All animal experiments in this protocol were approved by the Institutional Animal Care and Use Committee at the University of Michigan, Ann Arbor. All mice were&#160;housed at a 12:12 light cycle in pathogen-free rooms. See the Table of Materials for details related to all materials, animals, and equipment used in this protocol.
1. Construction of the silicone balloon catheter
NOTE: The silicone balloon is made as described previously13.
<ol>
	<li>Add 9.5 mL of distilled water, 14.2 mL of light corn syrup, and 33.8 g of sucrose to a 100 mL beaker. Heat and stir the solution until the sugar is completely dissolved.</li>
	<li>Prepare the dough by mixing 10 g of wheat flour and 5 g of water until an even consistency is achieved and allow it to rest for 10 min.</li>
	<li>Shape a small piece of dough into an oval-the &#34;head&#34;-and attach it to the end of a dry spaghetti strand. Then, dip this head into the sugar solution and slowly remove from the solution, as the head is now completely coated. 
	NOTE: The dough should be smooth and of even texture. The sizes of dough can be varied to generate different sizes of balloons, from 5 mm (short diameter) to 7 mm (long diameter). Try to cover them with a thin film of sugar solution.</li>
	<li>Suspend the spaghetti strand on a polystyrene foam block, or other holders, to form a glossy cover evenly over the head and dry overnight.</li>
	<li>Dip the mold into silicone dispersion (silicone elastomer dispersed in xylene). Place the spaghetti strand back into the polystyrene foam block at 37 &#176;C for 2 h or until dry. Repeat this step once. 
	NOTE: It is essential to prevent the silicone dispersion gel from oxidizing due through air exposure, as this will generate an uneven balloon thickness.</li>
	<li>Place the mold into the water to separate and collect the balloon. Store the balloon in 0.02% sodium azide.</li>
	<li>Cut a two-blunt-end tip from a 22 G needle; mount one blunt end to the silicone balloon and another blunt end to PE tubing. Use 4-0 silk to tie the balloon in place on the needle. 
	&#8203;NOTE: Test the balloon integrity by injecting water into it. Once the balloon is filled, press the balloon softly to test if the balloon maintains tension inside. Use a new balloon if it is leaking. The mounted balloons can be stored for future use.</li>
</ol>
2. Preparation of the heart perfusion system
<ol>
	<li>Make 1 L of Krebs-Henseleit (KH) perfusion buffer and transfer it to the water reservoir Langendorff system.</li>
	<li>Connect the air tube to the water reservoir and turn on the airflow to balance the KH buffer with 5% CO2 and 95% O2 for at least 30 min.</li>
	<li>Set the water bath at 41.5 &#176;C and circulate the water in the outer layer of the Langendorff system to warm up the system and KH buffer. 
	&#8203;NOTE: The water bath temperature requires optimization for each system. For this system, the water bath temperature will maintain the KH at 37-37.5 &#176;C when perfusing into the heart.</li>
</ol>
3. Isolation, mounting, and cannulating of the mouse heart
<ol>
	<li>For anticoagulation, adminster 200 units of heparin in Saline by intraperitoneal (i.p.)&#160;injection into the right-hand quadrant of C57/B6 mouse abdomen.&#160;Aspirate the syringe before injection to confirm the bevel of the needles&#160;is not in the bladder or lumen of the GI tract.&#160;Use at least four mice in each experimental condition (but also consider the size of the treatment effect).
	<ol>
		<li>After 30 min, administer 80 mg/kg ketamine and 10 mg/kg of xylazine i.p. to anesthetize the mouse. Check if the anesthetized mouse is unconscious by performing a toe pinch and ensuring no response is observed.&#160;Acepromazine 2mg/kg can be added&#160;to the ket/xyl cocktail if the used strain&#160;of mice does&#160;not reach&#160;an adequate level of anesthesia with ket/xyl only.</li>
		<li>Make an incision right below the sternum. Use scissors to open the chest by cutting the diaphragm and the ribs. Fold the anterior chest wall to fully expose the chest. Cut at the descending aorta (closed to the aorta arch). Transfer the heart, lungs, and thymus of the mouse to cold histidine-tryptophan-ketoglutarate (HTK) buffer. Isolate the organs under ice-cold HTK buffer. Expose the aorta by removing any connective tissues. 
		NOTE: Maximize the aorta length by including both the ascending aorta and aortic arch region in the excision to allow enough space to connect to a needle.</li>
	</ol>
	</li>
	<li>Connect the end of the aorta to a 22 G needle, and tie with a 6-0 silk suture. Ensure the cannula is above the aortic root so as not to interfere with the aortic valve. Perfuse the aorta with 10 mL of cold (4 &#176;C) HTK buffer over approximately 10 min. 
	NOTE: It takes less than 15 min from heart removal to cannulation of the aorta; However, it is important to keep the perfusion speed at the appropriate level. Injections that are too fast and vigorous can generate high pressures and cause vascular/cardiac damage.</li>
	<li>Store the heart in a 50 mL tube with ice-cold HTK for 8 h or immediately perform the perfusion (do not store the control) and avoid direct contact with ice. 
	NOTE: Direct contact of the heart tissue with ice may lead to cold injury.</li>
	<li>Connect the needle-mounted heart to the cannula in the Langendorff apparatus and tie it with a silk suture. 
	NOTE: To standardize the procedure, wait for a total of 3 min for the cannulation process before perfusion.</li>
	<li>Start the perfusion with a constant flow mode at 3 mL/min; then, change to constant pressure mode at 70-80 mmHg and adjust the heart to ~6 mL/min. 
	NOTE: Palpating the heart softly can help accelerate cardiac reanimation. If the perfusion flow rate is much higher than 6 mL/min at constant pressure mode, there could be a leak in the cannulation or the aorta valve may not be working properly. Adjust the connections to fix the leak. The constant flow mode overrides the vascular tone self-regulation of the heart. The constant pressure mode allows the heart to regulate its coronary perfusion flow. Therefore, the constant pressure mode will precisely measure the cardiac function and preservation quality of the heart.</li>
	<li>Connect a deflated, water-filled balloon to a pressure transducer and a water-filled syringe with a three-way tap. After a 15-20 min equilibration period, cut the right atrium (RA) and insert the balloon to the RV through the RA. Use tape to hold the balloon inside the RV. Minimize the open area of the RA to help constrain the balloon in the ventricle (see Figure 1 for the setup). 
	NOTE: A period of equilibration is necessary, as the cardiac contraction and relaxation are not stable at the beginning, and the measure is less accurate and representative. If the AV node is damaged during the opening of the RA, the heart will display frequent arrhythmias.</li>
	<li>After 20 min of RV functional data collection, cut the left atrium (LA) and insert a deflated water-filled balloon to the LV through the LA. Use tape to hold the balloon inside the LV. 
	&#8203;NOTE: The heart should maintain stable hemodynamics for more than 1.5 h.</li>
</ol>
4. Functional data recording
<ol>
	<li>Calibration of the pressure transducer
	<ol>
