Multiplex Analysis of Cardiac Hypertrophic Signaling: Reduced in vivo Phosphorylation of Glycogen Synthase Kinase-3β and Proline-Rich Akt Substrate (PRAS40)

Jens      R. Larsen; Morten      Smerup; Rikke      Norregaard; Jane      H. Christensen; Kim      Sivesgaard; Sara      D. Christensen; Karin      Christensen; Erik      Sloth; Peter      Torp; Niels      Gregersen; J.      Michael Hasenkam

Abstract

The serine/threonine kinase Akt (PKB) is activated in response to growth factors, cytokines and other growth promoting stimuli and is involved in the regulation of a number of cellular processes including metabolism, cell growth, proliferation and survival. To study intracellular metabolic effects of left ventricular hypertrophy (LVH) induced by chronic aortic banding in a recently established swine model, protein arrays can be used to analyze changes in cytosolic protein phosphorylation levels such as cell signal transduction and protein kinase pathways. Miniaturized sandwich immunoassays allow the simultaneous analysis of several parameters in a single experiment. Bead-based protein array systems or suspension microarrays are well-established multiplex sandwich immunoassay formats. Cytosolic proteins were extracted from pig hearts. Using the bead-based Luminex system, multiplexed sandwich immunoassays have been developed to analyze the phosphorylated proteins IR[pYpY1162/1163], IGF-1R[pYpY1135 /1136], IRS-1[pS312], Akt[pS473], PRAS40[pT246], p70S6K[pTpS421/424], and GSK-3β[pS9], which were measured simultaneously in extracted myocardial tissue homogenates with the Akt Pathway Phospho 7-Plex Panel using a Luminex 100 IS system. Using this assay, tissue homogenates derived from myocardial tissue samples from 28 individual porcine in vivo model experiments in normal hearts (n=17) and LVH hearts (n=11) were analyzed for phosphorylated protein concentrations. Results obtained allowed grouping of myocardial tissue samples into a LVH and a normal group. Two candidate molecules: PRAS40 (normal vs. LVH two-tailed probability P= 0.0022) and GSK-3β (P=0.003), had lower concentrations in LVH pigs than in normal animals, suggesting loss of GSK-3β inhibitory activity and contributing to speculation of altered insulin sensitivity associated with LVH.

Keywords: Cardiac hypertrophy, akt/protein kinase B, glycogen synthase (GSK)-3β, proline-rich akt substrate (PRAS40), multiplex analysis, animal model, phosphatidyli-nositol 3-kinase (PI3K) pathway, sandwich immunoassay flow cytometry, sevoflurane anesthesia, IR, IGF-1R, IRS-1, Akt, PRAS40, p70S6K, GSK-3, IR[pYpY1162/1163], IGF-1R[pYpY1135/1136], IRS-1[pS312], Akt[pS473], pT246, GSK-3[pS9], Ambulatory transthoracic ultrasound examinations