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.

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

<ol>
	<li>Neely, J. R., Morgan, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+carbohydrate+and+lipid+metabolism+and+the+energy+balance+of+heart+muscle.">Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle.</a> Annu Rev Physiol. 36, 413-459 (1974).</li><li>Sen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+regulation+of+load-induced+mTOR+signaling+and+ER+stress+in+mammalian+heart.">Glucose regulation of load-induced mTOR signaling and ER stress in mammalian heart.</a> J Am Heart Assoc. 2, e004796 (2013).</li><li>Young, M. E., McNulty, P., Taegtmeyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+and+maladaptation+of+the+heart+in+diabetes:+Part+II:+potential+mechanisms.">Adaptation and maladaptation of the heart in diabetes: Part II: potential mechanisms.</a> Circulation. 105, 1861-1870 (2002).</li><li>Stanley, W. C., Recchia, F. A., Lopaschuk, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+substrate+metabolism+in+the+normal+and+failing+heart.">Myocardial substrate metabolism in the normal and failing heart.</a> Physiol Rev. 85, 1093-1129 (2005).</li><li>Fillmore, N., Lopaschuk, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+mitochondrial+oxidative+metabolism+as+an+approach+to+treat+heart+failure.">Targeting mitochondrial oxidative metabolism as an approach to treat heart failure.</a> Biochim Biophys Acta. 1833, 857-865 (2013).</li><li>Jaswal, J. S., Keung, W., Wang, W., Ussher, J. R., Lopaschuk, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+fatty+acid+and+carbohydrate+oxidation--a+novel+therapeutic+intervention+in+the+ischemic+and+failing+heart.">Targeting fatty acid and carbohydrate oxidation--a novel therapeutic intervention in the ischemic and failing heart.</a> Biochim Biophys Acta. 1813, 1333-1350 (2011).</li><li>Taegtmeyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+metabolism+as+a+target+for+the+treatment+of+heart+failure.">Cardiac metabolism as a target for the treatment of heart failure.</a> Circulation. 110, 894-896 (2004).</li><li>Taegtmeyer, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Cardiac+Metabolism:+A+Scientific+Statement+From+the+American+Heart+Association.">Assessing Cardiac Metabolism: A Scientific Statement From the American Heart Association.</a> Circ Res. , (2016).</li><li>Barr, R. L., Lopaschuk, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+measuring+in+vitro/ex+vivo+cardiac+energy+metabolism.">Methodology for measuring in vitro/ex vivo cardiac energy metabolism.</a> J Pharmacol Toxicol Methods. 43, 141-152 (2000).</li><li>Neely, J. R., Liebermeister, H., Battersby, E. J., Morgan, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pressure+development+on+oxygen+consumption+by+isolated+rat+heart.">Effect of pressure development on oxygen consumption by isolated rat heart.</a> Am J Physiol. 212, 804-814 (1967).</li><li>Taegtmeyer, H., Hems, R., Krebs, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+energy-providing+substrates+in+the+isolated+working+rat+heart.">Utilization of energy-providing substrates in the isolated working rat heart.</a> Biochem J. 186, 701-711 (1980).</li><li>Liao, R., Podesser, B. K., Lim, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+continuing+evolution+of+the+Langendorff+and+ejecting+murine+heart:+new+advances+in+cardiac+phenotyping.">The continuing evolution of the Langendorff and ejecting murine heart: new advances in cardiac phenotyping.</a> Am J Physiol Heart Circ Physiol. 303, H156-H167 (2012).</li><li>Cingolani, O. H., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-volume+relation+analysis+of+mouse+ventricular+function.">Pressure-volume relation analysis of mouse ventricular function.</a> Am J Physiol Heart Circ Physiol. 301, H2198-H2206 (2011).</li><li>Pacher, P., Nagayama, T., Mukhopadhyay, P., Batkai, S., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cardiac+function+using+pressure-volume+conductance+catheter+technique+in+mice+and+rats.">Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats.</a> Nat Protoc. 