The studies described here have been funded by grants awarded to P.S Macdonald from the National Health and Medical Research Council Australia, the St Vincent&#39;s Clinic Foundation, and the NSW Health Cardiovascular Research Capacity Senior Investigator Grant.

All animals received humane care in compliance with the National Health and Medical Research Council (Australia) guidelines. All animal procedures were approved by the Animal Ethics Committee of the Garvan Institute of Medical Research (Sydney, Australia).
1. Preparation of Krebs-Henseleit (KH) buffer
<ol>
	<li>Prepare 2 L of a 20x stock solution of KH buffer containing 2.36 M NaCl, 0.094 M KCl, 0.024 M MgSO4, 0.024 M KH2PO4. Aliquot into 50 ...

Optimization of Donor Heart Preservation Using Working Rat Heart Perfusion Model

<ol>
	<li>Kwan, J. C., Gao, L., Macdonald, P. S., Hicks, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardio-protective+signalling+by+glyceryl+trinitrate+and+cariporide+in+a+model+of+donor+heart+preservation.">Cardio-protective signalling by glyceryl trinitrate and cariporide in a model of donor heart preservation.</a> Heart Lung Circ. 24 (3), 306-318 (2015).</li><li>Watson, A. 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=Enhanced+preservation+of+the+rat+heart+after+prolonged+hypothermic+ischemia+with+erythropoietin-supplemented+Celsior+solution.">Enhanced preservation of the rat heart after prolonged hypothermic ischemia with erythropoietin-supplemented Celsior solution.</a> J Heart Lung Transplant. 32 (6), 633-640 (2013).</li><li>Chew, H. 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=Outcomes+of+donation+after+circulatory+death+heart+transplantation+in+Australia.">Outcomes of donation after circulatory death heart transplantation in Australia.</a> J Am Coll Cardiol. 73 (12), 1447-1459 (2019).</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>Joshi, Y., 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=Heart+transplantation+from+DCD+donors+in+Australia:+Lessons+learned+from+the+first+74+cases.">Heart transplantation from DCD donors in Australia: Lessons learned from the first 74 cases.</a> Transplantation. 107 (2), 361-371 (2023).</li><li>Villanueva, J. 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=Functional+recovery+after+dantrolene-supplementation+of+cold+stored+hearts+using+an+ex+vivo+isolated+working+rat+heart+model.">Functional recovery after dantrolene-supplementation of cold stored hearts using an ex vivo isolated working rat heart model.</a> PLoS One. 13 (10), e205850 (2018).</li><li>Villanueva, J. 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+cardioprotective+potential+of+the+sodium-glucose+cotransporter+2+inhibitor+empagliflozin+in+donor+heart+preservation.">The cardioprotective potential of the sodium-glucose cotransporter 2 inhibitor empagliflozin in donor heart preservation.</a> J Heart Lung Transplant. 39 (10), 1151-1153 (2020).</li><li>Gao, L., 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=Critical+role+of+the+STAT3+pathway+in+the+cardioprotective+efficacy+of+zoniporide+in+a+model+of+myocardial+preservation+-+the+rat+isolated+working+heart.">Critical role of the STAT3 pathway in the cardioprotective efficacy of zoniporide in a model of myocardial preservation - the rat isolated working heart.</a> Br J Pharmacol. 162 (3), 633-647 (2011).</li><li>Cropper, J. R., Hicks, M., Ryan, J. B., Macdonald, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotection+by+cariporide+after+prolonged+hypothermic+storage+of+the+isolated+working+rat+heart.">Cardioprotection by cariporide after prolonged hypothermic storage of the isolated working rat heart.</a> J Heart Lung Transplant. 22 (8), 929-936 (2003).</li><li>Gao, L., 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=Enhanced+functional+recovery+of+the+heart+donated+after+circulatory+death+determination+with+antemortem+heparin.">Enhanced functional recovery of the heart donated after circulatory death determination with antemortem heparin.</a> J Heart Lung Transplant. 39 (6), 607-609 (2020).</li><li>Villanueva, J. 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+effect+of+increasing+donor+age+on+myocardial+ischemic+tolerance+in+a+rodent+model+of+donation+after+circulatory+death.">The effect of increasing donor age on myocardial ischemic tolerance in a rodent model of donation after circulatory death.</a> Transplant Direct. 7 (6), e699 (2021).</li><li>Kumarasinghe, G., 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=Improved+heart+function+from+older+donors+using+pharmacologic+conditioning+strategies.">Improved heart function from older donors using pharmacologic conditioning strategies.</a> J Heart Lung Transplant. 35 (5), 636-646 (2016).</li></ol>