		<li>Fill a 10 mL syringe with warm saline and connect the syringe to the dome through a three-way tap. Open the tap and slowly fill the dome with saline, and then close all taps and remove the syringe. Attach the filled dome to the transducer; connect the pressure gauge to the third end of the three-way tap.</li>
		<li>In the recording software, select the Bridge Amp from the dropdown menu of the channel connecting to the transducer. Rename the channel as Perfused pressure. Click Zero to zero the transducer.</li>
		<li>Start the recording by clicking Start so that the transducer is now reading 0 mmHg. After several seconds of recording, slowly push the syringe and increase the pressure to 100. Click stop to stop recording.</li>
		<li>In the Units conversion dialog, select an area of recording for 0 mmHg and click the arrow to Point 1 and type 0 mmHg. Select area of recording for 100 mmHg, click the arrow to Point 2, and type 100 mmHg. Click OK to calibrate the transducer.</li>
	</ol>
	</li>
	<li>Rename the channel corresponding to the pressure transducer with the balloon as Ventricle pressure. Start the recording when the heart is connected to the system. After inserting the balloon into the ventricle, adjust the water volume in the balloon using a micrometer syringe through the three-way tap to maintain the end-diastolic pressure at 5-10 mmHg. 
	NOTE: The end-diastolic measure could decrease during the measurement, preferably starting at close to 10 mmHg.</li>
	<li>Rename an empty channel as dP/dt. From the dropdown menu, select Derivative | source channel as Ventricle Pressure. The channel will record the ratio of pressure change in the ventricular cavity during the contraction period.</li>
	<li>Select a stable period of measure, then click Setting on the Blood pressure module.
	<ol>
		<li>Select the Ventricle pressure as the input channel and click selection for a period of calculation | OK.</li>
		<li>Click Classifier view to remove the outlier cardiac cycle (e.g., abnormal cycle time or pressure).</li>
		<li>Click table view to generate the tables of the average of max dP/dt (contraction) and min dP/dt (relaxation) for the selected period. 
		NOTE: Store the recording file for each sample and save the table of average heart function for statistical analysis.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kim, I. C., Youn, J. C., Kobashigawa, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past,+present+and+future+of+heart+transplantation.">The past, present and future of heart transplantation.</a> Korean Circulation Journal. 48 (7), 565-590 (2018).</li><li>Gaffey, A. 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=Transplantation+of+&amp;#34;high-risk&amp;#34;+donor+hearts:+Implications+for+infection.">Transplantation of &amp;#34;high-risk&amp;#34; donor hearts: Implications for infection.</a> The Journal of Thoracic and Cardiovascular Surgery. 152 (1), 213-220 (2016).</li><li>Hsich, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matching+the+market+for+heart+transplantation.">Matching the market for heart transplantation.</a> Circulation: Heart Failure. 9 (4), 002679 (2016).</li><li>Piperata, 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=Heart+transplantation+in+the+new+era+of+extended+donor+criteria.">Heart transplantation in the new era of extended donor criteria.</a> Journal of Cardiac Surgery. 36 (12), 4828-4829 (2021).</li><li>Huckaby, L. V., Hickey, G., Sultan, I., Kilic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+the+utilization+of+marginal+donors+for+orthotopic+heart+transplantation.">Trends in the utilization of marginal donors for orthotopic heart transplantation.</a> Journal of Cardiac Surgery. 36 (4), 1270-1276 (2021).</li><li>Singh, S. S. A., Dalzell, J. R., Berry, C., Al-Attar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+graft+dysfunction+after+heart+transplantation:+a+thorn+amongst+the+roses.">Primary graft dysfunction after heart transplantation: a thorn amongst the roses.</a> Heart Failure Reviews. 24 (5), 805-820 (2019).</li><li>Bell, R. M., Mocanu, M. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+heart+perfusion:+the+Langendorff+technique+of+isolated+heart+perfusion.">Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion.</a> Journal of Molecular and Cellular Cardiology. 50 (6), 940-950 (2011).</li><li>Rossello, X., Hall, A. R., Bell, R. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Langendorff+perfused+isolated+mouse+heart+model+of+global+ischemia-reperfusion+injury:+impact+of+ischemia+and+reperfusion+length+on+infarct+size+and+LDH+release.">Characterization of the Langendorff perfused isolated mouse heart model of global ischemia-reperfusion injury: impact of ischemia and reperfusion length on infarct size and LDH release.</a> Journal of Cardiovascular Pharmacology and Therapeutics. 21 (3), 286-295 (2016).</li><li>Matsuura, 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=Positive+inotropic+effects+of+ATP+released+via+the+maxi-anion+channel+in+Langendorff-perfused+mouse+hearts+subjected+to+ischemia-reperfusion.">Positive inotropic effects of ATP released via the maxi-anion channel in Langendorff-perfused mouse hearts subjected to ischemia-reperfusion.</a> Frontiers in Cell Development Biology. 9, 597997 (2021).</li><li>Tse, G., Hothi, S. S., Grace, A. A., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+arrhythmogenesis+following+slowed+conduction+in+heptanol-treated,+Langendorff-perfused+mouse+hearts.">Ventricular arrhythmogenesis following slowed conduction in heptanol-treated, Langendorff-perfused mouse hearts.</a> The Journal of Physiological Sciences. 62 (2), 79-92 (2012).</li><li>Kobashigawa, 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=Report+from+a+consensus+conference+on+primary+graft+dysfunction+after+cardiac+transplantation.">Report from a consensus conference on primary graft dysfunction after cardiac transplantation.</a> The Journal of Heart and Lung Transplantation. 33 (4), 327-340 (2014).</li><li>Lei, I., 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=Differential+inflammatory+responses+of+the+native+left+and+right+ventricle+associated+with+donor+heart+preservation.">Differential inflammatory responses of the native left and right ventricle associated with donor heart preservation.</a> Physiological Reports. 9 (17), 15004 (2021).</li><li>Miller, A., Wright, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+murine+ventricular+balloons+for+the+Langendorff+heart+preparation.">Fabrication of murine ventricular balloons for the Langendorff heart preparation.</a> Journal of Biotechnology &amp; Biomaterials. 1 (101), (2011).</li><li>Madsen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+immunology+of+heart+transplantation.">Advances in the immunology of heart transplantation.</a> The Journal of Heart and Lung Transplantation. 36 (12), 1299-1305 (2017).</li><li>Voelkel, N. F., 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=Right+ventricular+function+and+failure:+report+of+a+National+Heart,+Lung,+and+Blood+Institute+working+group+on+cellular+and+molecular+mechanisms+of+right+heart+failure.">Right ventricular function and failure: report of a National Heart, Lung, and Blood Institute working group on cellular and molecular mechanisms of right heart failure.</a> Circulation. 114 (17), 1883-1891 (2006).</li></ol>

