Quantitative Analysis of the Cardiac Phosphoproteome in Response to Acute β-Adrenergic Receptor Stimulation In Vivo.

Alican Güran,Kuan-Ting Pan,Christof Lenz,Pan Fang,Metin Avkiran,Henning Urlaub,Yanlong Ji

doi:10.3390/ijms222212584

Abstract

β-adrenergic receptor (β-AR) stimulation represents a major mechanism of modulating cardiac output. In spite of its fundamental importance, its molecular basis on the level of cell signalling has not been characterised in detail yet. We employed mass spectrometry-based proteome and phosphoproteome analysis using SuperSILAC (spike-in stable isotope labelling by amino acids in cell culture) standardization to generate a comprehensive map of acute phosphoproteome changes in mice upon administration of isoprenaline (ISO), a synthetic β-AR agonist that targets both β1-AR and β2-AR subtypes. Our data describe 8597 quantitated phosphopeptides corresponding to 10,164 known and novel phospho-events from 2975 proteins. In total, 197 of these phospho-events showed significantly altered phosphorylation, indicating an intricate signalling network activated in response to β-AR stimulation. In addition, we unexpectedly detected significant cardiac expression and ISO-induced fragmentation of junctophilin-1, a junctophilin isoform hitherto only thought to be expressed in skeletal muscle. Data are available via ProteomeXchange with identifier PXD025569.

Highlights

Using a customized mass spectrometry-based workflow (Figure 1), our study identified hundreds of phosphosites that are significantly regulated by β-adrenergic receptor (β-AR) stimulation, including both novel phosphoproteins and phosphosites that have not been previously identified as downstream effectors of β-AR stimulation and established downstream components of cyclic adenosine monophosphate (cAMP)/protein kinase (PKA) and CaMKII
The effectiveness of the acute β-AR stimulation protocol was tested by monitoring the ECG before and after injection, which revealed a significant increase in heart rate after ISO but not saline injection (Figure 2A)
To investigate the diversity and potential signature of cardiac protein phosphorylation events resulting from acute β-AR stimulation, we analysed the amino acid sequences flanking phosphorylation sites that displayed significantly increased phosphorylation in response to ISO

Summary

Introduction

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Β-adrenergic receptor (β-AR) stimulation represents a powerful mechanism for acutely enhancing cardiac output, which is a central component of the fight-or-flight response [1]. Activation of stimulatory G (Gs) protein-coupled β-ARs leads to increased activity of adenylate cyclase and subsequent production of cyclic adenosine monophosphate (cAMP). Increased cAMP concentration in the pertinent subcellular compartment(s) initiates downstream signalling pathways, primarily through activation of cAMP-dependent protein kinase (PKA), a serine/threonine kinase, and via non-kinase effectors such as Epac, a guanine nucleotide exchange protein directly activated by cAMP [2]. Activation of the cAMP/PKA pathway in cardiomyocytes leads to an increase in intracellular Ca2+ , which in turn activates another serine/threonine kinase, Ca2+ /calmodulin-dependent protein kinase

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