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Quantitative non-canonical amino acid tagging based proteomics identifies distinct patterns of protein synthesis rapidly induced by hypertrophic agents in cardiomyocytes, revealing new aspects of metabolic remodeling

Quantitative non-canonical amino acid tagging based proteomics identifies distinct patterns of protein synthesis rapidly induced by hypertrophic agents in cardiomyocytes, revealing new aspects of metabolic remodeling
Quantitative non-canonical amino acid tagging based proteomics identifies distinct patterns of protein synthesis rapidly induced by hypertrophic agents in cardiomyocytes, revealing new aspects of metabolic remodeling
Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. Pathological myocardial growth is a risk factor for cardiac failure to which faster protein synthesis is a major driving element. We aimed 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 due to 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 non-dividing primary cells. To address this, an optimized QuaNCAT LC/MS method was used to selectively quantify newly synthesized proteins in such cells. The study showed both classical (phenylephrine; PE) and more recent (insulin) pathological cardiac hypertrophic agents increased the synthesis of proteins involved in glycolysis, the Krebs cycle / 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 upregulated by signaling through mTORC1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was upregulated in rat hearts following TAC. This isoform possesses specific regulatory properties that may be involved in metabolic remodeling and as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly, PKM2 and eEF1 were not up-regulated in pregnancy or exercise induced CH, suggesting them as pathological CH specific markers. The study methods may be of utility to the examination of protein synthesis in primary cells.
1535-9476
3170-3189
Liu, Rui
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Kenney, Justin W.
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Manousopoulou, Antigoni
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Johnston, Harvey E.
dbd189e4-c79b-4a6e-8447-925d5c409eab
Kamei, Makoto
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Woelk, Christopher H.
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Xie, Jianling
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Schwarzer, Michael
55bcfb34-cfc6-4d79-a865-739716b775f5
Garbis, Spiros D.
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Proud, Christopher G.
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Liu, Rui
30b6a59b-759f-4b14-ad92-fb2f02d6491a
Kenney, Justin W.
a498bbd6-750d-4778-bd72-6ea336c883e8
Manousopoulou, Antigoni
9a5e4e75-cea9-4d0b-91c8-0fa2af02632f
Johnston, Harvey E.
dbd189e4-c79b-4a6e-8447-925d5c409eab
Kamei, Makoto
01a72f61-a26d-40fb-8617-6ac52f2094ed
Woelk, Christopher H.
4d3af0fd-658f-4626-b3b5-49a6192bcf7d
Xie, Jianling
5cf770b2-e2c2-40cc-b460-969b4f09fb4e
Schwarzer, Michael
55bcfb34-cfc6-4d79-a865-739716b775f5
Garbis, Spiros D.
7067fd19-50c9-4d42-9611-f370289470bd
Proud, Christopher G.
5832db56-9069-4617-a80d-08b069093dba

Liu, Rui, Kenney, Justin W., Manousopoulou, Antigoni, Johnston, Harvey E., Kamei, Makoto, Woelk, Christopher H., Xie, Jianling, Schwarzer, Michael, Garbis, Spiros D. and Proud, Christopher G. (2016) Quantitative non-canonical amino acid tagging based proteomics identifies distinct patterns of protein synthesis rapidly induced by hypertrophic agents in cardiomyocytes, revealing new aspects of metabolic remodeling. Molecular & Cellular Proteomics, 15 (10), 3170-3189. (doi:10.1074/mcp.M115.054312). (PMID:27512079)

Record type: Article

Abstract

Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. Pathological myocardial growth is a risk factor for cardiac failure to which faster protein synthesis is a major driving element. We aimed 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 due to 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 non-dividing primary cells. To address this, an optimized QuaNCAT LC/MS method was used to selectively quantify newly synthesized proteins in such cells. The study showed both classical (phenylephrine; PE) and more recent (insulin) pathological cardiac hypertrophic agents increased the synthesis of proteins involved in glycolysis, the Krebs cycle / 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 upregulated by signaling through mTORC1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was upregulated in rat hearts following TAC. This isoform possesses specific regulatory properties that may be involved in metabolic remodeling and as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly, PKM2 and eEF1 were not up-regulated in pregnancy or exercise induced CH, suggesting them as pathological CH specific markers. The study methods may be of utility to the examination of protein synthesis in primary cells.

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More information

Accepted/In Press date: 9 August 2016
e-pub ahead of print date: 9 August 2016
Published date: 1 October 2016
Organisations: Faculty of Medicine

Identifiers

Local EPrints ID: 399402
URI: http://eprints.soton.ac.uk/id/eprint/399402
ISSN: 1535-9476
PURE UUID: f01beae0-6ad2-45ce-ab70-3464b3c9f812
ORCID for Spiros D. Garbis: ORCID iD orcid.org/0000-0002-1050-0805

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Date deposited: 15 Aug 2016 14:21
Last modified: 07 Oct 2020 01:58

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