The authors do not have any conflicts of interest to disclose.

This protocol describes the retrograde perfusion Langendorff method via aortic cannulation. This technique can be used to evaluate the LV and RV function of murine hearts after cold storage. The results show that the prolonged cold storage of donor hearts leads to reduced cardiac function in both the LV and RV using this protocol. 
The studies of acute and chronic rejection after heart transplantation widely focus on immunobiology14. The effects of native cells on PGD during cold storage are less well examined. PGD occurs in ~10%-20% of heart transplants and accounts for 66% of early death within 30 days following transplantation. In particular, the incidence of PGD affecting the LV versus the RV differ after transplantation11. Without the contribution of recipient cellular responses, this ex vivo method focuses on the contributions of native cardiac cells to PGD after cold preservation of donor hearts. Further studies may incorporate recipient responses in a murine heart transplant model. 
In this protocol, the Langendorff perfusion of cold preserved donor hearts focused on the native cardiac responses to warm crystalloid perfusion without infiltrating cellular immunity. To achieve reproducible results, several critical steps were standardized. The mouse hearts were arrested using HTK solution and stored in ice-cold HTK, similar to clinical practice. The perfusion volume and infusion time of the HTK solution for every heart was closely monitored with a timer. The donor heart was kept in prechilled tubes on ice containing HTK in a 4 &#176;C room. The cannulation time waas standardized to ~3 min prior to perfusion. All these steps ensured that cold preservation duration was the major variable in the study. 
A period of irregular cardiac contractility for ~20 min was commonly seen at the beginning of perfusion. This equilibration and recovery period was facilitated by gradual warming and oxygenation of cardiac tissues. A relatively stable period was expected after the initial 20 min. The balloon was inserted into the ventricle cavity at ~18 min after the initial equilibration period. We started recording hemodynamics after the heart was stable for ~25 min, once the balloon was inserted. Perfusion with KH buffer maintained stable cardiac performance for ~1.5-2 h. We therefore elected to record hemodynamics for 20 min in each of the left and right ventricles.
There are several limitations of retrograde perfusion for studying the PGD of hearts after cold storage. First, due to the balloon size and a lack of space in each ventricular cavity (in particular, the RV), the simultaneous insertion of two balloons into both the LV and RV is very challenging. Thus, we measure the function of RV and LV sequentially. It is important to note that the interventricular septum contributes significantly to both left and right ventricular function. The septum contributes to ~50% of right ventricular function, so there is interventricular dependence15. It is also important to note that, while the procedures for reperfusion of the murine heart in the Langendorff device take ~3 min, surgical implantation of the human heart in the relatively warm surgical field takes ~45 min. In comparison, the murine heart in this Langendorff system incurs less ischemic time. This should be taken into account when considering clinical translation.
Since we used KH buffer to perfuse the heart without blood, this may also have less efficiency in oxygen delivery. However, the heart function is relatively stable through the initial 1.5-2 h of perfusion, thus allowing reliable hemodynamic measurements. Unfortunately, there are currently no viable working heart perfusion models for these smaller murine hearts, and the effect of ventricular loading cannot be evaluated in this system. Despite this, the perfusion system is highly reproducible and less labor-intensive and time-consuming than transplant models. It is also less costly than transplant studies, which may make it more suitable for screening different therapeutic options and various molecular pathways. With modifications to preservation solutions by adding candidate drugs, this platform can be used to evaluate the effects of pharmacological agents on reducing PGD in both the LV and RV.

