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.

<ol>
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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 affected by preexisting conditions such as heart failure and diabetes. In addition, the heart of genetically-modified rodents can be used to interrogate the effect of a specific protein on cardiac metabolism and function. Last, the effect of drugs and other circulating factors on these parameters can be individually tested. Because the flux of glucose through glycolysis does not always correlate with the rate of glucose oxidation, it may be informative to follow the rates of glucose flux through both pathways in a single experimental setting.
The rates of glycolysis can be determined by adding [5-3H]glucose to the perfusion buffer, because the release of 3H2O is determined by the activity of the enolase step of the glycolysis20. Alternatively [2-3H]glucose, which leads to the production of 3H2O at the phosphoglucose isomerase step of the glycolytic pathway, can be used to interrogate the rates of glucose uptake by the heart muscle16,17,21. The possibility to choose from multiple radiolabeled tracers to investigate the fate of glucose at different levels of its catabolism is another example of the versatility of the technique. However, because all the flux measurements rely on the quantitative recovery of either 3H2O or 14CO2 from the coronary effluent, only two radiolabeled tracers (one 3H-labeled and the other one labeled with 14C) can be used in the perfusion buffer at a time.
Methodological considerations
There are some inherent limitations to the measurement of cardiac metabolic flux and function using this protocol. Although the use of KH buffer allows for a total control over the composition of the perfusate, the physiological relevance of this experimental setup may be questionable. First, the use of a crystalloid solution over blood is known to negatively impact cardiac hemodynamics and resistance to ischemia22,23. Second, the perfusion buffer we describe here lacks a variety of hormones and nutrients that can directly modulate cardiac metabolism and consequently impact the heart&#39;s response to a simulated stress condition. Indeed, the mammalian heart is a metabolic omnivore and can, besides glucose and fatty acids, utilize other substrates to fulfill its energy requirements (lactate, pyruvate, ketone bodies, branched-chain amino acids). There is mounting evidence that some of these substrates play an important role in the metabolic remodeling of the diseased heart24,25. It is entirely possible for the investigator to add these substrates to the perfusion buffer, to modulate their concentration in order to mimic more closely different physiological conditions (e.g. fed state versus fasted state) or disease states, and to follow their rates of oxidation using the appropriate radiolabeled tracers26,27. The metabolism of triglycerides can also be traced, and in that case it is recommended to avoid the use of heparin prior to excision of the heart as this will induce the release of lipoprotein lipase from the endothelium and significantly decrease the extraction of triglyceride from the buffer28. With careful selection of the radiolabeled tracers, the incorporation of radioactivity in various tissue metabolites can also be measured after perfusion to determine parameters such as de novo triglyceride synthesis, glycogen turnover, or rates of protein synthesis18,29,30. In other words, the investigator can work with a wide variety of substrates/hormones combinations and concentrations. The choice of the buffer composition will mostly depend on the objectives of the experiment. Because these controlled buffer conditions create a &#34;simplified&#34; physiological model, results from ex vivo working heart perfusion experiments should be interpreted with caution and if possible the data should be further validated in vivo.
Choice of the anesthetic
The anesthetic used on rats prior to excision of the heart should be carefully selected based on its known effects on cardiopulmonary function and metabolism to ensure that this will not interfere with the experiment to be performed. Both injectable and inhalational anesthetics rapidly induce hyperglycemia and variable degrees of glucose intolerance that may affect cardiac metabolism during perfusion31,32. Compared to other agents, the short-term effects of barbiturates (Pentobarbital and thiobutabarbital) on glucose homeostasis are relatively negligible31. However, intraperitoneal pentobarbital anesthesia prior to heart excision has been shown to decrease cardiac output, impair left ventricular function, and increase myocardial lactate release in an ex vivo protocol of ischemia-reperfusion33. The negative inotropic effect that pentobarbital has on the isolated rat heart is suggested to be caused by tissue accumulation of the drug34. Conversely the use of inhalational anesthetics, which are more rapidly washed out from the cardiac tissue, is associated with better contractile function ex vivo33. Volatile anesthetics may actually provide cardioprotection during ischemia-reperfusion through a mechanism involving the opening of the mitochondrial KATP channels and increased binding of hexokinase to mitochondria32. In summary, the type of anesthetic used in an isolated working heart perfusion protocol is a significant variable in the assessment of both functional and metabolic parameters that may directly impact the outcomes of the experiment. Therefore, the type and the dose of anesthetic used should always be reported in the methods section of an experiment, and these parameters should not be changed between experiments that are meant to be compared.
Radioisotopes in heart perfusion
