This work was supported by grants from the Canadian National Transplant Research Program. SH is the recipient of a Faculty of Medicine and Dentistry Motyl Graduate Studentship in Cardiac Sciences. DHF is a recipient of a Collaborative Research Projects (CHRP) grant in aid from the National Sciences and Engineering Research Council and Canadian Institutes of Health Research.

<ol>
	<li>Ardehali, A., 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=Ex-vivo+perfusion+of+donor+hearts+for+human+heart+transplantation+(PROCEED+II):+a+prospective,+open-label,+multicentre,+randomised+non-inferiority+trial.">Ex-vivo perfusion of donor hearts for human heart transplantation (PROCEED II): a prospective, open-label, multicentre, randomised non-inferiority trial.</a> Lancet. 385 (9987), 2577-2584 (2015).</li><li>Collins, M. J., Moainie, S. L., Griffith, B. P., Poston, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+and+evaluating+hearts+with+ex+vivo+machine+perfusion:+An+avenue+to+improve+early+graft+performance+and+expand+the+donor+pool.">Preserving and evaluating hearts with ex vivo machine perfusion: An avenue to improve early graft performance and expand the donor pool.</a> European Journal of Cardiothoracic Surgery. 34 (2), 318-325 (2008).</li><li>Freed, D. H., White, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+heart+preservation:+Straight+up,+or+on+the+rocks?.">Donor heart preservation: Straight up, or on the rocks?.</a> Lancet. 385 (9987), 2552-2554 (2015).</li><li>Guibert, E. 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=Organ+preservation:+Current+concepts+and+new+strategies+for+the+next+decade.">Organ preservation: Current concepts and new strategies for the next decade.</a> Transfusion Medicine and Hemotherapy. 38 (2), 125-142 (2011).</li><li>Collins, M. J., 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=Use+of+diffusion+tensor+imaging+to+predict+myocardial+viability+after+warm+global+ischemia:+Possible+avenue+for+use+of+non-beating+donor+hearts.">Use of diffusion tensor imaging to predict myocardial viability after warm global ischemia: Possible avenue for use of non-beating donor hearts.</a> Journal of Heart and Lung Transplantation. 26 (4), 376-383 (2007).</li><li>White, C. W., 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=A+cardioprotective+preservation+strategy+employing+ex+vivo+heart+perfusion+facilitates+successful+transplant+of+donor+hearts+after+cardiocirculatory+death.">A cardioprotective preservation strategy employing ex vivo heart perfusion facilitates successful transplant of donor hearts after cardiocirculatory death.</a> Journal of Heart and Lung Transplantation. 32 (7), 734-743 (2013).</li><li>Iyer, A., 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=Normothermic+ex+vivo+perfusion+provides+superior+organ+preservation+and+enables+viability+assessment+of+hearts+from+DCD+donors.">Normothermic ex vivo perfusion provides superior organ preservation and enables viability assessment of hearts from DCD donors.</a> American Journal of Transplantation. 15 (2), 371-380 (2015).</li><li>Peltz, M., 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=Perfusion+preservation+maintains+myocardial+ATP+levels+and+reduces+apoptosis+in+an+ex+vivo+rat+heart+transplantation+model.">Perfusion preservation maintains myocardial ATP levels and reduces apoptosis in an ex vivo rat heart transplantation model.</a> Surgery. 138 (4), 795-805 (2005).</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> American Journal of Physiology-Heart and Circulatory Physiology. 303 (2), H156-H167 (2012).</li><li>Rivard, L., Gallegos, R., Ogden, I., Bianco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+Preservation+of+the+Donor+Heart:+Basic+Science+to+Pre-Clinical.">Perfusion Preservation of the Donor Heart: Basic Science to Pre-Clinical.</a> Journal of Extra Corporeal Technology. 41 (3), 140-148 (2009).</li><li>Dhital, K. K., 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=Adult+heart+transplantation+with+distant+procurement+and+ex+vivo+preservation+of+donor+hearts+after+circulatory+death:+A+case+series.">Adult heart transplantation with distant procurement and ex vivo preservation of donor hearts after circulatory death: A case series.</a> Lancet. 385 (9987), 2585-2591 (2015).</li><li>Messer, S., Ardehali, A., Tsui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normothermic+donor+heart+perfusion:+Current+clinical+experience+and+the+future.">Normothermic donor heart perfusion: Current clinical experience and the future.</a> Transplant International. 28 (6), 634-642 (2015).</li><li>White, C. W., 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=Assessment+of+donor+heart+viability+during+ex+vivo+heart+perfusion.">Assessment of donor heart viability during ex vivo heart perfusion.</a> Canadian Journal of Physiology and Pharmacology. 93 (10), 893-901 (2015).</li><li>Messer, S. J., 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=Functional+assessment+and+transplantation+of+the+donor+heart+after+circulatory+death.">Functional assessment and transplantation of the donor heart after circulatory death.</a> Journal of Heart and Lung Transplantation. 35 (12), 1443-1452 (2016).</li><li>Hatami, 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=Endoplasmic+reticulum+stress+in+ex+vivo+heart+prfusion:+A+comparison+between+working+vs+non-working+modes.">Endoplasmic reticulum stress in ex vivo heart prfusion: A comparison between working vs non-working modes.</a> Canadian Journal of cardiology. 33 (10), (2017).</li><li>White, C. W., 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=Ex+vivo+perfusion+in+a+loaded+state+improves+the+preservation+of+donor+heart+function.">Ex vivo perfusion in a loaded state improves the preservation of donor heart function.</a> Canadian Journal of cardiology. 31 (10), s202 (2015).</li><li>White, C. W., 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=A+wholeblood-based+perfusate+provides+superior+preservation+of+myocardial+function+during+ex+vivo+heart+perfusion.">A wholeblood-based perfusate provides superior preservation of myocardial function during ex vivo heart perfusion.</a> Journal of Heart and Lung Transplantation. (14), (2014).</li><li>Lips, D. J., 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=Left+ventricular+pressure-volume+measurements+in+mice:+comparison+of+closed-chest+versus+open-chest+approach.">Left ventricular pressure-volume measurements in mice: comparison of closed-chest versus open-chest approach.</a> Basic Research in Cardiology. 99 (5), 351-359 (2004).</li><li>Morita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+there+a+crystal+ball+for+predicting+the+outcome+of+cardiomyopathy+surgery?+Preload+recruitable+stroke+work,+may+be+a+possible+candidate.">Is there a crystal ball for predicting the outcome of cardiomyopathy surgery? Preload recruitable stroke work, may be a possible candidate.</a> Journal of Cardiology. 71 (4), 325-326 (2018).</li><li>Hatami, 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=.">.</a> Canadian Society for Transplantation. , (2017).</li><li>Anthony, C., 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=Ex+vivo+coronary+angiographic+evaluation+of+a+beating+donor+heart.">Ex vivo coronary angiographic evaluation of a beating donor heart.</a> Circulation. 130 (25), e341-e343 (2014).</li><li>Sandha, J. K., 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=Steroids+Limit+Myocardial+Edema+During+Ex+vivo+Perfusion+Of+Hearts+Donated+After+Circulatory+Death.">Steroids Limit Myocardial Edema During Ex vivo Perfusion Of Hearts Donated After Circulatory Death.</a> Annals of Thoracic Surgery. , (2018).</li></ol>