3, 1422-1434 (2008).</li><li>Abraham, D., Mao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Pressure-Volume+Loop+Analysis+Using+Conductance+Catheters+in+Mice.">Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice.</a> J Vis Exp. , (2015).</li><li>Harmancey, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+improves+metabolic+and+contractile+efficiency+in+stressed+rat+heart.">Insulin resistance improves metabolic and contractile efficiency in stressed rat heart.</a> FASEB J. 26, 3118-3126 (2012).</li><li>Harmancey, R., Vasquez, H. G., Guthrie, P. H., Taegtmeyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+long-chain+fatty+acid+oxidation+impairs+postischemic+recovery+of+the+insulin-resistant+rat+heart.">Decreased long-chain fatty acid oxidation impairs postischemic recovery of the insulin-resistant rat heart.</a> FASEB J. 27, 3966-3978 (2013).</li><li>Goodwin, G. W., Taylor, C. S., Taegtmeyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+energy+metabolism+of+the+heart+during+acute+increase+in+heart+work.">Regulation of energy metabolism of the heart during acute increase in heart work.</a> J Biol Chem. 273, 29530-29539 (1998).</li><li>Lopaschuk, G. D., Ussher, J. R., Folmes, C. D., Jaswal, J. S., Stanley, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+fatty+acid+metabolism+in+health+and+disease.">Myocardial fatty acid metabolism in health and disease.</a> Physiol Rev. 90, 207-258 (2010).</li><li>Neely, J. R., Denton, R. M., England, P. J., Randle, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+increased+heart+work+on+the+tricarboxylate+cycle+and+its+interactions+with+glycolysis+in+the+perfused+rat+heart.">The effects of increased heart work on the tricarboxylate cycle and its interactions with glycolysis in the perfused rat heart.</a> Biochem J. 128, 147-159 (1972).</li><li>Katz, J., Dunn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-2-t+as+a+tracer+for+glucose+metabolism.">Glucose-2-t as a tracer for glucose metabolism.</a> Biochemistry. 6, 1-5 (1967).</li><li>Gillis, A. M., Kulisz, E., Mathison, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+electrophysiological+variables+in+blood-perfused+and+buffer-perfused,+isolated,+working+rabbit+heart.">Cardiac electrophysiological variables in blood-perfused and buffer-perfused, isolated, working rabbit heart.</a> Am J Physiol. 271, H784-H789 (1996).</li><li>Qiu, Y., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+ischemic+vulnerability+and+responsiveness+to+cardioplegic+protection+in+crystalloid-perfused+versus+blood-perfused+hearts.">Comparison of ischemic vulnerability and responsiveness to cardioplegic protection in crystalloid-perfused versus blood-perfused hearts.</a> J Thorac Cardiovasc Surg. 103, 960-968 (1992).</li><li>Cotter, D. G., Schugar, R. C., Crawford, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketone+body+metabolism+and+cardiovascular+disease.">Ketone body metabolism and cardiovascular disease.</a> Am J Physiol Heart Circ Physiol. 304, H1060-H1076 (2013).</li><li>Huang, Y., Zhou, M., Sun, H., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branched-chain+amino+acid+metabolism+in+heart+disease:+an+epiphenomenon+or+a+real+culprit?.">Branched-chain amino acid metabolism in heart disease: an epiphenomenon or a real culprit?.</a> Cardiovasc Res. 90, 220-223 (2011).</li><li>Buse, M. G., Biggers, J. F., Friderici, K. H., Buse, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+branched+chain+amino+acids+by+isolated+hearts+and+diaphragms+of+the+rat.+The+effect+of+fatty+acids,+glucose,+and+pyruvate+respiration.">Oxidation of branched chain amino acids by isolated hearts and diaphragms of the rat. The effect of fatty acids, glucose, and pyruvate respiration.</a> J Biol Chem. 247, 8085-8096 (1972).</li><li>Liepinsh, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heart+is+better+protected+against+myocardial+infarction+in+the+fed+state+compared+to+the+fasted+state.">The heart is better protected against myocardial infarction in the fed state compared to the fasted state.</a> Metabolism. 63, 127-136 (2014).