An improved capillary electrophoresis method for measuring tissue metabolites associated with cellular energy state

Casey, Tammy M.; Dufall, Kitiya; Arthur, Peter G.

doi:10.1046/j.1432-1327.1999.00324.x

Cited by 32 publications

(20 citation statements)

References 15 publications

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“…Isolation and Culture of Neonatal Cardiomyocytes-Cardiomyocytes from neonatal (1-day-old) Sprague-Dawley rats were isolated as described previously (20). Cells were resuspended in Dulbecco's modified Eagle's medium:M199 (4:1) containing 10% (v/v) horse serum, 5% (v/v) heat-inactivated fetal bovine serum, 10 M cytosine ␤-D-arabinofuranoside, 5 mM creatine, and 80 units/ml penicillin/ streptomycin and preplated for 45 min to remove non-cardiomyocyte cells.…”

Section: Methodsmentioning

confidence: 99%

Proteomic Analysis Reveals Different Protein Changes during Endothelin-1- or Leukemic Inhibitory Factor-induced Hypertrophy of Cardiomyocytes in Vitro

Casey

Arthur

Bogoyevitch

2005

Molecular & Cellular Proteomics

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Proteomic analyses are being increasingly used to identify protein changes accompanying changes in cellular function. An advantage of this approach is that it is largely unbiased by prior assumptions on the importance of each protein in the process under investigation. Here we have evaluated the protein changes that accompany the enlargement, or hypertrophy, of cardiomyocytes in culture. We have taken the additional step of comparing the changes that accompany a concentric hypertrophic phenotype stimulated by endothelin-1 exposure and an eccentric hypertrophic phenotype stimulated by leukemic inhibitory factor exposure. Following separation of the protein extracts by two-dimensional gel electrophoresis and staining with colloidal Coomassie Brilliant Blue, we identified 15 protein spots representing 12 proteins that changed in response to endothelin-1. In comparison, 17 protein spots representing 17 proteins changed in response to leukemic inhibitory factor, and 35 protein spots representing 28 proteins did not change under these conditions. Importantly the well established marker of cardiac pathology, atrial natriuretic factor, was identified as a protein up-regulated by both endothelin-1 and leukemic inhibitory factor (2.4 ؎ 0.8-and 2.2 ؎ 0.3-fold, respectively). However, nine of the observed protein changes occurred for only endothelin-1, whereas 11 of the changes occurred only with leukemic inhibitory factor exposure. These two different stimuli are therefore able to elicit unique changes in the protein expression profile of cardiac myocytes. This is consistent with the differences in morphologies noted as well as the different signaling pathways utilized by these different stimuli. Cardiac hypertrophy is a compensatory response of the heart to increased hemodynamic load. This enables the normal maintenance of cardiac output through an increase in the size of the individual cardiomyocytes and an overall increase in cardiac mass (1). Two morphologically different forms of hypertrophy are produced in response to increased hemodynamic load. Pressure overload, as observed during hypertension and aortic stenosis, produces a "concentric" form of hypertrophy that is characterized by the parallel addition of new sarcomeres. This results in increased cardiomyocyte length, increased cardiomyocyte width, and increased ventricular wall thickness (2). In contrast, volume overload as induced by chronic aortic regurgitation and mitral regurgitation produces an "eccentric" form of hypertrophy. Volume overload-induced eccentric hypertrophy is characterized by the addition of new sarcomeres in series, resulting in an increase in individual cardiomyocyte length and ventricular dilation (3).Changes in cell morphology resembling those observed during increased pressure and volume load can be induced in vitro by stimulating cultured neonatal cardiomyocytes with various growth factors and cytokines (4). These cultures therefore provide a robust system in which the effects of various hypertrophic agents can be studied. G-protein-cou...