Given the sensitive nature of heart baseline function, care must be taken to maintain the perfusion rig clean and with compatible components. For example, the correct PVC tubing must be used. Some tubing materials, such as silicone, can contribute to lower aortic flow and contractility, which may be due to loss of oxygen via the tubing. While there are isolated working rat heart perfusion circuits commercially available, most of these are primarily used for Langendorff perfusion only. Generally, functional measurements c...

The success of a heart transplant can be greatly influenced by the preservation method used for the donor heart. Donor hearts are subjected to ischemic insults throughout the organ retrieval, transport, and transplantation process. The degree of ischemic damage to the donor organ can be mitigated by selecting an appropriate donor heart preservation strategy. Cold static storage (CSS) remains the most feasible and common method for donor heart preservation; however, CSS may not necessarily be the best option for all heart donation pathways. For example, hearts procured via the donation after circulatory death (DCD) pathway are maintained and assessed for viability using normothermic machine perfusion, whereas hearts procured via the donation after brain death (DBD) pathway can be preserved using CSS or hypothermic machine perfusion. Another important determinant for the preservation strategy used is the amount of either warm ischemia (for DCD) or cold ischemia (for DBD) the heart is subjected to during organ procurement and transportation.
Many centers use additional pharmacological supplementation of the heart preservation solution to increase ischemic tolerance and improve post-transplant heart function. Rodent studies from our laboratory showed that supplementation of cardioplegia with erythropoietin and glyceryl trinitrate was able to significantly improve cardiac functional recovery after prolonged cold storage of the donor heart1,2. These studies then progressed to porcine studies of donor heart preservation and provided the pre-clinical groundwork that led to the incorporation of erythropoietin and glyceryl trinitrate into the cardioplegia solution of hearts procured via a DCD pathway utilizing normothermic machine perfusion3,4,5. Currently, supplementation with erythropoietin and glyceryl trinitrate is also used in Australian transplant units for clinical DBD heart retrievals incorporating CSS. The overall goal of the isolated working rat heart (IWRH) perfusion protocol outlined below is to test donor heart preservation strategies in a laboratory setting and test their efficacy for preserving donor heart function after reperfusion. The isolated working rat heart protocol described has been used for over two decades within our laboratory and has proven extremely useful in screening for potential supplements and/or strategies that can improve donor heart preservation2,7,8,9.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL colorless Eppendorff tube, 1000 per box</td>
			<td>Bio Strategy Pty Ltd</td>
			<td>0030.125.150</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL cent/tubes (S) Sleeve/25 PP ctn/500</td>
			<td>Sigma-aldrich Pty Ltd</td>
			<td>CLS430791-500ea</td>
			<td></td>
		</tr>
		<tr>
			<td>BP Transducer/Cable kit</td>
			<td>ADInstruments</td>
			<td>MLT1199</td>
			<td>Data Acquisition</td>
		</tr>
		<tr>
			<td>Bridge Amp</td>
			<td>ADInstruments</td>
			<td>FE221</td>
			<td>Data Acquisition</td>
		</tr>
		<tr>
			<td>Carbogen 555G2</td>
			<td>BOC</td>
			<td>BOC002</td>
			<td></td>
		</tr>
		<tr>
			<td>Checktemp1 thermometer</td>
			<td>Hanna Instruments</td>
			<td>HI98509</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Clamp Pinch 1/4-7/16 PK 12</td>
			<td>Thomas Scientific</td>
			<td>2848Y40</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Clamp W/Extension Stem Med.</td>
			<td>Thomas Scientific</td>
			<td>8847T08</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Clamp W/Extension Stem Sm.</td>
			<td>Thomas Scientific</td>
			<td>8847T02</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Clips, Vessel, 60 g Pressure</td>