All flux measurements performed with the preceding protocol are performed in the non-recirculating mode, meaning that the coronary effluent is discarded instead of being returned to the buffer reservoir (Figure 1). The investigator may rather choose to use the perfusion apparatus in the recirculating mode, which has the advantage of requiring a much smaller volume of buffer and hence radioactivity to operate. However, recycling of the coronary effluent compromises the steady state composition of the perfusion buffer through recovery of byproducts originating from the metabolic activity of the heart (including lactate, but also 3H2O and H14CO3-), which complicates the determination of metabolic fluxes and may even alter cardiac function over time35. These issues are prevented in the non-recirculating mode. Due to the amount of radioactivity and to the large volume of perfusion buffer used in a single experiment, the investigator should be particularly cautious when using the working rat heart in metabolic flux analyses. When using a perfusion apparatus for the first time, it is recommended to perform several mock experiments with non-radioactive buffer to become familiar with the different steps of the protocol and with the safety procedures. The mounting of the heart on the cannulas is a particularly sensitive step because it requires establishing a close contact with the apparatus and because buffer may leak or shoot out from one of the heart&#39;s severed blood vessels. The perfusion apparatus described by Taegtmeyer and colleagues11 has the advantage of including a Langendorff reservoir that is entirely independent from the rest of the perfusion apparatus, and can therefore be filled with non-radioactive KH buffer to prevent spills of radioactive material during the heart&#39;s cannulation. Regardless of the system used, the experiments should be performed in a dedicated area with restricted access. The room should include a sink and a supply of ultrapure water to perform buffer preparation and cleaning of contaminated equipment in the same area. Investigators should work closely with their institution&#39;s radiation safety office to establish specific containment, control and decontamination procedures.
Cleaning of the perfusion apparatus
The post experimental cleaning of the perfusion apparatus is a critical step that is too often overlooked and, if not carefully executed, responsible for most of the issues related to poor experimental reproducibility. The nutrient-rich perfusion buffer and the high temperature of the perfusion system provide ideal conditions for bacterial and fungal growth. The utilization of proteins (insulin, BSA) and fatty acids in the perfusion buffer adds another degree of complexity to the cleaning procedure because these compounds will stick to the surface of the tubing and glassware, making them hard to flush out of the apparatus. The use of radioisotopes will also impose additional constraints such as the creation of a dedicated containment area in the laboratory for manipulation of the contaminated equipment and disposal of solid and liquid radioactive wastes. Therefore, the adequate cleaning procedure will mostly depend on both the composition of the perfusion buffer and on the type of perfusion apparatus used. If using a commercial apparatus, check the manufacturer&#39;s website or contact the customer service to obtain information on how to perform cleaning without damaging any of the components of the apparatus. In general, all glassware and plastic parts can be washed with a wide range of soaps. Enzyme-active powered detergents are particularly useful to remove fatty acid-BSA residue. Most contaminants will also be removed by running 0.1 M HCl or 0.1 M HNO3 through the apparatus for several hours. We obtain satisfactory cleaning results by flushing the system first with 0.1 M HCl, then with a fresh 1% (w:v) solution of enzyme-active powered detergent prepared in warm water, and finally by rinsing the system with ultrapure water. When using a custom-built perfusion apparatus as the one originally described by Taegtmeyer and colleagues11, it may be easier and more effective to completely dismantle the apparatus at the end of each experimental day. The glassware is cleaned by a 15 min immersion into a concentrated bath of a chromic acid and sulfuric acid mixture. The plastic parts and the PVC tubing can be washed with a residue-free detergent. We found it is actually faster and more reliable to entirely replace the PVC tubing every day, because it limits the manipulation of material contaminated by radiochemicals and because it prevents leaving residues of cleaning agents behind. In any case, the investigator should apply the following rules: 1- Clean the perfusion apparatus and all dirty equipment immediately after completing the perfusion experiment. Waiting will only make the cleaning more difficult and time consuming. 2- Check the circulating bath of the water jacket system regularly for contaminations. Use of a water conditioner will increase the interval between cleaning of the water bath. 3- After cleaning, thoroughly rinse all glassware and tubing with tap water several times and finish rinsing with ultrapure water to remove residues from cleaning agents. 4- Two heart preparations consecutively failing when the dissection was carefully executed and the perfusion buffer carefully prepared are likely to indicate a contamination of the apparatus (either by bacteria or by solvent residues). If this happens, the washing and rinsing procedures should be entirely repeated.
Scaling down to the mouse heart
Although the preceding protocol focuses on the determination of metabolic rates in an isolated working rat heart, the same procedures can be applied to the mouse heart with a few modifications to the perfusion apparatus and data acquisition equipment36. Due to its smaller size the isolated working mouse heart is technically more challenging, but because of its significantly lower flow rate it has the advantage of requiring a much lower volume of perfusion buffer. The large number of genetically modified mouse models that are already available also makes the isolated working mouse heart very attractive to study the function of a single gene product on cardiac metabolic and functional remodeling. However, the recent technological advances in genome editing and a rapidly growing catalog of transgenic and knockout rats are rapidly closing the gap between the two rodent species. Moreover, the rat has long been praised as a superior animal model to investigate cardiovascular disease because its pathophysiology closely mimics human conditions37. For these reasons, we believe that the isolated working rat heart still hold an important place in the modern era of cardiovascular research.

