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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

Published: September 28th, 2016

DOI:

10.3791/54497

1Department of Physiology and Biophysics, Mississippi Center for Obesity Research, Cardiovascular-Renal Research Center, University of Mississippi Medical Center

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

Clinical Relevance

In the mammalian heart, there is a strong positive relationship between the flux of substrates through oxidative metabolic pathways, ATP generation and cardiac work1. Over the past two decades, the investigation of the intricate link between cardiac metabolism and function has led to recognize that alterations in cardiac metabolism are a cause for contractile dysfunction and possibly pathological structural remodeling in the setting of different types of heart disease2-4.Therefore, it is expected that our understanding of the mechanisms governing metabolic remodeling of the stressed heart will lead t....

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NOTE: All animal procedures were performed according to the NIH Public Health Service Policy on the Human Care and Use of Animals and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. All procedures involving the use of radioisotopes were approved and performed according to the guidelines set by the radiation safety office of the University of Mississippi Medical Center.

1. Preparation of Stock Buffer Solutions and Reagents

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Two representative experiments are described in the figures below. In both cases, the heart of a 16 week old male Sprague Dawley rat was isolated and perfused in the working mode with KH buffer prepared according to the preceding protocol. In each experiment, the heart was subjected to a stress condition to affect cardiac work. Cardiac contractile function was assessed by continuous recording of pulse pressure through insertion of a pressure transducer in the aortic line and by determinat.......

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The preceding protocol details the methods to simultaneously quantify the flux of substrate through glucose oxidation and fatty acid oxidation in the isolated working rat heart. The measurements can then be superimposed to the recorded cardiac functional parameters to determine the relationship between substrates metabolism and cardiac work under baseline and stress conditions (change in workload, ischemia-reperfusion, etc…). It is also possible to evaluate how the metabolism/contraction relationship is af.......

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This work was supported by National Institutes of Health Grants R00 HL112952 (to R. H.), R01 HL108618 (to J.P.G.), P01 HL051971, and P20 GM104357. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Name Company Catalog Number Comments
Sodium Chloride (NaCl) Fisher Scientific BP358
Potassium Chloride (KCl) Fisher Scientific BP366
Potassium Phosphate Monobasic (KH2PO4) Fisher Scientific P284
Magnesium Sulfate Heptahydrate (MgSO4*7H2O) Fisher Scientific M63
Sodium Bicarbonate (NaHCO3) Fisher Scientific S233
Calcium Chloride (CaCl2) Sigma-Aldrich C5670
AG 1-X8 resin, chloride form, 100-200 dry mesh size, 500 g Bio-Rad 1401441 This item can be replaced by purchasing directly the hydoxide form  (see reference below), but this will cost almost 8 times more
AG 1-X8 resin, hydroxide form, 100-200 dry mesh size, 100 g Bio-Rad 1432445 Purchasing this item allows to bypass the conversion of the anion exchange resin from the chloride form to the hydroxide form (See section 1.2 of protocol)
Glass Microanalysis Vacuum Filter Holder Fisher Scientific 09-753-2
Sodium Hydroxide (NaOH) Fisher Scientific S318 Corrosive. Consult the product MSDS for appropriate handling and storage.
Gas Dispersion Tube with Fritted Cylinder Fisher Scientific 11-138B
Probumin Bovine Serum Albumin Fatty Acid Free, Powder EMD Millipore 820027 We recommend the use of a charcoal-defatted BSA, as other purification process such as cold ethanol fractionation may leave residues toxic for the heart.
Sodium Oleate Sigma-Aldrich O7501
Oleic Acid, [9,10-3H(N)]- PerkinElmer NET289005MC Radioactive material. Follow your Institution's radiation safety office guidelines for ordering and handling.
Dialysis Membrane Tubing, 29 mm diameter Fisher Scientific 08-667E
D-(+)-Glucose Sigma-Aldrich G7021
Glucose, D-[14C(U)]- PerkinElmer NEC042B005MC Radioactive material. Follow your Institution's radiation safety office guidelines for ordering and handling.
Humulin R U-100 Eli Lilly and Company NDC 0002-8215-01 (HI-210)
Inactin Hydrate Sigma-Aldrich T133 Controlled substance on USDEA Schedule III
3-0 Silk Black Braid Roboz Surgical SUT-15-3
10X Hyamine Hydroxide PerkinElmer 6003005 Highly toxic and causes severe burns. Consult the product MSDS for appropriate handling and storage
20 mL Glass Scintillation Vials Fisher Scientific 03-341-25E Use glass vials for quantitative recovery of 14CO2
20 mL HDPE Scintillation Vials Fisher Scientific 03-337-23B Use HDPE vials for quantitative recovery of 3H2O
Red Rubber Sleeve Stoppers Fisher Scientific 14-126DD Fit 20 mL scintillation vials; Reusable
BD PrecisionGlide Needle 23G x 40 mm BD 305194 Use to inject perchloric acid through the rubber sleeve stopper of the CO2 trap
Perchloric Acid, 60% Fisher Scientific A228 Highly corrosive and may act as an oxidizer and/or cause an explosion hazard. Consult the product MSDS for appropriate handling and storage
Ultima Gold, Scintillation Cocktail PerkinElmer 6013327
Glass Wool Fisher Scientific AC38606
Decon Dri-Clean Detergent Powder Fisher Scientific 04-355 For cleaning of glassware, plastic parts, and tubing
Alconox Tergazyme Enzyme-Active Powered Detergent Fisher Scientific 16-000-115 For cleaning of "hard to reach" surfaces (tubing, glassware) contaminated by fatty acid-BSA residue

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