			<td>Coherent Scientific</td>
			<td>14121</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Closed Connector</td>
			<td>Thomas Scientific</td>
			<td>8847E25</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Covidien Sofsilk 2-0 black precut 45 cm box of 24</td>
			<td>Specialist Medical Supplies</td>
			<td>S195</td>
			<td>Surgical consumables</td>
		</tr>
		<tr>
			<td>Custom Made 250 mL Jacketed Degasser</td>
			<td>Custom Blown Glassware Pty Ltd</td>
			<td>N/A</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Custom Made 750 mL Reservoir</td>
			<td>Custom Blown Glassware Pty Ltd</td>
			<td>N/A</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose Anhydrous SigmaUltra</td>
			<td>Sigma-aldrich Pty Ltd</td>
			<td>G7528-1Kg</td>
			<td>To make Krebs Buffer</td>
		</tr>
		<tr>
			<td>Dual Channel Console</td>
			<td>ADInstruments</td>
			<td>TS402</td>
			<td>Data Acquisition</td>
		</tr>
		<tr>
			<td>Erlenmyer flasks 2 L&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Microfibre type GF/C glass fibre 47 mm, 100</td>
			<td>Bio-strategy Pty Ltd</td>
			<td>1822047</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, 15 cm, 0.3 mm, CRVD</td>
			<td>Coherent Scientific</td>
			<td>14114</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Four Prong Clamps with 9 mm x 115 mm long arm for holding 2-70 mm diam objects. Vinyl coated</td>
			<td>Met-App Australia Pty Ltd</td>
			<td>1352</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Heater Circulator. Digital Solid State Control. (1020 Watts/240 Volts)</td>
			<td>Thermoline Scientific</td>
			<td>TU3</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Heparin 5000 U/5 mL box 50 Pfizer 02112115</td>
			<td>Clffird Hallam Healthcare Pty Ltd</td>
			<td>1258693</td>
			<td>Drugs</td>
		</tr>
		<tr>
			<td>Ilium Xylazil 20 Inj 50 mL</td>
			<td>Cenvet Australia Pty Ltd</td>
			<td>X5010</td>
			<td>Anaesthetic</td>
		</tr>
		<tr>
			<td>Johns Hopkins Bulldog Clamp</td>
			<td>Coherent Scientific&#160;</td>
			<td>CS-WPI-14117</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 100 mg/50 mL</td>
			<td>Provet (NSW) Pty Ltd</td>
			<td>KETAI1</td>
			<td>Anaesthetic</td>
		</tr>
		<tr>
			<td>Magnesium Sulphate heptahydrate AR 500 g Chemsupply</td>
			<td>Bio Strategy Pty Ltd</td>
			<td>MA048-500g</td>
			<td>To make Krebs Buffer</td>
		</tr>
		<tr>
			<td>Male/Female Hinged Adapter</td>
			<td>Thomas Scientific</td>
			<td>8847V08</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Masterflex L/S Easy-Load Head for Precision Tubing, PPS, CRS Rotor</td>
			<td>John Morris</td>
			<td>1015164</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Metzenbaum scissors, 11.5 cm curved</td>
			<td>Coherent Scientific</td>
			<td>WPI-501748</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Metzenbaum scissors, 14.5 cm straight</td>
			<td>Coherent Scientific</td>
			<td>WPI-501252</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Mounting Hardware F/2-HEADS SS</td>
			<td>John Morris</td>
			<td>1014414</td>
			<td>Mounting screws for pump heads</td>
		</tr>
		<tr>
			<td>Open-sided connector</td>
			<td>Thomas Scientific</td>
			<td>8847E05</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Paraformadehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148-500G</td>
			<td>Sample processing</td>
		</tr>
		<tr>
			<td>Potassium Chloride (AnalaR NORMAPUR) 500 g</td>
			<td>VWR Chemicals&#160;</td>
			<td>26764.26</td>
			<td>To make Krebs Buffer</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic&#160;</td>
			<td>Sigma-Aldrich Pty Ltd</td>
			<td>P5379-500g</td>
			<td>To make Krebs Buffer</td>
		</tr>
		<tr>
			<td>Powerlab 2/26, 2 channel recorder + Labchart software</td>
			<td>ADInstruments</td>
			<td>ML826</td>
			<td>Computer Hardware and Software</td>
		</tr>
		<tr>
			<td>Precision XN Inline Flowsensor, 3.2 mm (1/8&#34;)ID ME4PXN-KR37 XF</td>
			<td>ADInstruments</td>
			<td>ME4PXN</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Scalpel with handle disposable #11 pkt/10</td>
			<td>BSN Medical (Aust) Pty Ltd</td>
			<td>73252-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Gasket for Swinnex 47 mm 5/PK</td>
			<td>Merck Millipore</td>
			<td>SX0004701</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Silicone O-Ring 5-329 10/PK</td>
			<td>Merck Millipore</td>
			<td>XX1104707</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Single Buret Clamp</td>
			<td>Thomas Scientific</td>
			<td>8847T32</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Slip-on inlet Filter pore size 10 &#181;m (bubbler)</td>
			<td>Sigma-aldrich</td>
			<td>59277</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Sm. 360 Rotation Connector</td>
			<td>Thomas Scientific</td>
			<td>8847E35</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich Pty Ltd</td>
			<td>S6297 - 250g</td>
			<td>To make Krebs Buffer</td>
		</tr>
		<tr>
			<td>Swinnex Filter Holder, 47 mm</td>
			<td>Merck Millipore</td>
			<td>SX0004700</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>syringes 1 mL box/100</td>
			<td>Becton Dickinson Pty Ltd</td>
			<td>302100</td>
			<td></td>
		</tr>
		<tr>
			<td>Three Prong Clamps with 9 mm diameter x 125 mm long arm and twin screw for holding 5-80 mm</td>
			<td>Met-App Australia Pty Ltd</td>
			<td>1356</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Tubing Flowmeter Module TS410</td>
			<td>ADInstruments</td>
			<td>TS410</td>
			<td>Data Acquisition</td>
		</tr>
		<tr>
			<td>Tubing PVC 6.35 mm ID x 9.52 mm OD 50ft Roll 15.24m, clear , DEHP phthalate free, food grade meets REAC</td>
			<td>Thermo Fisher</td>
			<td>NAL 8701-0600</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Tubing PVC 7.94mm ID x 11.1mm OD 50ft Roll 15.24 m, clear , DEHP phthalate free, food grade meets REAC</td>
			<td>Thermo Fisher</td>
			<td>NAL 8701-0900</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Tubing PVC 9.52 mm ID x 12.7 mm OD 100ft Roll</td>
			<td>Thermo Fisher</td>
			<td>NAL 8701-4120</td>
			<td>Rig Construction</td>
		</tr>
		<tr>
			<td>Vannas scissors, 8.5 cm, Straight, 7 mm Blades</td>
			<td>Coherent Scientific</td>
			<td>WPI-500-086</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Water Bath 30 Litre with Suspended Tray</td>
			<td>Thermoline Scientific</td>
			<td>TLWB-30</td>
			<td>Rig Construction</td>
		</tr>
	</tbody>
</table>

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

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, transportation, and transplantation. Previous work from our laboratory showed that the addition of glyceryl trinitrate and erythropoietin to donor heart preservation solutions could significantly improve cardiac functional recovery after prolonged cold storage and in donation after circulatory death hearts. This supplementation protocol has been implemented in clinical use in transplant units around Australia, Belgium, and the United Kingdom. Here, we outline a protocol for testing supplementation strategies using an ex vivo isolated working rat heart (IWRH) perfusion circuit. Using this methodology, supplementation strategies can be tested in the context of prolonged cold static storage, donation after brain death, and donation after circulatory death donor heart preservation. Cardiac functional recovery, measured by aortic flow, coronary flow, cardiac output, pulse pressure, and heart rate, can be used to determine whether a particular preservation strategy can minimize ischemia-reperfusion injury of the donor heart.

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

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

The results from the baseline perfusion will determine whether the initial experiment (pre-storage) was successful. The aortic flow displayed during Langendorff mode should be between 14 - 22 mL/min. Langendorff flow is displayed as a negative value on the flow meter due to the retrograde perfusion of the aorta. An example of an acceptable Langendorff trace is shown in Figure 4A.
Once switched to working mode, the aortic flow should be at least 30 mL/min once the ...

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.

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, ...

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

Abstract