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 remodelling
A paper by Dr Spiros Garbis, Dr Christopher Woelk, Professor Chris Proud et al. that has been accepted for publication in the journal Molecular and Cellular Proteomics.
Mol Cell Proteomics. 2016 Aug 9. pii: mcp.M115.054312. [Epub ahead of print]
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
Liu R1, Kenney JW2, Manousopoulou A3, Johnston HE3, Kamei M4, Woelk CH3, Xie J4, Schwarzer M5, Garbis SD3, Proud CG6.
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 remodelling 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.
KEYWORDS: Cardiac hypertrophy; Cardiovascular disease; Energy metabolism; PKM2; Protein Synthesis*; QuaNCAT; Ribosomes*; Signal Transduction*; mTORC1; pSILAC
PMID: 27512079
This inclusion in Molecular and Cellular Proteomics demonstrates how the IFLS continues to collaborate with world class institutions in Australia, Canada and Germany.