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Section: Methodsmentioning

confidence: 99%

Proteomic Analysis Reveals Different Protein Changes during Endothelin-1- or Leukemic Inhibitory Factor-induced Hypertrophy of Cardiomyocytes in Vitro

Casey

Arthur

Bogoyevitch

2005

Molecular & Cellular Proteomics

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“…Adenylates were measured on a Beckman PACE/MDQ capillary electropheris system following the adapted methods of Casey et al (1999). The capillary was uncoated fused silica, 50µm ID, 50 cm long.…”

Section: Biochemical Analysismentioning

confidence: 99%

Geographical variation in thermal tolerance within Southern Ocean marine ectotherms

Morley

Hirse

Pörtner

et al. 2009

Comparative Biochemistry and Physiology Part A: Molecular &

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“…9 Estimation of Oxygen Consumption by Protein Synthesis, RNA Synthesis, and Na ؉ ,K ؉ -ATPase Activity…”

Section: Isolation Of Cardiomyocytesmentioning

confidence: 99%

Hypoxia Causes Downregulation of Protein and RNA Synthesis in Noncontracting Mammalian Cardiomyocytes

Casey

Pakay

Guppy

et al. 2002

Circulation Research

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Abstract-The aim was to identify energy-consuming processes, other than contraction, downregulated during moderate hypoxia (Ϸ5 mol/L, 0.5% O 2 ) and severe hypoxia (Ͻ0.5 mol/L, Ͻ0.05% O 2 ) in isolated neonatal cardiomyocytes. The metabolic response of cardiomyocytes to moderate and severe hypoxia was assessed by measuring rates of energy consumption and energetic status of cells maintained under these conditions. We found that the rates of energy production were decreased during both forms of hypoxia. Decreased rates of energy production under moderate hypoxia were associated with reduced energy wastage through a downregulation of proton leak in the mitochondria. Cellular protein synthesis and RNA synthesis, major energy-consuming pathways, were downregulated only during severe hypoxia, when oxygen concentrations were low enough to induce energetic stress (quantitatively defined as being any situation in which phosphocreatine concentrations had fallen by Ն40% 3 has also been demonstrated experimentally in a variety of animal models. 4,5 These studies collectively suggest that hibernating cardiomyocytes maintain metabolic stability by reducing their rates of energy utilization to levels that match rates of energy production. It is not clear whether contraction is the only process downregulated during myocardial hibernation or whether any of the other energy-using processes are also shut down.The response of isolated contracting cardiomyocytes 6,7 to hypoxia (Ϸ8 to Ϸ29 mol/L, 0.8% to 3% O 2 ) is similar to the phenomenon observed in hibernating regions of myocardium, in that contractile activity is reduced and recovers with reoxygenation. We have previously shown that isolated noncontracting cardiomyocytes are also capable of reversibly downregulating metabolic activity during hypoxia (Ͻ5 mol/L, Ͻ0.5% O 2 ). 8 In the present study, we have assessed the effect of hypoxia on cellular energy consumption not associated with contraction by measuring the rates of protein synthesis, RNA synthesis, Na ϩ ,K ϩ -ATPase activity, and proton leak. Whereas hibernation was previously thought to be due only to reduced rates of contractile activity, 4 -7 in the present study, we show that rates of protein synthesis, RNA synthesis, and proton leak are also downregulated by noncontracting cardiomyocytes exposed to reduced oxygen availability.

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An improved capillary electrophoresis method for measuring tissue metabolites associated with cellular energy state

Cited by 32 publications

References 15 publications

Proteomic Analysis Reveals Different Protein Changes during Endothelin-1- or Leukemic Inhibitory Factor-induced Hypertrophy of Cardiomyocytes in Vitro

Proteomic Analysis Reveals Different Protein Changes during Endothelin-1- or Leukemic Inhibitory Factor-induced Hypertrophy of Cardiomyocytes in Vitro

Geographical variation in thermal tolerance within Southern Ocean marine ectotherms

Hypoxia Causes Downregulation of Protein and RNA Synthesis in Noncontracting Mammalian Cardiomyocytes

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