polticelli, Antonio Zaza, Giuseppe Faggian, Gianluigi Condorelli, Daniele Catalucci. 1. From Humanitas Clinical and Research Center, Rozzano, Milan, Italy (F.R., P. Ceriotti, M.M., P. Carullo, N.S., E.D.P., P.K., M.-L.B., G.C., D.C.); Institute of Genetic and Biomedical Research UOS Milan National Research Council, Milan, Italy (F.R., P. Carullo, N.S., E.D.P., M.-L.B., D.C.); Department of Biotechnologies and Biosciences, University of Milan-Bicocca, Milan, Italy (M.R., A.Z.); Departments of Life Sciences (S.R.) and Clinical and Experimental Medicine (M.M.), University of Parma, Parma, Italy; University Hospital of Verona, Division of Cardiac Surgery, Verona, Italy (M.T., G.F.); Department of Sciences, University of Roma Tre, Rome, Italy (S.C., F.P.); University of Montpellier, CNRS UMR 5203, INSERM, Department of Neuroscience, Institute for Functional Genomics, LabEx Ion Channel Science and Therapeutics, Montpellier, France (M.C., J.C.); and National Institute of Nuclear Physics, Rome Tre Section, Rome, Italy (F.P.).
Abstract
BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavβ2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavβ2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavβ2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavβ2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavβ2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavβ2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavβ2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.
BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS:Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavβ2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavβ2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathypatients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavβ2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavβ2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavβ2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavβ2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavβ2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.
Authors: Tania Zaglia; Paola Ceriotti; Antonio Campo; Giulia Borile; Andrea Armani; Pierluigi Carullo; Valentina Prando; Raffaele Coppini; Vladimiro Vida; Tomas O Stølen; Wisløff Ulrik; Elisabetta Cerbai; Giovanni Stellin; Giuseppe Faggian; Diego De Stefani; Marco Sandri; Rosario Rizzuto; Fabio Di Lisa; Tullio Pozzan; Daniele Catalucci; Marco Mongillo Journal: Proc Natl Acad Sci U S A Date: 2017-10-09 Impact factor: 11.205
Authors: Simone Pickel; Yiliam Cruz-Garcia; Sandra Bandleon; Katalin Barkovits; Cornelia Heindl; Katharina Völker; Marco Abeßer; Kathy Pfeiffer; Alice Schaaf; Katrin Marcus; Petra Eder-Negrin; Michaela Kuhn; Erick Miranda-Laferte Journal: Front Cardiovasc Med Date: 2021-07-07
Authors: Iacopo Gesmundo; Michele Miragoli; Pierluigi Carullo; Letizia Trovato; Veronica Larcher; Elisa Di Pasquale; Mara Brancaccio; Marta Mazzola; Tania Villanova; Matteo Sorge; Marina Taliano; Maria Pia Gallo; Giuseppe Alloatti; Claudia Penna; Joshua M Hare; Ezio Ghigo; Andrew V Schally; Gianluigi Condorelli; Riccarda Granata Journal: Proc Natl Acad Sci U S A Date: 2017-10-25 Impact factor: 11.205