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
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Rattus Norvegicus (rat)
TISSUE(S): Heart
SUBMITTER: 
Antigoni Manousopoulou
LAB HEAD: Spiro Garbis
PROVIDER: PXD004593 | Pride | 2018-08-13
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 12AUG14_InsRapa_1.mzid | Mzid | |||
| 12AUG14_InsRapa_2.mzid | Mzid | |||
| 12AUG14_InsRapa_2.raw | Raw | |||
| 12AUG14_InsRapa_3.mzid | Mzid | |||
| 12AUG14_InsRapa_3.raw | Raw |
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Project description: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.
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Project description:The aim of this project was to quantify the effects of different hypertrophic stimuli on synthesis of specific proteins in adult rat ventricular cardiomyocytes
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2017-05-18 | E-GEOD-69047 | biostudies-arrayexpress
Project description:Cardiac hypertrophy is an independent risk for heart failure (HF) and sudden death. Deciphering signalling pathways dependent on extracellular calcium (Ca(2+)) influx that control normal and pathological cardiac growth may enable identification of novel therapeutic targets. The objective of the present study is to determine the role of the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1 and stromal interaction molecule 1 (Stim1) in postnatal cardiomyocyte store operated Ca(2+) entry (SOCE) and impact on normal and hypertrophic postnatal cardiomyocyte growth. Employing a combination of siRNA-mediated gene silencing, cultured neonatal rat ventricular cardiomyocytes together with indirect immunofluorescence, epifluorescent Ca(2+) imaging and site-specific protein phosphorylation and real-time mRNA expression analysis, we show for the first time that both Orai1 and Stim1 are present in cardiomyocytes and required for SOCE due to intracellular Ca(2+) store depletion by thapsigargin. Stim1-KD but not Orai1-KD significantly decreased diastolic Ca(2+) levels and caffeine-releasable Ca(2+) from the sarcoplasmic reticulum (SR). Conversely, Orai1-KD but not Stim1-KD significantly diminished basal NRCM cell size, anp and bnp mRNA levels and activity of the calcineurin (CnA) signalling pathway although diminishing both Orai1 and Stim1 proteins similarly attenuated calmodulin kinase II (CamKII) and ERK1/2 activity under basal conditions. Both Orai1- and Stim1-KD completely abrogated phenylephrine (PE) mediated hypertrophic NRCM growth and enhanced natriuretic factor expression by inhibiting G(q)-protein conveyed activation of the CamKII and ERK1/2 signalling pathway. Interestingly, only Orai1-KD but not Stim1-KD prevented Gq-mediated CaN-dependent prohypertrophic signalling. This study shows for the first time that both Orai1 and Stim1 have a key role in cardiomyocyte SOCE regulating both normal and hypertrophic postnatal cardiac growth in vitro.
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Project description:Recent work has provided compelling evidence that increased levels of acetylcholine (ACh) can be protective in heart failure, whereas reduced levels of ACh secretion can cause heart malfunction. Previous data show that cardiomyocytes themselves can actively secrete ACh, raising the question of whether this cardiomyocyte derived ACh may contribute to the protective effects of ACh in the heart. To address the functionality of this non-neuronal ACh machinery, we used cholinesterase inhibitors and a siRNA targeted to AChE (acetylcholinesterase) as a way to increase the availability of ACh secreted by cardiac cells. By using nitric oxide (NO) formation as a biological sensor for released ACh, we showed that cholinesterase inhibition increased NO levels in freshly isolated ventricular myocytes and that this effect was prevented by atropine, a muscarinic receptor antagonist, and by inhibition of ACh synthesis or vesicular storage. Functionally, cholinesterase inhibition prevented the hypertrophic effect as well as molecular changes and calcium transient alterations induced by adrenergic overstimulation in cardiomyocytes. Moreover, inhibition of ACh storage or atropine blunted the anti-hypertrophic action of cholinesterase inhibition. Altogether, our results show that cardiomyocytes possess functional cholinergic machinery that offsets deleterious effects of hyperadrenergic stimulation. In addition, we show that adrenergic stimulation upregulates expression levels of cholinergic components. We propose that this cardiomyocyte cholinergic signaling could amplify the protective effects of the parasympathetic nervous system in the heart and may counteract or partially neutralize hypertrophic adrenergic effects.
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Project description:AimsHypertrophic cardiomyopathy (HCM) is characterized by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM.Methods and resultsHuman cardiomyocytes derived from control-induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy.ConclusionThis study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing.
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Project description:Backgroundα-1-syntrophin (SNTA1), a protein encoded by SNTA1, is highly expressed in human cardiomyocytes. Mutations in SNTA1 are associated with arrhythmia and cardiomyopathy. Previous research on SNTA1 has been based on non-human cardiomyocytes. This study was designed to identify the phenotype of SNTA1-deficiency using human cardiomyocytes.MethodsSNTA1 was knocked out in the H9 embryonic stem cell line using the CRISPR-Cas9 system. H9SNTA1KO cells were then induced to differentiate into cardiomyocytes using small molecule inhibitors. The phenotypic discrepancies associated with SNTA1-deficient cardiomyocytes were investigated.ResultsSNTA1 was truncated at the 149th amino acid position of PH1 domain by a stop codon (TGA) using the CRISPR-Cas9 system. SNTA1-deficiency did not affect the pluripotency of H9SNTA1KO, and they retain their in vitro ability to differentiate into cardiomyocytes. However, H9SNTA1KO derived cardiomyocytes exhibited hypertrophic phenotype, lower cardiac contractility, weak calcium transient intensity, and lower level of calcium in the sarcoplasmic reticulum. Early treatment of SNTA1-deficient cardiomyocytes with ranolazine improved the calcium transient intensity and cardiac contractility.ConclusionSNTA1-deficient cardiomyocytes can be used to research the etiology, pathogenesis, and potential therapies for myocardial diseases. The SNTA1-deficient cardiomyocyte model suggests that the maintenance of cardiac calcium homeostasis is a key target in the treatment of myocardial-related diseases.
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