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 Ca 2+ -ATPase 2A (SERCA2A) Ca 2+ 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.phospholamban | proteomic | bioinformatics | heart disease | signaling C ardiovascular diseases (CVDs) leading to systolic/diastolic heart failure (HF), such as hypertensive/diabetic heart disease, stroke, and vascular atherosclerosis, are leading causes of death in the developed world (1). Many CVDs are associated with genetic predispositions. For example, in humans, the arginine to cysteine (R9C) substitution in phospholamban (PLN) has been shown to result in dilated cardiomyopathy (DCM) presenting in adolescence, leading to rapid deterioration of heart function and premature death (2). However, the etiology and molecular mechanisms of progression of DCM and other CVDs leading to HF are complex and still poorly understood, further complicating clinical assessment and management. From a biological and clinical perspective, the identification and characterization of clinically relevant, potentially druggable, pathways driving the maladaptive response in affected heart tissue are key challenges to improved diagnostic and therapeutic tools for earlier detection and preventative treatment of both inherited and chronic CVDs.Cardiac muscle contraction is controlled by Ca 2+ flux and signaling relays, which are perturbed in HF. Internal stores of Ca 2+ required for the proper functioning of cardiomyocytes (CMs) are normally maintained through the function of the sarco(endo)plasmic reticulum Ca 2+ -ATPase 2 ...

A preliminary study of the effect of a high-salt diet on transcriptome dynamics in rat hypothalamic forebrain and brainstem cardiovascular control centers

“…Hence, the downregulation of this gene in SHRs being fed with HS diet seem to corelate with the development salt-sensitive HPN. Furthermore, this gene was also found in kidney transcriptome as well as haemoglobin where both studies were related to HPN (Marques et al, 2017;Zhang et al, 2014), and it has also been reported that Pi4k2a act via WNT-signaling pathway (Kuzmanov et al, 2016).…”

REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects

“…Two-week post LAD induced MI hearts were induced and isolated as described previously 54 . Finally, 16-week-old PLN R9C mutant mice, a model of human DCM were analyzed as described 55 .…”

Phospho‐Proteomic Analysis of Cardiac Dyssynchrony and Resynchronization Therapy

“…Generally, HF dys is hyper-phosphorylated by Ser/Thr kinases compared to Con and CRT hearts, which agrees with previous studies that suggest broad kinase activation and hyper-phosphorylation occurs during heart failure. [26][27][28][29] Importantly, CRT reduced and thus restored overall phosphorylation. While the clinical efficacy of CRT has been known for some time, it was nonetheless unexpected that it should reverse such a broad fingerprint of heart failure.…”