title: Metabolic Tracing Using Stable Isotope-Labeled Substrates and Mass Spectrometry in the Perfused Mouse Heart.
abstract: There has been a resurgence of interest for the field of cardiac metabolism catalyzed by evidence demonstrating a role of metabolic dysregulation in the pathogenesis of heart disease as well as the increased need for new therapeutic targets for patients with these diseases. In this regard, measuring substrate fluxes is critical in providing insight into the dynamics of cellular metabolism and in delineating the regulation of metabolite production and utilization. This chapter provides a comprehensive description of concepts, guidelines, and tips to assess metabolic fluxes relevant to energy substrate metabolism using (13)C-labeled substrates and (13)C-isotopomer analysis by gas chromatography-mass spectrometry (GC-MS), and the ex vivo working heart as study model. The focus will be on the mouse and on flux parameters, which are commonly assessed in the field, namely, those relevant to substrate selection for energy metabolism, specifically the relative contribution of carbohydrate (glucose, lactate, and pyruvate) and fatty acid oxidation to acetyl-CoA formation for citrate synthesis, glycolysis, as well as anaplerosis. We provide detailed procedures for the heart isolation and perfusion in the working mode as well as for sample processing for metabolite extraction and analysis by GC-MS and subsequent data processing for calculation of metabolic flux parameters. Finally, we address practical considerations and discuss additional applications and future challenges.
authors.affiliation: Department of Nutrition, Université de Montréal, Montreal, Quebec, Canada; Montreal Heart Institute, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Ruiz
authors.affiliation: Montreal Heart Institute, Université de Montréal, Montreal, Quebec, Canada; Department of Medicine, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Roselle
authors.initials: R
authors.lastname: Gélinas
authors.affiliation: IHU Institut de Rythmologie et Modélisation Cardiaque, Fondation Bordeaux, Université de Bordeaux, Bordeaux, France; Inserm U1045 Centre de Recherche Cardio-Thoracique de Bordeaux, Université de Bordeaux, Bordeaux, France.
authors.forename: Fanny
authors.initials: F
authors.lastname: Vaillant
authors.affiliation: Institut du thorax, Université de Nantes, Nantes, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lauzier
authors.affiliation: Department of Nutrition, Université de Montréal, Montreal, Quebec, Canada; Montreal Heart Institute, Université de Montréal, Montreal, Quebec, Canada; Department of Medicine, Université de Montréal, Montreal, Quebec, Canada. Electronic address: christine.des.rosiers@umontreal.ca.
authors.forename: Christine
authors.initials: C
authors.lastname: Des Rosiers
mesh_terms: D000818:Animals; D002247:Carbon Isotopes; D004734:Energy Metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D000067268:Isolated Heart Preparation; D007553:Isotope Labeling / Q000379:methods*; D051379:Mice; D009206:Myocardium / Q000378:metabolism*; D019289:Pyruvic Acid
chemical_list: D002247:Carbon Isotopes; D019289:Pyruvic Acid
keywords: Anaplerosis; Citric acid cycle; Ex vivo working mouse heart; Glycolysis; Substrate oxidation