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 determination of cardiac power. The consequences of each stress condition on the utilization of energy providing substrates were simultaneously determined by measurement of the rates of glucose and oleate oxidation.
In the first experiment, an acute increase in workload was simulated after 20 min perfusion at near-physiological workload by increasing the afterload by 40% (accomplished by manually raising the aortic overflow chamber) and by adding epinephrine (1 &#181;mol/L) to the perfusion buffer (Figure 3A). Heart rate, which can be determined by measuring the interval between the peak systolic pressure values on the pulse pressure trace, increased immediately by more than 50% upon workjump induction (Figure 3B). Cardiac power increased by more than 40% at workjump, which was accompanied by an increase in both rates of glucose and oleate oxidation (Figure 3C). The results show that, under the stress of an acute increase in workload, the heart rapidly adapts to increase energy generation for contraction from both glucose and fatty acid oxidation. However, the relative increase in rate of oxidation was more important for glucose (~2.8-fold) than it was for oleate (~1.3 fold). Increased reliance on carbohydrates oxidation to cope with the extra workload is a typical feature of the pressure-overloaded mammalian heart18.
In the second experiment, the heart was perfused under baseline conditions for 20 min followed by 15 min total, global, normothermic ischemia (induced by closing both the atrial and aortic lines) and 30 min reperfusion (Figure 4A). Under these experimental conditions, a near-normal pulse pressure was restored between 2 and 3 min after the beginning of reperfusion (Figure 4B). Cardiac power rapidly increased above pre-ischemia values during the first 10 min of reperfusion before falling back to near-baseline values (Figure 4C). Compared to baseline, the rate of oleate oxidation increased during reperfusion whereas glucose oxidation rapidly returned to pre-ischemic levels. By simultaneously measuring contractile function and both rates of substrate oxidation one can clearly appreciate that, in the present experimental conditions, the variation in the rates of fatty acid oxidation is the main determinant for changes in the amount of work generated at reperfusion. The higher switch toward fatty acid utilization post ischemia is a well-estalished feature of the reperfused heart19.
<img alt="Figure 1" src="/files/ftp_upload/54497/54497fig1.jpg" /> 
Figure 1: Simplified Scheme of the Isolated Working Rat Heart Apparatus. The Krebs-Henseleit buffer supplemented with the fatty acid-BSA solution is oxygenated by passing through a membrane oxygenator. Typical cardiac preload and afterload values for a rat heart perfused in near-physiological conditions are indicated. The settings can be modified by adjusting the heights of the atrial reservoir and aortic overflow chamber. The tubes of graduated syringes have been placed underneath the heart chamber and the aortic overflow chamber in order to measure coronary flow and aortic flow, respectively. The flow of buffer can be stopped (when measuring the aortic and coronary flows) or rerouted (when switching from the Langendorff to the working mode) through the use of three-way stopcocks. Red arrows indicate the optimal positioning for in-line oxygen microlelectrodes to determine the &#34;arteriovenous&#34; oxygen difference. In-line flow probes and pressure transducers can be placed in the preload and afterload lines to measure both atrial and aortic flow rates and pressures (green arrows). Left ventricular pressures and volumes can also be recorded with a PV catheter inserted through the apex of the left ventricle. Not shown on the figure is the water jacket surrounding all the glassware elements and the perfusion tubing in the dark blue color. These water-jacketed elements are all connected serially via tubing to a circulating water bath set at 37 &#186;C. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54497/54497fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54497/54497fig2.jpg" /> 
Figure 2: Representation of the Different Steps Involved in the Setup of the CO2 Trap. (A) Add 1 ml of 10x concentrated hydroxide of hyamine. (B) Add a 0.5 ml sample contained in a capless tube. (C) Seal the vial. (D) Inject 200 &#181;l of perchloric acid (60% w:w%). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54497/54497fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54497/54497fig3.jpg" /> 
Figure 3: Representative Results of a Workjump Experiment. (A) Continuous recording of pulse pressure. (B) Short interval recording depicting the change in pulse pressure at the time of workjump initiation. (C) Cardiac rates of glucose oxidation (red trace with circles) and oleate oxidation (blue trace with squares) were determined by analysis of the coronary effluent at a 5 min interval. Cardiac power (black trace with triangles) was determined by measurement of the cardiac output at the same time the coronary effluent samples were recovered. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54497/54497fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54497/54497fig4.jpg" /> 
Figure 4: Representative Results of an Ischemia-reperfusion Experiment. (A) Continuous recording of pulse pressure. (B) Short interval recording depicting the recovery of contractile function during the first 5 min of reperfusion. (C) Cardiac rates of glucose oxidation (red trace with circles) and oleate oxidation (blue trace with squares) were determined by analysis of the coronary effluent at a 5 min interval, except during the first 5 min of reperfusion where analyzes were performed at a 1 min interval. Cardiac power (black trace with triangles) was determined by measurement of the cardiac output at the same time the coronary effluent samples were recovered. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54497/54497fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart at JoVE.com