DHF holds patents on ex situ organ perfusion technology and methods. DHF and JN are founders and major shareholders of Tevosol, Inc.

Successful perfusion is defined according to the aims of the study; however, this should include uninterrupted ESHP for the desired amount of time and complete collection of the data on cardiac function during the perfusion. For this purpose, a few critical steps in the protocol must be followed.
The heart is an organ with high oxygen and energy demands, and minimizing the ischemic time before cannulation and perfusion is an important principle that must be followed. The process of procurement...

Clinical relevance
While most aspects of cardiac transplantation have evolved significantly since the first heart transplant in 1967, cold storage (CS) remains the standard for donor heart preservation1. CS exposes the organ to a period of cold ischemia that limits the safe preservation interval (4&#8211;6 hours) and increases the risk of primary graft dysfunction2,3,4. Due to the static nature of CS, assessments of function or therapeutic interventions are not possible in the time between the organ procurement and transplantation. This is a particular limitation in extended criteria donors including hearts donated after circulatory death (DCD), creating an obstacle for overcoming the considerable gap between demand and the current donor pool5,6. To address this limitation, ex situ heart perfusion has been proposed as a novel, semi-physiologic method of preserving donated hearts, minimizing exposure to cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart during preservation time1,7,8.
Ex situ heart perfusion
One of the most frequently used methods for ex situ examination of the isolated heart is Langendorff perfusion. In this method, introduced by Oskar Langendorff in 1895, the blood flows into the coronary arteries and out the coronary sinus of the isolated heart, with the heart in an empty and beating state9,10. Clinical ESHP in a Langendorff mode with the Transmedics Organ Care System apparatus (OCS) has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts1, and has facilitated the clinical transplantation of DCD hearts11. However, there are concerns about the ability of the device to evaluate organ viability, as a number of donor hearts initially thought to be transplantable were discarded after perfusion on the OCS3. The OCS supports the heart in the Langendorff (non-working) mode, and thus possesses a limited capacity for evaluation of the pumping function of the heart3,12. A growing body of evidence suggests that functional parameters offer a better way to assess organ viability, suggesting that assessments of cardiac function may become a reliable tool for the evaluation and selection of hearts for transplantation during ESHP3,12,13,14, Furthermore, our studies on ex situ perfused porcine hearts suggest that ESHP in working mode provides enhanced functional preservation of the heart during the perfusion interval15,16.
An ESHP apparatus capable of preserving the heart in a working mode must possess a level of automation to safely and precisely maintain preload, afterload and flow rates. Also, such a system should possess the flexibility to facilitate comprehensive assessments of cardiac function to be undertaken. The ESHP apparatus used here is equipped with custom software that 1) provides and maintains desired aortic (Ao) and left atrial (LA) pressure/flow and 2) provides real-time analysis of functional parameters and visual evaluation of pressure waveforms with minimal need for supervision. Pressure data is acquired with standard fluid-filled pressure transducers, and flow data is acquired with transit-time doppler flow probes. These signals are digitized with a bridge and analog input, respectively. The heart is positioned horizontally with a slight elevation to the great vessels on a soft silicone membrane. The cannulation attachments pass through the membrane, incorporating a compliance chamber for dampening ventricular ejection. The goal of this work is to provide researchers in the field of cardiac transplantation with a protocol for ex situ perfusion and evaluation of the heart, under normothermic, semi-physiologic conditions in working mode, in a large mammal (Yorkshire pig) model.

<table><tbody><tr><td>Debakey-Metzenbaum dissecting scissors</td><td>Pilling</td><td>342202</td><td></td></tr><tr><td>MAYO dissecting scissors</td><td>Pilling</td><td>460420</td><td></td></tr><tr><td>THUMB forceps</td><td>Pilling</td><td>465165</td><td></td></tr><tr><td>Debakey straight vascular tissue forceps&amp;nbsp;</td><td>Pilling</td><td>351808</td><td></td></tr><tr><td>CUSHING Gutschdressing forceps</td><td>Pilling</td><td>466200</td><td></td></tr><tr><td>JOHNSON needle holder</td><td>Pilling</td><td>510312</td><td></td></tr><tr><td>DERF needle holder</td><td>Pilling</td><td>443120</td><td></td></tr><tr><td>Sternal saw</td><td>Stryker</td><td>6207</td><td></td></tr><tr><td>Sternal retractor</td><td>Pilling</td><td>341162</td><td></td></tr><tr><td>Vorse tubing clamp</td><td>Pilling</td><td>351377</td><td></td></tr><tr><td>MORRIS ascending aorta clamp</td><td>Pilling</td><td>353617</td><td></td></tr><tr><td>Surgical snare (tourniquet) set</td><td>Medtronic</td><td>CVR79013</td><td></td></tr><tr><td>2-0 SILK black 12&quot; x 18&quot; strands</td><td>ETHICON</td><td>A185H</td><td></td></tr><tr><td>3-0 PROLENE blue 18&quot; PS-2 cutting</td><td>ETHICON</td><td>8687H</td><td></td></tr><tr><td>Biomedicus pump drive (modified)</td><td>Medtronic</td><td>540</td><td>Modified to allow remote electronic control of pump speed</td></tr><tr><td>Biomedicus pump</td><td>Maquet</td><td>BPX-80</td><td></td></tr><tr><td>Membrane oxigenator D 905</td><td>SORIN GROUP</td><td>50513</td><td></td></tr><tr><td>Tubing flow module&amp;nbsp;&amp;nbsp;</td><td>Transonic</td><td>Ts410</td><td></td></tr><tr><td>PXL clamp-on flow sensor</td><td>Transonic</td><td>ME9PXL-BL37SF</td><td></td></tr><tr><td>TruWave pressure transducer</td><td>Edwards</td><td>VSYPX272</td><td></td></tr><tr><td>Intercept tubing 3/8&quot; x 3/32&quot; xX 6&#039;</td><td>Medtronic</td><td>3506</td><td></td></tr><tr><td>Intercept tubing 1/4&quot; x 1/16&quot; x 8&#039;</td><td>Medtronic</td><td>3108</td><td></td></tr><tr><td>Heated/Refrigerated Bath Circulator&amp;nbsp;</td><td>Grant</td><td>TX-150</td><td></td></tr><tr><td>ABL 800 FLEX Blood Gas Analyzer</td><td>Radiometer</td><td>989-963</td><td></td></tr><tr><td>DLP cardioplegia cannula (aortic root cannula)</td><td>Medtronics</td><td>20613994495406</td><td></td></tr><tr><td>5F Ventriculr straight pigtail cathter</td><td>CORDIS</td><td>534550S</td><td></td></tr><tr><td>5F AVANTI+ Sheath Introducer</td><td>CORDIS</td><td>504605A</td><td></td></tr><tr><td>Emerald Amplatz Guidewire</td><td>CORDIS</td><td>502571A</td><td></td></tr><tr><td>Dual chamber pace maker</td><td>Medtronic</td><td>5388</td><td></td></tr><tr><td>Defibrilltor</td><td>CodeMaster</td><td>M1722B</td><td></td></tr><tr><td>Infusion pump</td><td>Baxter</td><td>AS50</td><td></td></tr><tr><td>Surgical electrocautery device</td><td>Kls Martin</td><td>ME411</td><td></td></tr><tr><td>Gas mixer</td><td>SECHRIST</td><td>3500 CP-G</td><td></td></tr><tr><td>Medical oxygen tank</td><td>praxair</td><td>2014408</td><td></td></tr><tr><td>Cabon dioxide tank</td><td>praxair</td><td>5823115</td><td></td></tr><tr><td>Bovine serum albumin</td><td>MP biomedicals</td><td>218057791</td><td></td></tr></tbody></table>