</li><li>Niu, Y. G., Hauton, D., Evans, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+triacylglycerol-rich+lipoproteins+by+the+working+rat+heart:+routes+of+uptake+and+metabolic+fates.">Utilization of triacylglycerol-rich lipoproteins by the working rat heart: routes of uptake and metabolic fates.</a> J Physiol. 558, 225-237 (2004).</li><li>Goodwin, G. W., Arteaga, J. R., Taegtmeyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+turnover+in+the+isolated+working+rat+heart.">Glycogen turnover in the isolated working rat heart.</a> J Biol Chem. 270, 9234-9240 (1995).</li><li>Sender, P. M., Garlick, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+rates+of+protein+in+the+Langendorff-perfused+rat+heart+in+the+presence+and+absence+of+insulin,+and+in+the+working+heart.">Synthesis rates of protein in the Langendorff-perfused rat heart in the presence and absence of insulin, and in the working heart.</a> Biochem J. 132, 603-608 (1973).</li><li>Hindlycke, M., Jansson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+tolerance+and+pancreatic+islet+blood+flow+in+rats+after+intraperitoneal+administration+of+different+anesthetic+drugs.">Glucose tolerance and pancreatic islet blood flow in rats after intraperitoneal administration of different anesthetic drugs.</a> Ups J Med Sci. 97, 27-35 (1992).</li><li>Zuurbier, C. J., Keijzers, P. J., Koeman, A., Van Wezel, H. B., Hollmann, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia's+effects+on+plasma+glucose+and+insulin+and+cardiac+hexokinase+at+similar+hemodynamics+and+without+major+surgical+stress+in+fed+rats.">Anesthesia's effects on plasma glucose and insulin and cardiac hexokinase at similar hemodynamics and without major surgical stress in fed rats.</a> Anesth Analg. 106, 135-142 (2008).</li><li>Oguchi, T., Kashimoto, S., Yamaguchi, T., Nakamura, T., Kumazawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+pentobarbital+appropriate+for+basal+anesthesia+in+the+working+rat+heart+model?.">Is pentobarbital appropriate for basal anesthesia in the working rat heart model?.</a> J Pharmacol Toxicol Methods. 29, 37-43 (1993).</li><li>Segal, J., Schwalb, H., Shmorak, V., Uretzky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+anesthesia+on+cardiac+function+and+response+in+the+perfused+rat+heart.">Effect of anesthesia on cardiac function and response in the perfused rat heart.</a> J Mol Cell Cardiol. 22, 1317-1324 (1990).</li><li>Webster, I., Smith, A., Lochner, A., Huisamen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sanguinarine+non-+versus+re-circulation+during+isolated+heart+perfusion--a+Jekyll+and+Hyde+effect?.">Sanguinarine non- versus re-circulation during isolated heart perfusion--a Jekyll and Hyde effect?.</a> Cardiovasc Drugs Ther. 28, 489-491 (2014).</li><li>Belke, D. D., Larsen, T. S., Lopaschuk, G. D., Severson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+and+fatty+acid+metabolism+in+the+isolated+working+mouse+heart.">Glucose and fatty acid metabolism in the isolated working mouse heart.</a> Am J Physiol. 277, R1210-R1217 (1999).</li><li>Iannaccone, P. M., Jacob, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rats!+.">Rats! .</a> Dis Model Mech. 2, 206-210 (2009).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 (KH2PO4)</td>
			<td>Fisher Scientific</td>
			<td>P284</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate (MgSO4*7H2O)</td>
			<td>Fisher Scientific</td>
			<td>M63</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Fisher Scientific</td>
			<td>S233</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</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&#160; (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, [9,10-3H(N)]-</td>
			<td>PerkinElmer</td>
			<td>NET289005MC</td>
			<td>Radioactive material. Follow your Institution&#39;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&#39;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 14CO2</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 3H2O</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 CO2 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 &#34;hard to reach&#34; 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...

