title: Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy.
abstract: Phospholamban (PLN) plays a central role in Ca2+homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1.|Ted Rogers Centre for Heart Research, University of Toronto, Toronto, ON, Canada M5G 1M1.
authors.forename: Uros
authors.initials: U
authors.lastname: Kuzmanov
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1.
authors.forename: Hongbo
authors.initials: H
authors.lastname: Guo
authors.affiliation: Department of Physiology, University of Toronto, Toronto, ON, Canada M5S 3E1.
authors.forename: Diana
authors.initials: D
authors.lastname: Buchsbaum
authors.affiliation: Department of Physiology, University of Toronto, Toronto, ON, Canada M5S 3E1.
authors.forename: Jake
authors.initials: J
authors.lastname: Cosme
authors.affiliation: Department of Physiology, University of Toronto, Toronto, ON, Canada M5S 3E1.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Abbasi
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1.
authors.forename: Ruth
authors.initials: R
authors.lastname: Isserlin
authors.affiliation: Department of Physiology, University of Toronto, Toronto, ON, Canada M5S 3E1.
authors.forename: Parveen
authors.initials: P
authors.lastname: Sharma
authors.affiliation: Ted Rogers Centre for Heart Research, University of Toronto, Toronto, ON, Canada M5G 1M1; anthony.gramolini@utoronto.ca andrew.emili@utoronto.ca.|Department of Physiology, University of Toronto, Toronto, ON, Canada M5S 3E1.
authors.forename: Anthony O
authors.initials: AO
authors.lastname: Gramolini
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1; anthony.gramolini@utoronto.ca andrew.emili@utoronto.ca.
authors.forename: Andrew
authors.initials: A
authors.lastname: Emili
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002311:Cardiomyopathy, Dilated / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D006801:Humans; D008822:Mice, Transgenic; D009154:Mutation; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D010750:Phosphoproteins; D020543:Proteome; D000099211:Phospholamban
keywords: bioinformatics; heart disease; phospholamban; proteomic; signaling