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 ...

methods-for-determination-rates-glucose-fatty-acid-oxidation-isolated

Universidad San Sebastian

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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

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

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 ...

Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to investigate metabolic capability. To obtain the measures of mitochondrial respiration, fresh biological samples must be used, and the entire laboratory procedure must be completed within approximately 2 h. Furthermore, multiple pieces of specialized equipment are required to perform these laboratory assays. This creates a challenge for measuring mitochondrial respiration in the tissues of wild animals living far from physiology laboratories as live tissue cannot be preserved for very long after collection in the field. Moreover, transporting live animals over long distances induces stress, which can alter mitochondrial energetics.
This manuscript introduces the Auburn University (AU) MitoMobile, a mobile mitochondrial physiology laboratory that can be taken into the field and used on-site to measure mitochondrial metabolism in tissues collected from wild animals. The basic features of the mobile laboratory and the step-by-step methods for measuring isolated mitochondrial respiration rates are presented. Additionally, the data presented validate the success of outfitting the mobile mitochondrial physiology laboratory and making mitochondrial respiration measurements. The novelty of the mobile laboratory lies in the ability to drive to the field and perform mitochondrial measurements on the tissues of animals captured on site.

We designed and constructed a mobile laboratory to measure respiration rates in isolated mitochondria of wild animals captured at field locations. Here, we describe the design and outfitting of a mobile mitochondrial laboratory and the associated laboratory protocols.

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to ...

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are ...

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

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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

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

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

Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro