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CaMKIId As a Nodal Mediator of Insulin Signaling and Adrenergic Signaling in Cardiomyocytes – a Signaling Network Map Profiling by Quantitative Phospho‐proteomics
Author(s) -
Dewan Sukriti,
Campos Alex,
Wu Shuangding,
Stowe Jennifer,
Wolf Alex,
Bers Donald M,
Brill Laurence M,
Brown Joan H,
McCulloch Andrew
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1273.3
Subject(s) - phosphoproteomics , signal transduction , proteomics , gene isoform , microbiology and biotechnology , biology , kegg , proteome , insulin receptor , phosphorylation , chemistry , bioinformatics , endocrinology , insulin , transcriptome , biochemistry , gene expression , protein kinase a , protein phosphorylation , insulin resistance , gene
Background Calcium and calmodulin mediated protein kinase II (CaMKIId) plays a key role in regulating cardiac physiology. Upregulated expression and activation of CaMKIId is reported in HF. While cardiac specific over‐expression of CaMKIIdc isoform leads to progressive HF in mice, cardiac specific knockout (KO) of CaMKIId isoform develop hypertrophy but do not progress into end‐stage HF. To elucidate the exact role of CaMKIId as a cardioprotective target, it is pertinent to investigate the signaling mechanisms altered by CaMKIId targets and study the pleiotropic effects of these interactions. Aim The purpose of this study is to identify novel CaMKIId signaling targets and interactions thereof, using a sensitive, wide‐range and quantitative, mass spectrometry‐based phosphoproteomics approach in acutely stressed isolated cardiomyocytes. Methods To identify putative downstream CaMKIId targets, mice were divided into two groups ‐ CaMKIId KO and control group. Adult cardiomyocytes were freshly isolated by the Langendorff method. A total of 3 biological replicates (pooled from 12 animals) were used per genotype for the present study. Isolated cardiomyocytes were treated with 10uM of isoproterenol for 5 minutes to induce CaMKIId activation via beta‐adrenergic stimulation. Enriched phosphopeptides from CaMKIId KO and control mice samples were differentially quantified high‐resolution liquid chromatography mass spectrometry (LC‐MS/MS). All mass spectra from Phospho‐ and Total samples were analyzed with MaxQuant software. For pathway analysis, proteins with differentially regulated phospho‐sites were keyed in ClueGo (Cytoscape plug‐in) and Ingenuity Pathway Analysis (IPA); and the resulting enriched signaling pathways were functionally grouped by parsing through KEGG, Reactome and Wiki Signaling Pathway databases. These functional signaling groups were then visualized as a comprehensive interconnected network. An interaction map of all enriched signaling pathways with differentially regulated phospho‐proteins was generated. Flourimetric glucose uptake assays and western blots were used to quantify insulin resistance under stress conditions and mechanisms thereof. Results Key network hubs and pathways that were predicted to be involved in CaMKIId signaling are cardiac contraction, cAMP signaling, beta‐adrenergic signaling and insulin signaling. Specifically we were able to identify Insulin signaling pathway as a novel pathway being regulated by CaMKIId inhibition. Conclusion CaMKIId is a key mediator of contractility, electrophysiology, calcium handling and insulin signaling within cardiomyocytes. Support or Funding Information NIH grant 1R01HL105242