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Proc Natl Acad Sci U S A. 2016 Nov 1;113(44):12592-12597. Epub 2016 Oct 14.

Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy.

Author information

1
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1.
2
Ted Rogers Centre for Heart Research, University of Toronto, Toronto, ON, Canada M5G 1M1.
3
Department of Physiology, University of Toronto, Toronto, ON, Canada M5S 3E1.
4
Ted Rogers Centre for Heart Research, University of Toronto, Toronto, ON, Canada M5G 1M1; anthony.gramolini@utoronto.ca andrew.emili@utoronto.ca.
5
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada M5S 3E1; anthony.gramolini@utoronto.ca andrew.emili@utoronto.ca.

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.

KEYWORDS:

bioinformatics; heart disease; phospholamban; proteomic; signaling

PMID:
27742792
PMCID:
PMC5098613
DOI:
10.1073/pnas.1606444113
[Indexed for MEDLINE]
Free PMC Article

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