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.

HIF1 mediates a switch in pyruvate kinase isoforms after myocardial infarction

“…Pkm2 expression has also been observed in other models of heart disease. In a transverse aortic constriction model, Pkm2 was found to be upregulated in rat hearts (25). Long-term administration of sunitinib, a tyrosine kinase inhibitor used in cancer treatment, also increased expression of Pkm2 and other glycolytic genes, which may indicate a shift toward fetal-like cardiac metabolism (35).…”

“…These changes corresponded with increased HIF1 expression, which bound to a hypoxia response element in the promoter of PKM, indicating a direct link between HIF1 and Pkm2 expression. Increased Pkm2 expression has been noted in other forms of chronic heart disease and has also been associated with heart failure (25,35). It appears that enhanced Pkm2 expression during acute MI is an initial response to injury that is sustained for weeks after injury.…”

Cardiomyocyte Proteome Remodeling due to Isoproterenol‐Induced Cardiac Hypertrophy during the Compensated Phase

“…Regulation of their expression is associated with altered blood pressure and heart failure. Other proteomic study reported the regulation of HNRNPA1, HNRNPA2 and ALDOA as a consequence of metabolic remodeling due to cardiac hypertrophic agents [31].…”

Maf1 ameliorates cardiac hypertrophy by inhibiting RNA polymerase III through ERK1/2

“…At the molecular level, cardiac hypertrophy is characterized by the re-expression of the fetal gene program and an imbalance between protein synthesis and degradation 1-3. As previously shown, prolonged external hypertrophic stimuli markedly activate the process of gene transcription, resulting in excessive protein accumulation in cardiomyocytes and leading to the development of cardiac hypertrophy 4. Pathological hypertrophy has been associated with abnormalities leading to the enhancement of the IGF1-phosphoinositide 3-kinase (PI3K)-Akt pathway, mitogen activated protein kinases (MAPKs), and Gαq signaling.…”