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 ...

The overall goal of this procedure is to obtain simultaneous real time measures of myocardial rates of glucose oxidation and fatty acid oxidation, as well as cardiac contractual function in an isolated working rat heart. This method can help answer key questions in the cardiovascular field. Such as how the heart metabolically adapts to acute changes in energy demands and how these responses affected in pathological conditions.The main advantage of this technique is that it provides the investigator total control over the environmental variables such as the nature and the concentrations of substrates, drugs, or hormones delivered to the heart. The most would be Bhavisha Bakrania a post doctoral fellow from the cardiovascular Reno resource center at the University of Mississippi Medical Center. To begin, start the pump to prime the perfusion apparatus and to oxygenate the buffer.After preparing the materials, anesthetize a rat and prepare it for surgery. Clip the abdomen free of hair and perform a mid line incision with lateral extension. Move the stomach and intestine aside to access the inferior vena cava and inject 200 USP units of Heparin.Wait five to 10 seconds, then using scissors cut the diaphragm and the sides of the rib cage to expose the contents of the chest cavity. Next, delicately grab the heart using the hand and excise both the heart and lungs together. Cut at the level of the descending aorta, being careful not to damage the aortic arch and ascending aorta in the process.Immediately transfer the heart and lungs to a dissection dish filled with ice cold KH buffer. After the heart stops beating, learn to work quickly so it can be restarted within a minute or two. Now submerge the heart and lungs in a new dish of ice cold KH buffer.Quickly trim off any large pieces of lung tissue, then cut the descending aorta right above the aortic arch. For the following steps, take the necessary precautions for use of radioactives. Next, using forceps carefully open the aorta and slide the heart up onto the cannula.Be very careful not pass the aortic route with a cannula, as this may cause hypoperfusion of the heart and damage to the aortic valves. Now, secure the junction with a micro clip and initiate the Lagendorff perfusion of the heart. Without rushing, secure the aorta to the aortic cannula using a 3 0 silk suture and remove the micro clip.Then locate the pulmonary vein and trim away non cardiac tissues as needed. Leave enough tissue to anchor the left atrium to the cannula. The cannulation of the left atrium is the second most critical step of the protocol as improper insertion of the cannula or the presence of a leak will weaken contractions and compromise the quantification of metabolic rates.Next, prepare the left atrial line cannula, just as done for the right cannula. Then with forceps, delicately grab the opening of the pulmonary vein and slide the heart up on the atrial cannula. Move the tissues as needed without bending the aorta or overly straining the heart.Then secure the cannula with a 3 0 silk suture. Finally, open the atrial line while simultaneously switching the aortic line to working mode. If buffer leaks out from the left atrium, close the atrial line and switch back to Lagendorff mode.Then use more sutures to better secure the cannula and try switching to working mode again. If the experiment includes direct measurement of LV function with a catheter, then begin by puncturing the LV apex using a 26 gauge needle, introduce a pressure volume catheter and align the shaft with the LV longitudinal axis. Then position the distal electrode E1 just below the aortic valve, adjacent to the endocardial border and position the proximal electrode E4 just inside the ventricular wall.Now seal the heart in a water jacketed chamber. Then start monitoring functional parameters in the software. Once baseline parameters have been stable for at least five minutes, start recording data.To measure the coronary flow, record the time it takes to collect 20 milliliters of effluent. Then take two millimeters of the effluent into a syringe via the three way stopcock and discard the remainder. Immediately transfer the two milliliter sample into a micro centrifuge tube and take