title: Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling.
abstract: Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory properties, so this finding indicates it may be involved in metabolic remodeling and also serve as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly those two candidates were not up-regulated in pregnancy or exercise induced CH, indicating PKM2 and eEF1 were pathological CH specific markers. We anticipate that the methodologies described here will be valuable for other researchers studying protein synthesis in primary cells.
authors: School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, United Kingdom; §South Australian Health & Medical Research Institute, North Terrace, Adelaide, SA 5000, Australia.
authors: Rui
authors: R
authors: Liu
authors: School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, United Kingdom.
authors: Justin W
authors: JW
authors: Kenney
authors: From the ‡Center for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, United Kingdom; ¶Clinical and Experimental Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK.
authors: Antigoni
authors: A
authors: Manousopoulou
authors: From the ‡Center for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, United Kingdom; ‖Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK.
authors: Harvey E
authors: HE
authors: Johnston
authors: §South Australian Health & Medical Research Institute, North Terrace, Adelaide, SA 5000, Australia.
authors: Makoto
authors: M
authors: Kamei
authors: ¶Clinical and Experimental Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK.
authors: Christopher H
authors: CH
authors: Woelk
authors: §South Australian Health & Medical Research Institute, North Terrace, Adelaide, SA 5000, Australia.
authors: Jianling
authors: J
authors: Xie
authors: **Department of Cardiovascular Surgery, Jena University Hospital-Friedrich Schiller University of Jena, Erlanger Allee 101, 07747 Jena, Germany.
authors: Michael
authors: M
authors: Schwarzer
authors: From the ‡Center for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, United Kingdom; ¶Clinical and Experimental Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK; ‖Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK; christopher.proud@sahmri.com S.D.Garbis@soton.ac.uk.
authors: Spiros D
authors: 0000-0002-1050-0805
authors: SD
authors: Garbis
authors: From the ‡Center for Proteomic Research, Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, United Kingdom; §South Australian Health & Medical Research Institute, North Terrace, Adelaide, SA 5000, Australia; School of Biological Sciences, University of Adelaide, Adelaide, SA5005, Australia christopher.proud@sahmri.com S.D.Garbis@soton.ac.uk.
authors: Christopher G
authors: CG
authors: Proud
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D013058:Mass Spectrometry; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology*; D020543:Proteome / Q000187:drug effects* / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D007328:Insulin; D020543:Proteome; D010656:Phenylephrine