Heart transplantation improves survival and the quality of life in patients with end-stage heart failure1. Unfortunately, the shortage of heart donors limits the number of patients who could benefit from this therapy and limits the ability of clinicians to optimally match donors with recipients2,3,4. Furthermore, the new allocation system has contributed to longer ischemic times and significantly increased the use of marginal donors since 20185. Consequently, the mean age of heart donors and the ischemic time is increasing over time, leading to a higher rate of primary graft dysfunction (PGD) despite significant improvements in the strategies for heart preservation 6.
PGD can affect the left, the right, or both ventricles, and remains a life-threatening complication that represents the leading cause of early deaths after heart transplantation. Investigating the mechanisms of PDG and the development of strategies for better heart preservation are important considerations, given the potential life-saving impact on heart recipients. Therefore, experimental models that allow a robust and reliable assessment of donor heart function after a prolonged storage time are essential to increase our understanding of PGD and facilitate the development of novel therapies. The ability to accurately assess cardiac function in the mouse heart allows access to a vast repertoire of transgenic murine models that can accurately identify PGD mechanisms.
In physiologic and pharmacologic studies, the Langendorff retrograde perfusion model is widely used to assess heart function7. Specifically, cardiac performance is detected by a silicone balloon connected to a pressure transducer within the left ventricular (LV) cavity. A key feature of PGD is the inadequate contraction and relaxation of the ventricular muscle. Prior Langendorff studies have focused on using an LV balloon to produce reliable and reproducible results in LV functional assessment8,9,10. However, the use of an intracavitary balloon to assess right ventricular (RV) function using the balloon system is less well recognized.
Given a significant PGD rate involving the RV after transplantation11, experimental methods to study both LV and RV function would help determine the molecular and physiological mechanisms that contribute to RV PGD. This protocol shows that intracavitary silicone balloons can provide reliable assessments of LV and RV function in the same murine heart12. To evaluate the potential use of the Langendorff system in the PGD study, we examined the heart functions with different periods of storage and found decreased cardiac function in contraction and relaxation with the prolonged cold storage of murine hearts. Interestingly, the LV has a higher functional reduction than the RV. In summary, the protocol described here can be used for assessing the effect of a candidate drug and molecular pathways on both LV and RV function. The ability to use this method on murine hearts will facilitate the performance of detailed mechanistic studies.

