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.
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.
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.
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 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.
2. Preparation of the heart perfusion system
3. Isolation, mounting, and cannulating of the mouse heart
4. Functional data recording
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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 °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.
The authors have nothing to disclose.
None.
4-0 silk suture | Braintree Scientific | SUTS108 | |
6-0 Silk suture | Braintree Scientific | SUTS104 | |
All purpose flour | Kroger | ||
BD General Use and PrecisionGlide Hypodermic Needles 22 G | Fisher scientific | 14-826-5A | |
BD Syringe with Luer-Lok Tips (Without Needle) | Fisher scientific | 14-823-16E | |
Corn Syrup | Kroger | ||
Custodiol HTK Solution | Essential Pharmaceuticals LLC | ||
Dissecting Scissors | World Precision Instruments | 14393/14394 | |
Falcon 50 mL conical tubes | Fisher scientific | 14-959-49A | |
Heparin sodium salt from porcine intestinal mucosa | Sigma | H4784 | |
Krebs Henseleit buffer | Sigma | K3753 | |
Nusil silicone dispersions | Avantor | ||
Perfusion system | Radnoti | 130101BEZ | |
PowerLab | ADInstruments | PL3508 | |
Sodium azide | Sigma | S2002 | |
Sodium bicarbonate | Sigma | S5761 | |
Sucrose | Sigma | S0389 | |
Sucrose | Sigma | S0389 | |
Xylazine | Sigma | X1126 |