normothermic ex situ heart perfusion in working mode: assessment of cardiac function and metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

At the start of the perfusion (in non-working mode), the heart will normally resume a sinus rhythm when the temperature of the system and perfusate approaches normothermia. When entering working mode, as the LA pressures are approaching the desired values, ejection on the Ao pressure tracing should be observed and the LA flow (a reflection of cardiac output) should increase gradually. In a Yorkshire pig model (35&#8211;50 kg) and a starting heart weight of 180&#8211;220 grams, the initial...

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Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

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13.0K Views.  University of Alberta. Deze methode kan helpen bij het beantwoorden van belangrijke vragen over het optimaal behoud van donorharten, evenals hun functionele en metabole evaluatie voor latere transplantatie geschiktheid. Het belangrijkste voordeel van deze techniek is het mogelijk maakt een functionele evaluatie van het donorhart dat niet alleen is gebaseerd op indirecte metabole beoordelingen. De implicaties van deze techniek breiden zich uit tot de therapie van disfunctionele donorharten, omdat de hartfunctie in d...

Video: Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

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

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

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 methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

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.

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 ...

Biology

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the allocation of a growing number of donor hearts with extended criteria. These marginal grafts are associated with a high risk of primary graft failure and may benefit from ex situ perfusion before transplant. This technology allows for extended organ preservation using warm oxygenated blood perfusion with continuous metabolic monitoring. The only NESP device currently available for clinical practice perfuses the organ in an unloaded non-working state, which does not allow for functional assessment of the beating heart. We therefore developed an original platform of NESP in working mode conditions with adjustment of left ventricular preload and afterload. This protocol was applied in porcine hearts. Ex situ functional assessment of the heart was achieved with intracardiac conductance catheterization and surface echocardiography. Along with a description of the experimental protocol, we herein report the main results, as well as pearls and pitfalls associated with the acquisition of pressure-volume loops and myocardial power during NESP. Correlations between hemodynamic findings and ultrasound variables are of major interest, especially for further rehabilitation of donor hearts before transplantation. This protocol aims to improve the assessment of donor hearts to both increase the donor pool and reduce the incidence of primary graft failure.

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

A reliable noninvasive approach for functional assessment of the donor heart during normothermic ex situ heart perfusion (NESP) is lacking. We herein describe a protocol for ex situ assessment of myocardial performance using the epicardial echocardiography and conductance catheter method.