<table><tbody><tr><td>4-0 silk suture</td><td>Braintree Scientific</td><td>SUTS108</td><td></td></tr><tr><td>6-0 Silk suture</td><td>Braintree Scientific</td><td>SUTS104</td><td></td></tr><tr><td>All purpose flour</td><td>Kroger</td><td></td><td></td></tr><tr><td>BD General Use and PrecisionGlide Hypodermic Needles 22 G</td><td>Fisher scientific</td><td>14-826-5A</td><td></td></tr><tr><td>BD Syringe with Luer-Lok Tips (Without Needle)</td><td>Fisher scientific</td><td>14-823-16E</td><td></td></tr><tr><td>Corn Syrup</td><td>Kroger</td><td></td><td></td></tr><tr><td>Custodiol HTK Solution</td><td>Essential Pharmaceuticals LLC</td><td></td><td></td></tr><tr><td>Dissecting Scissors&amp;nbsp;</td><td>World Precision Instruments</td><td>14393/14394</td><td></td></tr><tr><td>Falcon 50 mL conical tubes</td><td>Fisher scientific</td><td>14-959-49A</td><td></td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>H4784</td><td></td></tr><tr><td>Krebs Henseleit buffer</td><td>Sigma</td><td>K3753</td><td></td></tr><tr><td>Nusil silicone dispersions</td><td>Avantor</td><td></td><td></td></tr><tr><td>Perfusion system</td><td>Radnoti</td><td>130101BEZ</td><td></td></tr><tr><td>PowerLab</td><td>ADInstruments</td><td>PL3508</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Xylazine</td><td>Sigma</td><td>X1126</td><td></td></tr></tbody></table>

assessment of ex vivo murine biventricular function in a langendorff model

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation is an important risk factor for PGD, and reliable evaluation of cardiac function is essential to study the functional responses of the donor heart after cold preservation. The accompanying video describes a technique to assess murine right and left ventricular function using ex vivo perfusion based in a Langendorff model after cold preservation for different durations. In brief, the heart is isolated and stored in a cold histidine-tryptophan-ketoglutarate (HTK) solution. Then, the heart is perfused with a Kreb buffer in a Langendorff model for 60 min. A silicone balloon is inserted into the left and right ventricle, and cardiac functional parameters are recorded (dP/dt, pressure-volume relationships). This protocol allows the reliable evaluation of cardiac function after different heart preservation protocols. Importantly, this technique allows the study of cardiac preservation responses specifically in native cardiac cells. The use of very small murine hearts allows access to an enormous array of transgenic mice to investigate the mechanisms of PGD.

Adult C57Bl/6 mouse hearts, 3 months of age, were harvested and mounted to the Langendorff system. The donor heart was stored in HTK for 0 and 8 h, and then perfused with oxygenated KH buffer. A silicone balloon connecting to a pressure transducer was used to measure the contraction and relaxation of LV and RV function.
The aortic pressures were maintained in the 70-80 mmHg range. The heart rate was comparable in mouse hearts with 0 and 8 h of storage. The LV and RV function were examined by measuring systolic and diastolic pressure. dP/dt, a derivative to calculate the ratio of pressure change, was calculated to determine the pressure dynamics. The absolute number of max dP/dt and min dP/dt could represent the level of muscular contraction and relaxation. At 0 h of storage, the LV had higher systolic pressure compared to the RV (Figure 2C and Figure 3A). The LV showed more muscular contraction and relaxation than the RV after perfusion of 0 h storage (Figure 2C and Figure 3B,C). However, after 8 h of cold storage, both the LV and RV showed a significant functional reduction compared to a 0 h baseline (Figure 2A-D and Figure 3B,C). The decreases in cardiac contraction were more severe in the LV. After 8 h of storage, the contraction and relaxation of the LV was 25.1% and 30.7% of the 0 h baseline, while the RV had 32.5% and 29.1% of function compared to the 0 h baseline (Figure 3B,C). These results showed that the PGD of the LV after prolonged storage had a more significant cardiac contraction reduction than the RV.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64384/64384fig01.jpg" /> 
Figure 1: Mounting and cannulation of the mouse heart. (A) Overall setup of the perfusion setup. 1. Perfusion reservoir. 2. Oxygenation chamber. 3. Air trap chamber. 4. Heart chamber. 5. Value switch for constant flow and pressure. 6 and 7. Oxygen inflow. (B) Cannulated hearts with the RV in the front. (C) Position of the RV to cut for opening its cavity. (D) Tap the balloon tube with the cannula. Abbreviation: RV = right ventricle. <a href="https://www.jove.com/files/ftp_upload/64384/64384fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64384/64384fig02.jpg" /> 
Figure 2: Comparing the function of the LV versus RV. (A) Tracing record of max and min dP/dt in the RV and LV in the donor heart with 0 h of storage. (B) The record of max and min dP/dt in the RV and LV in the donor heart with 8 h of storage. (C,D) Details of dP/dt, LV pressure, heart rate, and perfusion pressure in the LV and RV at 0 h and 8 h. Abbreviations: RV = right ventricle; LV = left ventricle; dP/dt = pressure-time relationship. <a href="https://www.jove.com/files/ftp_upload/64384/64384fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64384/64384fig03.jpg" /> 
Figure 3: Comparing the function of the LV versus RV after storage and perfusion. (A) Systolic and diastolic pressure of the LV and RV after 0 h and 8 h of storage. (B) Max dP/dt and (C) Min dP/dt of the LV and RV after perfusion with 0 h and 8 h of storage. This figure is from Lei et al.12. <a href="https://www.jove.com/files/ftp_upload/64384/64384fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model at JoVE.com

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system.