a half milliliter aliqout to determine the rate of glucose oxidation.Store the rest on ice. Continue taking coronary effluence samples periodically and before finishing, inject 10 micro liters of hypertonic saline into the atrial line. Just before it attaches to the cannula.Use the response to this bolus to calculate the parallel conductance for accurate determination of cardiac volume. Next, unseal the heart chamber and remove the catheter in the LV.Then cut the heart off the cannulas and drop it into ice cold KH buffer. Now, quickly dry the heart on a paper towel and measure its wet weight.Once weighed, proceed to dissect the required tissue samples and then freeze the remaining tissue using tongs cooled on dry ice. In preparation, load glass scintillation vials with one milliliter of 10x hyamine hydroxide. Prepare one for each coronary effluence sample and two for controls.As soon as a sample vial is loaded, place it directly into a loaded scintillation vial. For the analysis, delicately cap the vial with a rubber sleeve stopper and use parafilm to seal the vial. Now, using a one milliliter syringe with a long 23 gauge needle, inject 200 micro liters of 60%perchloric acid through the rubber sleeve.Let all the sample vials sit overnight. The next day, carefully retrieve each sample tube with tweezers and wash their bottoms off over the open vials with one milliliter of scintillation cocktail to retrieve all of the hydroxide of hyamine. Then add nine more milliliters of scintillation cocktail to each vial.To get the specific activity, add 0.5 milliliters of profusion buffer into two of the vials. Now shake the vials vigorously and let the vial settle for at least six hours before taking measurements. To determine the myocardial oleate oxidation rates, first prepare an anionic exchange resin column for each sample.Just load the tips of three milliliters syringe tubes with glass wool. Then add the resin water slurry up to the two milliliter mark in each column using a transfer pipette. Next, wash the resin by passing two milliliters of ultra pure water through each column three times.Then place scintillation vials under the columns and load the columns with a half milliliter of coronary effluent. For the background measurement, use perfusion buffer instead. Now, elute the columns successively with one half milliliter, one milliliter, and finally two milliliters of ultra pure water.Discard the columns. Then add 13 milliliters of scintillation cocktail to the vials. Add 0.5 milliliters of perfusion buffer directly to two vials filled with 13 milliliters of liquid scintillation cocktail for determination of the specific activity.Vigorously shake the vials, let them settle for six plus hours and take measurements. Hearts were isolated from two 16 week rats and subjected to different stress conditions. For one heart, an acute increase in work load was simulated by increasing the after load by 40%and by adding epinephrine to the perfusion buffer.The heart rate immediately increased by more than 50%with the work jump induction. The cardiac power increased by more than 40%at the work jump, which was accompanied by an increase in both rates of glucose and oleate oxidation. The relative increase from base line for glucose oxidation was striking.The other heart was subjected to 15 minutes of global ischemia and 30 minutes of reperfusion. Under these experimental conditions, a near normal pulse pressure was restored a few minutes into the reperfusion. Within 10 minutes, cardiac power rapidly increased to above pre-ischemia values before returning to near baseline values.This resulted from increased oleate oxidation. Where as glucose oxidation was quick to resume to pre-ischemic activity. Once mastered, isolation of the heart to the initial reperfusion in the working mode can be done in less than five minutes if performed properly.Following this procedure, other methods such as RNA and protein expression analyses can be done in order to answer additional questions, such as the effect of a drug or stress condition on molecular signaling pathways. Don't forget that working with radio tracers can be extremely hazardous and precautions such as wearing full personal protective equipments should be always taken while performing this procedure.

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JoVE Journal

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14.5K vues.  University of Mississippi Medical Center. The overall goal of this procedure is to obtain simultaneous real time measures of myocardial rates of glucose oxidation and fatty acid oxidation, as well as cardiac contractual function in an isolated working rat heart. This method can help answer key questions in the cardiovascular field. Such as how the heart metabolically adapts to acute changes in energy demands and how these responses affected in pathological conditions.The main advantage of this technique is that it provides the...

Vidéo: 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...

investigating cardiac metabolism in the isolated perfused mouse heart with hyperpolarized [1-13c]pyruvate and 13c/31p nmr spectroscopy

Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

We describe an experimental setup for administrating hyperpolarized 13C-labeled metabolites in continuous perfusion mode to an isolated perfused mouse heart. A dedicated 13C-NMR acquisition approach enabled the quantification of metabolic enzyme activity in real-time, and a multiparametric 31P-NMR analysis enabled the determination of the tissue ATP content and...

Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy

determination of fatty acid oxidation and lipogenesis in mouse primary hepatocytes

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and...

measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated mouse hepatocytes

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

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...

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

evaluation of fatty acid oxidation in cultured bone cells

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells

The present protocol describes an assay to assess the capacity for fatty acid oxidation in cultures of primary bone cells or relevant cell lines.

the inverted heart model for interstitial transudate collection from the isolated rat heart

The Inverted Heart Model for Interstitial Transudate Collection from the Isolated Rat Heart

This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.

using an isolated working rat heart model to test donor heart preservation strategies

Author Spotlight: Enhancing Donor Heart Preservation Through Isolated Rat Heart Perfusion Studies

We describe an ex vivo isolated working rat heart protocol to test donor heart preservation strategies. This paper describes the protocol for use in static cold storage of rodent donor hearts; however, the protocol can also be used for donor hearts obtained after donation after circulatory death and brain death.

Using an Isolated Working Rat Heart Model to Test Donor Heart Preservation Strategies

an isolated working heart system for large animal models

An Isolated Working Heart System for Large Animal Models

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data...

Optimization of Donor Heart Preservation Using a Working Rat Heart Perfusion Model

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quantitative determination of de novo fatty acid synthesis in brown adipose tissue using deuterium oxide

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Here, we present an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo lipogenesis in brown adipose tissue in...

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

rodent working heart model for the study of myocardial performance and oxygen consumption

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various...

optimized incorporation of alkynyl fatty acid analogs for the detection of fatty acylated proteins using click chemistry

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass...

isolation of rat heart for testing donor heart preservation methods

Isolation of Rat Heart for Testing Donor Heart Preservation Methods

semi-minimal invasive method to induce myocardial infarction in rats and the assessment of cardiac function by an isolated working heart system

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart...

analysis of fatty acid content and composition in microalgae

Analysis of Fatty Acid Content and Composition in Microalgae

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete...

determination of the excitation and coupling rates between light emitters and surface plasmon polaritons

Determination of the Excitation and Coupling Rates Between Light Emitters and Surface Plasmon Polaritons

This protocol describes the instrumentation for determining the excitation and coupling rates between light emitters and Bloch-like surface plasmon polaritons arising from periodic arrays.

alternative in vitro methods for the determination of viral capsid structural integrity

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

nadh fluorescence imaging of isolated biventricular working rabbit hearts

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

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...

implantation of engineered tissue in the rat heart

Implantation of Engineered Tissue in the Rat Heart

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis...

quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in...

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an impending predator attack.  Although it is not known whether invertebrates posses an autonomic nervous system identical to that of vertebrates, evidence shows invertebrates have a  sympathetic-like  response to regulate the internal environment and ready the organism to act behaviorally to a given stimuli.  Furthermore, this physiological response can be feasibly measured and it acts as a biological index for the animal's internal state.  Measurements of the physiological response can be directly related to internal and external stressors through changes in the central nervous system controlled coordination of the cardio-vascular and respiratory systems.   More specifically, monitoring heart and ventilation rates provide quantifiable measures of the stress response not always behaviorally observed. Crayfish are good model organisms for heart and ventilatory rate measurements due to the feasibility of recording, as well as the rich history known of the morphology of the crayfish, dating back to Huxley in 1888, and the well-studied typical behaviors.

Invertebrates show an autonomic sympathetic-like response similar to that described for vertebrates. The coordination of the cardio-vascular and ventilatory systems allows for measurement of a biological index in which to assess an organism internal state.

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

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 ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.