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

Enhanced Cardiac Assessment in Ex Vivo Perfusion with Left Ventricular Loading

Cardiac Loading using Passive Left Atrial Pressurization and Passive Afterload for Graft Assessment

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. Despite the immense opportunity for assessment due to the beating state of the heart, current clinical practice depends on limited metabolic trends for graft evaluation. Hemodynamic measurements obtained from left ventricular loading have garnered significant attention within the field due to their potential as objective assessment parameters. In effect, this protocol provides an easy and effective manner of incorporating loading capabilities to established Langendorff perfusion systems through the simple addition of an extra reservoir. Furthermore, it demonstrates the feasibility of employing passive left atrial pressurization for loading, an approach that, to our knowledge, has not been previously demonstrated. This approach is complemented by a passive Windkessel base afterload, which acts as a compliance chamber to maximize myocardial perfusion during diastole. Lastly, it highlights the capability of capturing functional metrics during cardiac loading, including left ventricular pulse pressure, contractility, and relaxation, to uncover deficiencies in cardiac graft function after extended periods of preservation times (&#707;6 h).

Author Spotlight: Enhancing Graft Viability Assessment Through Quantitative Metrics and Innovative Reservoir Systems

The protocol describes a porcine ex vivo heart perfusion system in which direct loading of the left ventricle may serve as an assessment technique for graft health while simultaneously providing a holistic evaluation of graft function. A discussion of the system design and possible assessment metrics is also provided.

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. ...

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

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial information for understanding the mechanism of diseases and designing treatments. In this work, we describe procedures and methodologies for studying in-cell metabolic activity in a retrogradely perfused mouse heart in real-time. The heart was isolated in situ,&#160;in conjunction with cardiac arrest to minimize the myocardial ischemia and was perfused inside a nuclear magnetic resonance (NMR) spectrometer. While in the spectrometer and under continuous perfusion, hyperpolarized [1-13C]pyruvate was administered to the heart, and the subsequent hyperpolarized [1-13C]lactate and [13C]bicarbonate production rates served to determine, in real-time, the rates of lactate dehydrogenase and pyruvate dehydrogenase production. This metabolic activity of hyperpolarized [1-13C]pyruvate was quantified with NMR spectroscopy in a model free-manner using the product selective saturating-excitations acquisition approach. 31P spectroscopy was applied in between the hyperpolarized acquisitions to monitor the cardiac energetics and pH. This system is uniquely useful for studying metabolic activity in the healthy and diseased mouse heart.

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 pH.

Metabolism is the basis of important processes in cellular life. Characterizing how metabolic networks function in living tissues provides crucial ...

Optogenetic Activation of Intrinsic Cardiac Autonomic Neurons in Excised Perfused Mouse Hearts

A balance of cholinergic and catecholaminergic activation is necessary to maintain heart health. Interrogating the interaction between these pathways can be done using optogenetics through selective expression of channelrhodopsin-2 (ChR2) in cardiac autonomic neurons. Such cardiac applications of optogenetics allow for the study of the intrinsic release of neurotransmitters in a spatiotemporal manner. This method illustrates an ex vivo approach for specific optogenetic stimulation of cardiac neurons in perfused mouse hearts. Transgenic mice were bred to express ChR2 in either choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH) neurons throughout the body. A micro-LED (465 nm) encased in a silicone elastomer was prepared for stimulating the neurons of the right atrium that innervate the sinoatrial node. The micro-LED was connected to a function generator set to pulse waves at 10 Hz with a 30 ms pulse width. Hearts with confirmed expression were excised and retrogradely perfused on a Langendorff system circulating Krebs-Henseleit solution. Electrocardiogram (ECG), temperature, and coronary flow rate were recorded using the LabChart software. Once the heart stabilized, the micro-LED was placed on the right atrium and tested for optimal heart rate response. An application of this approach combines the intrinsic release of cholinergic neurotransmitter (acetylcholine) during optogenetic activation of a ChAT-ChR2 mouse heart simultaneously with increasing exogenous catecholaminergic neurotransmitter (norepinephrine) added to the perfusate. The resulting changes in heart rate during the simultaneous cholinergic and catecholaminergic activation are presented. This method describes a valuable experimental approach for investigating the kinetics of sudden intrinsic autonomic neuron activation in perfused hearts and the interactions between cardiac cholinergic and catecholaminergic activity.

This protocol illustrates a method for optogenetic stimulation of intrinsic cardiac neurons in transgenic mouse hearts. The approach described is used for investigating the kinetics of sudden cardiac neuron activation in ex vivo perfused hearts and the interactions between cholinergic and catecholaminergic activity.

A balance of cholinergic and catecholaminergic activation is necessary to maintain heart health. Interrogating the interaction between these pathways can ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

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