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

Primary graft dysfunction (PGD) remains the leading cause of early death following heart transplantation. Prolonged ischemic time during cold preservation ...

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1.9K Views.  University of Michigan, Ann Arbor. Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system. 

Video: Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

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

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

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

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

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 acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum of physiological, biochemical, morphological, and pharmacological parameters in centrally denervated hearts. Here, we describe a setup for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cyclic adenosine monophosphate (cAMP) dynamics. The intracardiac autonomic nervous system is modulated by the mechanical dissection of atrial fat pads-in which murine ganglia are located mainly&#8212;or by the usage of global as well as targeted pharmacological interventions. An octapolar electrophysiological catheter is introduced into the right atrium and the right ventricle, and epicardial-placed multi-electrode arrays (MEA) for high-resolution mapping are used to determine cardiac electrophysiology and arrhythmogenesis. F&#246;rster resonance energy transfer (FRET) imaging is performed for the real-time monitoring of cAMP levels in different cardiac regions. Neuromorphology is studied by means of antibody-based staining of whole hearts using neuronal markers to guide the identification and modulation of specific targets of the intracardiac autonomic nervous system in the performed studies. The ex vivo Langendorff setup allows for a high number of reproducible experiments in a short time. Nevertheless, the partly open nature of the setup (e.g., during MEA measurements) makes constant temperature control difficult and should be kept to a minimum. This described method makes it possible to analyze and modulate the intracardiac autonomic nervous system in decentralized hearts.

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Here, we present a protocol for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics using an ex vivo Langendorff setup.

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum ...

Enhancing CVD Research with Modified Langendorff Perfusion

Modified Langendorff Perfusion for Extended Perfusion Times of Rodent Cardiac Grafts

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and scientific advancement. As a result, innovation, improvement and/or repurposing of the available research toolset can provide improved testbeds for research advancement. Langendorff perfusion is an extremely valuable research technique for the field of CVD research that can be modified to accommodate a wide array of experimental needs. This tailoring can be achieved by personalizing a large number of perfusion parameters, including perfusion pressure, flow, perfusate, temperature, etc. This protocol demonstrates the versatility of Langendorff perfusion and the feasibility of achieving longer perfusion times (4 h) without graft function loss by utilizing lower perfusion pressures (30-35 mmHg). Achieving extended perfusion times without graft damage and/or function loss caused by the technique itself has the potential to eliminate confounding elements from experimental results. In effect, in scientific circumstances where longer perfusion times are relevant to the experimental needs (i.e., drug treatments, immunological response analysis, gene editing, graft preservation, etc.), lower perfusion pressures can be key for scientific success.

Author Spotlight: Advancing Cardiovascular Research &#8212; Tailored Langendorff Perfusion Techniques for Improved Experimental Outcomes

This article demonstrates the feasibility of achieving longer perfusion times (4 h) of murine cardiac grafts without function loss by employing lower (30-35 mmHg) than physiological (60-80 mmHg) perfusion pressures during Langendorff.

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and ...

Bioengineering

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. Assessment of RV function and morphology is complex, partly due to the complex shape of the RV. Currently, cardiac magnetic resonance (CMR) imaging is the golden standard for noninvasive assessment of RV function and morphology. The current protocol describes CMR imaging in a mouse model of RV pressure load induced by pulmonary artery banding (PAB). PAB is performed by placing a 6-0 suture around the pulmonary artery over a 23 G needle. The PAB gradient is determined using echocardiography at 2 and 6 weeks. At 6 weeks, the right and left ventricular morphology and function is assessed by measuring both end-systolic and end-diastolic volumes and mass by ten to eleven cine slices 1 mm thick using a 9.4 T magnetic resonance imaging scanner equipped with a 1,500 mT/m gradient. Representative results show that PAB induces a significant increase in RV pressure load, with significant effects on biventricular morphology and RV function. It is also shown that at 6 weeks of RV pressure load, cardiac output is maintained. Presented here is a reproducible protocol for the quantification of biventricular morphology and function in a mouse model of RV pressure load and may serve as a method for experiments exploring determinants of RV remodeling and dysfunction.

To understand the pathophysiology of right ventricular (RV) adaptation to abnormal loading, experimental models are crucial. However, assessment of RV dimensions and function is complex and challenging. This protocol provides a method to perform cardiac magnetic resonance imaging (CMR) as a noninvasive benchmark procedure in mice subjected to RV pressure load.

Right ventricular (RV) function and failure are major determinants of outcome in acquired and congenital heart diseases, including pulmonary hypertension. ...

Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) generated by recording both pressure and volume during cardiac catheterization are vital when assessing both systolic and diastolic cardiac function. Left and right heart function are closely related, reflected in ventricular interdependence. Thus, recording biventricular function in the same animal is important to get a complete assessment of cardiac function. In this protocol, a closed chest approach to cardiac catheterization consistent with the way catheterization is performed in patients is adopted in mice. While challenging, the closed chest strategy is a more physiological approach, because opening the chest results in major changes in preload and afterload that create artifacts, most notably a fall in systemic blood pressure. While high-resolution echocardiography is used to assess rodents, cardiac catheterization is invaluable, particularly when assessing diastolic pressures in both ventricles.
Described here is a procedure to perform invasive, closed chest, sequential left and right ventricular pressure-volume (PV) loops in the same animal. PV loops are acquired using&#160;admittance technology with a mouse pressure-volume catheter and pressure-volume system acquisition. The procedure is described, beginning with the neck dissection, which is required to access the right jugular vein and the right carotid artery, to the insertion and positioning of the catheter, and finally the data acquisition. Then, the criteria required to ensure the acquisition of high-quality PV loops are discussed. Finally, the analysis of the left and right ventricular PV loops and the different hemodynamic parameters available to quantify systolic and diastolic ventricular function are briefly described.

Presented here is a protocol to assess biventricular heart function in mice by generating pressure-volume (PV) loops from the right and left ventricle in the same animal using closed chest catheterization. The focus is on the technical aspect of surgery and data acquisition.

Assessment of cardiac function is essential to conduct cardiovascular and pulmonary-vascular preclinical research. Pressure-volume loops (PV loops) ...

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar Langendorff over a century ago. Although this technique has been applied to mice over the last 25 years, its use in this species has been limited to adult animals. Development of a successful method to consistently cannulate the neonatal murine aorta would allow for the systematic study of the isolated retrograde perfused heart during a critical period of cardiac development in a genetically modifiable and low-cost species. Modification of the Langendorff preparation enables cannulation and establishment of reperfusion in the neonatal murine heart while minimizing ischemic time. Optimization requires a two-person technique to permit successful cannulation of the newborn mouse aorta using a dissecting microscope and a modified commercially available needle. The use of this approach will reliably establish retrograde perfusion within 3 min. Because the fragility of the neonatal mouse heart and ventricular cavity size prevents direct measurement of intraventricular pressure generated using a balloon, use of a force transducer connected by a suture to the apex of the left ventricle to quantify longitudinal contractile tension is necessary. This method allows investigators to successfully establish an isolated constant-flow retrograde-perfused newborn murine heart preparation, permitting the study of developmental cardiac biology in an ex-vivo manner. Importantly, this model will be a powerful tool to investigate the physiological and pharmacological responses to ischemia-reperfusion in the neonatal heart.

The present protocol describes aortic cannulation and retrograde perfusion of the ex-vivo neonatal murine heart. A two-person strategy, using a dissecting microscope and a blunted small gauge needle, permits reliable cannulation. Quantification of longitudinal contractile tension is achieved using a force transducer connected to the apex of the left ventricle.

The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar ...

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human&#160;and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

Assessment of Ex Vivo Murine Biventricular Function in a Langendorff Model

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Chapters in this video

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

An Isolated Working Heart System for Large Animal Models

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Modified Langendorff Perfusion for Extended Perfusion Times of Rodent Cardiac Grafts

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Quantification of Biventricular Function and Morphology by Cardiac Magnetic Resonance Imaging in Mice with Pulmonary Artery Banding

Biventricular Assessment of Cardiac Function and Pressure-Volume Loops by Closed-Chest Catheterization in Mice

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk