Name: methods for the determination of rates of glucose and fatty acid oxidation in the isolated working rat heart
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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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The authors declare that they have no competing financial interests.

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&#8230;). It is also possible to evaluate how the metabolism/contraction relationship is af...

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 to the identification of therapeutic targets for the prevention or treatment of heart failure5-7. The recent publication of a scientific statement from the American Heart Association on &#34;Assessing Cardiac Metabolism&#34; emphasizes the growing interest of the scientific community for this field of research8. But while the technological advances in cardiac imaging now allow for a rapid and accurate evaluation of cardiac morphology and function, the in vivo study of cardiac metabolism remains limited and burdensome: Nuclear Magnetic Resonance (NMR) spectroscopy and Positron Emission Tomography (PET) imaging can be used to follow cardiac high energy phosphate metabolism and Krebs cycle activity, but these techniques are plagued by high operating costs and by their inability to determine the contribution of various substrates to oxidative metabolism in steady-state conditions9.To this date the ex vivo working heart preparation represents the sole and unique technique available to study, simultaneously and in real time, contractile function and flux of substrates into oxidative metabolic pathways7,9. The following protocol aims to provide guidelines in the preparation and utilization of reagents used to determine the rates of substrates utilization in the isolated working rat heart.
The Isolated Working Rodent Heart Apparatus
Although the technique is almost half a century old, the isolated working rat heart preparation remains a method of choice for cardiovascular research. As with the Langendorff heart preparation, the working rodent heart offers a relatively simple, reliable, and inexpensive way to measure a wide range of cardiac parameters independently from the confounding effects of other organs, neurohormonal and other circulating factors. But in contrast to the Langendorff-perfused heart, the working heart continues to perform near-physiological cardiac work, a prerequisite for the generation of oxidative metabolic flux to levels that are relevant to in vivo conditions. This is achieved by delivering the perfusion buffer to the left ventricle (LV) via a cannula connected to the left atrium, and as the LV fills and contracts, the buffer is ejected through the aortic line against a determined afterload hydrostatic pressure. The design of the perfusion apparatus originally described by Neely and colleagues10 was subsequently improved by Taegtmeyer, Hems and Krebs11, but has changed very little ever since. As described in the original apparatus, contractile function can be assessed through determination of cardiac output, using no more than graduated cylinders and a stopwatch to measure aortic and coronary flows10,11. Several vendors now offer complete working rodent heart perfusion systems. These commercially available apparatus can be acquired with flowprobes, pressure transducers, a pressure-volume catheter and all the equipment necessary for cardiac functional data acquisition and analysis. The vendors provide extensive documentation and training sessions to familiarize the new user with their equipment. Several review articles also detail protocols on the working heart instrumentation and on the use of catheters to measure cardiac function in rodents12-15. For this reason, we will only briefly mention the set-up of the perfusion apparatus and the recording equipment. The present protocol rather aims to complement the already available information with a description of the methods that can be implemented to simultaneously measure the rates of glucose and long-chain fatty acid oxidation, the two major energy providing substrates in the normal heart. We describe here all the steps involved in the use of radiolabeled energy substrates for the assessment of myocardial oxidative metabolism, from the preparation of reagents and buffers to the recovery and processing of samples, to the data analysis.
Principles of the Method
Cardiomyocytes generate the bulk of their energy for contraction from the oxidative phosphorylation of fatty acids (principally long-chain fatty acids) and carbohydrates (glucose and lactate). The heart has very limited energetic reserves and relies on a constant supply of these energy providing substrates from the circulation. The catabolism of glucose through the glycolytic pathway yields pyruvate which is then decarboxylated by the pyruvate dehydrogenase complex of the inner mitochondrial membrane. Long-chain fatty acids, extracted from circulating albumin or lipoprotein triglycerides, are first activated into acyl-CoA molecules in the cytosol and subsequently transported inside the mitochondrial matrix through the carnitine shuttle to enter the beta-oxidation pathway. The acetyl-CoA molecules produced by the catabolism of glucose and fatty acids fuel the Krebs cycle to generate the reducing equivalents (NADH and FADH2) which are used by the electron transport chain to build the proton-motive force across the inner mitochondrial membrane and generate ATP through the activity of the ATP synthase. Water and carbon dioxide are the end byproducts of the enzymatic reactions taking place inside the Krebs cycle. The supply of 14C- and 3H- radiolabeled substrates (such as 14C-radiolabeled glucose and 3H-radiolabeled oleic acid) to the isolated working heart will consequently lead to the production of 14CO2 and 3H2O which can be quantitatively recovered from the coronary effluent. The collection of 14CO2 is carried out by keeping the isolated perfused heart into a sealed chamber and by immediately recovering the coronary effluent as it exits the heart. A small anion exchange column is used to separate and recover 3H2O from the coronary effluent. The radioactivity from the processed samples is measured with a liquid scintillation counter, and 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 pathways16,17.

<table><tbody><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>BP366</td><td></td></tr><tr><td>Potassium Phosphate Monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>P284</td><td></td></tr><tr><td>Magnesium Sulfate Heptahydrate (MgSO&lt;sub&gt;4&lt;/sub&gt;*7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>M63</td><td></td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>AG 1-X8 resin, chloride form, 100 - 200 dry mesh size, 500 g</td><td>Bio-Rad</td><td>1401441</td><td>This item can be replaced by purchasing directly the hydoxide form&amp;nbsp; (see reference below), but this will cost almost 8 times more</td></tr><tr><td>AG 1-X8 resin, hydroxide form, 100 - 200 dry mesh size, 100 g</td><td>Bio-Rad</td><td>1432445</td><td>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)</td></tr><tr><td>Glass Microanalysis Vacuum Filter Holder</td><td>Fisher Scientific</td><td>09-753-2</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Fisher Scientific</td><td>S318</td><td>Corrosive. Consult the product MSDS for appropriate handling and storage.</td></tr><tr><td>Gas Dispersion Tube with Fritted Cylinder</td><td>Fisher Scientific</td><td>11-138B</td><td></td></tr><tr><td>Probumin Bovine Serum Albumin Fatty Acid Free, Powder</td><td>EMD Millipore</td><td>820027</td><td>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.</td></tr><tr><td>Sodium Oleate</td><td>Sigma-Aldrich</td><td>O7501</td><td></td></tr><tr><td>Oleic Acid, [9,10-&lt;sup&gt;3&lt;/sup&gt;H(N)]-</td><td>PerkinElmer</td><td>NET289005MC</td><td>Radioactive material. Follow your Institution&#039;s radiation safety office guidelines for ordering and handling.</td></tr><tr><td>Dialysis Membrane Tubing, 29 mm diameter</td><td>Fisher Scientific</td><td>08-667E</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Glucose, D-[14C(U)]-</td><td>PerkinElmer</td><td>NEC042B005MC</td><td>Radioactive material. Follow your Institution&#039;s radiation safety office guidelines for ordering and handling.</td></tr><tr><td>Humulin R U-100</td><td>Eli Lilly and Company</td><td>NDC 0002-8215-01 (HI-210)</td><td></td></tr><tr><td>Inactin Hydrate</td><td>Sigma-Aldrich</td><td>T133</td><td>Controlled substance on USDEA Schedule III</td></tr><tr><td>3-0 Silk Black Braid</td><td>Roboz Surgical</td><td>SUT-15-3</td><td></td></tr><tr><td>10x Hyamine Hydroxide</td><td>PerkinElmer</td><td>6003005</td><td>Highly toxic and causes severe burns. Consult the product MSDS for appropriate handling and storage</td></tr><tr><td>20 ml Glass Scintillation Vials</td><td>Fisher Scientific</td><td>03-341-25E</td><td>Use glass vials for quantitative recovery of &lt;sup&gt;14&lt;/sup&gt;CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>20 ml HDPE Scintillation Vials</td><td>Fisher Scientific</td><td>03-337-23B</td><td>Use HDPE vials for quantitative recovery of &lt;sup&gt;3&lt;/sup&gt;H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Red Rubber Sleeve Stoppers</td><td>Fisher Scientific</td><td>14-126DD</td><td>Fit 20 mL scintillation vials; Reusable</td></tr><tr><td>BD PrecisionGlide Needle 23G x 40 mm</td><td>BD</td><td>305194</td><td>Use to inject perchloric acid through the rubber sleeve stopper of the CO&lt;sub&gt;2&lt;/sub&gt; trap</td></tr><tr><td>Perchloric Acid, 60%</td><td>Fisher Scientific</td><td>A228</td><td>Highly corrosive and may act as an oxidizer and/or cause an explosion hazard. Consult the product MSDS for appropriate handling and storage</td></tr><tr><td>Ultima Gold, Scintillation Cocktail</td><td>PerkinElmer</td><td>6013327</td><td></td></tr><tr><td>Glass Wool</td><td>Fisher Scientific</td><td>AC38606</td><td></td></tr><tr><td>Decon Dri-Clean Detergent Powder</td><td>Fisher Scientific</td><td>04-355</td><td>For cleaning of glassware, plastic parts, and tubing</td></tr><tr><td>Alconox Tergazyme Enzyme-Active Powered Detergent</td><td>Fisher Scientific</td><td>16-000-115</td><td>For cleaning of &quot;hard to reach&quot; surfaces (tubing, glassware) contaminated by fatty acid-BSA residue</td></tr></tbody></table>

methods for the determination of rates of glucose and fatty acid oxidation in the isolated working rat heart

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.

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

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.6K Views.  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.

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

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Developmental Biology

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Medicine

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Biochemistry

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Immunology and Infection

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Bioengineering

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Neuroscience

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

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

Summary

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Chapters in this video

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate