Metabolic Tracing Using Stable Isotope-Labeled Substrates and Mass Spectrometry in the Perfused Mouse Heart

Methods Enzymol. 2015;561:107-47. doi: 10.1016/bs.mie.2015.06.026. Epub 2015 Aug 17.

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.

Keywords: Anaplerosis; Citric acid cycle; Ex vivo working mouse heart; Glycolysis; Substrate oxidation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Animals
  • Carbon Isotopes
  • Energy Metabolism*
  • Gas Chromatography-Mass Spectrometry / methods*
  • Isolated Heart Preparation*
  • Isotope Labeling / methods*
  • Mice
  • Myocardium / metabolism*
  • Pyruvic Acid

Substances

  • Carbon Isotopes
  • Pyruvic Acid