CaMKIId As a Nodal Mediator of Insulin Signaling and Adrenergic Signaling in Cardiomyocytes – a Signaling Network Map Profiling by Quantitative Phospho‐proteomics

Abstract

BackgroundCalcium 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.AimThe 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.MethodsTo 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.ResultsKey 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.ConclusionCaMKIId is a key mediator of contractility, electrophysiology, calcium handling and insulin signaling within cardiomyocytes.Support or Funding InformationNIH grant 1R01HL105242