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 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.
The field of cardiovascular research has seen important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs). However, despite the general decrease in incidence and mortality rates, CVDs remain the leading cause of death globally1,2. This alarming fact highlights the need for increased research and scientific advancement, which is undoubtedly dependent on the accuracy and predictability of the available research tools. As a result, there is a constant need for innovation, improvement and/or repurposing of the research toolset. For instance, retrograde or Langendorff heart perfusion, a technique available to the field for over a century, can be easily modified to cover a larger scope of scientific needs and achieve a broader range of applications.
The isolation of the cardiac graft from the rest of the organism during Langendorff perfusion provides an important degree of control over a wide array of experimental parameters, including temperature, circulating solution, coronary perfusion pressures, etc.3,4,5,6,7. The manipulation of these parameters facilitates the simulation of a large number of cardiac scenarios that can be leveraged to further scientific advancements5,8,9,10. Among these parameters, perfusion pressure is likely the most overlooked experimental setting11.
During Langendorff, perfusion pressures exhibit a direct correlation with heart rate, peak systolic/diastolic pressures, and oxygen consumption11. This correlation provides direct and precise control over the amount of work produced by the cardiac grafts, which can be adjusted to meet individual experimental needs. Despite this valuable control capability, the field has historically gravitated towards the use of higher perfusion pressures (60-80 mmHg), subjecting all cardiac grafts to high work demand irrespective of experimental needs8,12,13,14,15. The consequences of this unnecessarily high demand for work arise from the overarching principle that overwork tends to result in premature failure. This seems to be particularly true for cardiac grafts perfused via Langendorff, as the non-physiological nature of this method and the lack of recovery support present in vivo seem to exacerbate graft failure. This premature loss of graft function limits perfusion and experimental times significantly. In effect, in circumstances where longer perfusion times are more relevant to the experimental needs (i.e., drug treatments, immunological response analysis, gene editing, graft preservation, etc.), lower cardiac work can be afforded in exchange for increased graft durability.
This protocol demonstrates the feasibility of utilizing lower perfusion pressures (30-35 mmHg) during Langendorff, as well as the significant effect these pose for cardiac graft function over time when compared to higher perfusion pressures (60-80 mmHg). Furthermore, the findings in this manuscript highlight the importance of prioritizing the customization of the wide array of perfusion parameters to better meet experimental needs.
This study is conducted following the Institutional Animal Care and Use Committee (IACUC) of Massachusetts General Hospital.
1. System design
2. Perfusate preparation
3. Perfusion system setup
4. Cardiac graft procurement preparation
5. Cardiac graft procurement
6. Perfusion initiation
7. Intraventricular balloon:
8. Sampling
9. End/cleanup
Hearts from adult male Lewis rats (250-300 g body weight) were harvested and perfused at high (70-80 mmHg) or low (30-35 mmHg) perfusion pressures (n = 3 per group). The effects of perfusion pressure on overall cardiac function and health were determined by collecting heart rate, edema, and left ventricular function.
A clear correlation between heart rate and perfusion pressures was determined (Figure 2). Heart rate was statistically higher in high-pressure hearts when compared to low-pressure hearts for all time points, except the first one (60 min, Figure 2A,B). Interestingly, low-pressure hearts seem to undergo a period of adjustment at the beginning of perfusion, where it took about 30 min for the heart rate to stabilize and reach the levels that were maintained through the rest of the perfusion (Figure 2A). A large difference in left ventricular pulse pressure (LVPP) was also observed between the groups, with the LVPP of high-pressure hearts being statistically higher than low-pressure hearts at every timepoint (Figure 3B). This sustained high demand for work resulted in a progressive loss of function in high-pressure hearts with a statistical decrease in LVPP seen after 2 h of perfusion (Figure 3A,B). Alternatively, no loss of function was present in hearts perfused with low pressures, with LVPP remaining unchanged throughout perfusion time (Figure 3A,B). Similar to LVPP, high-pressure hearts exhibited higher cardiac muscle contraction (dP/dtmax) and relaxation (dP/dtmin) throughout the perfusion time when compared to low-pressure hearts (Figure 3C,D). In accordance, high-pressure hearts underwent a progressive loss of contractility and relaxation capacity, with both parameters being statistically higher 1 h into the perfusion time when compared to the last hour of perfusion. Differently, cardiac muscle contractility and relaxation capabilities were comparably low in the low-pressure group and remained unchanged over 4 h of perfusion time (Figure 3C,D). In addition to the functional effects, high perfusion pressures over extended periods of time also exacerbate interstitial fluid retention within the cardiac grafts, leading to edema. This edema was semi-quantified in percent weight change and resulted in high-pressure hearts having statistically higher weight gain when compared to hearts perfused at low pressures (Figure 2C).
Figure 1: Perfusion system setup. (A) Overall perfusion setup. Dashed lines represent the order in which the components of the system were connected to optimize perfusate circulation. Solid, colored lines represent the order in which the components were connected to optimize perfusate temperature. (B) The proper way of handling the heart after cannulation to avoid emptying of the catheter and introducing air into the coronaries. Please click here to view a larger version of this figure.
Figure 2: Effects of pressure on heart rate and edema. (A) Heart rate obtained from the intraventricular balloon measurements. The solid line is the median of the experimental groups. The shaded area is the interquartile range. (B) Area under the curve (AUC) of heart rate data for every hour of perfusion. (C) Percent weight gained after 4 h of perfusion at low and high pressures. All data are expressed as median ± interquartile range (IQR). *p < 0.01, **p < 0.05, ***p < 0.001. Please click here to view a larger version of this figure.
Figure 3: Effects of pressure on left ventricular function. (A) Maximum systolic pressure plotted over time, denoted as left ventricular pulse pressure (LVPP). The solid line is the median of the experimental groups. The shaded area is the interquartile range. (B) The area under the LVPP curve (AUC) for every hour of perfusion. (C) Cardiac muscle contractility quantified from the maximum derivative of the pressure pulse. (D) Cardiac muscle relaxation quantified from the minimum derivative of the pressure pulse. All data are expressed as median ± interquartile range. *p < 0.01, **p < 0.05, ***p < 0.001, ****p < 0.0001. Please click here to view a larger version of this figure.
Ion | Concentration (mmol/L) |
Na+ | 135–145 |
K + | <6.00 |
Ca +2 | 1.0–1.3 |
Cl – | 96–106 |
Table 1: Acceptable range of ion concentration in the perfusate.
Langendorff perfusion is an extremely pliable technique that allows impressive tailoring and adjustment to meet a wide array of experimental needs. This tailoring is permitted by the significant adjustability of most perfusion parameters, including perfusion pressures. Due to the retrograde nature of Langendorff, perfusion pressures are equivalent to coronary perfusion pressures, which play an essential role in cardiac function. Coronary perfusion pressures (CPP) are known to directly control cardiac work, as a wide array of cardiac indices (i.e., left ventricular pressure, contractility (dP/dtmax), wall tension, ventricular stiffness) are directly proportional to CPP16,17,18. Historically, the field has utilized perfusion pressures, and in effect CPP, between 60 mmHg and 80 mmHg in an attempt to mimic physiological conditions5,8,15,19,20,21. However, the non-physiological nature of retrograde ex vivo machine perfusion, in combination with the high demand for work, leads to an over-time loss of cardiac function (Figure 3). Alternatively, lower perfusion pressures (30-35 mmHg), despite not accurately replicating the physiological conditions of rat hearts in vivo, inherently decrease cardiac work demand and achieve extended perfusion times (4 h) without the over-time loss of function (Figure 3), and decreased graft edema (Figure 2C). The use of lower perfusion pressures, although it signifies a deviation from physiological CPP, seems to provide important advantages over the use of physiological perfusion pressures, as the elimination of existent technique-dependent loss of function during Langendorff perfusion improves the technique into a more accurate and predictable model system with significant potential to advance cardiovascular research. Particularly, the research areas that benefit and/or require extended perfusion times to reach scientific relevance (i.e., drug treatments, immunological response analysis, gene editing, normothermic graft preservation, etc.) are becoming increasingly important in the battle against CVDs.
Langendorff perfusion is undisputable an essential tool for the field of cardiovascular research. Therefore, along with the significant benefits this scientific technique poses to the research community, it comes with an important level of scientific complexity. In effect, there are several critical steps within this protocol that require careful standardization, primarily to avoid cardiac graft damage prior to, during, and immediately after initiating perfusion. The first chance of graft damage is inconspicuous during the portal vein flush. This flush with heparinized saline aims to remove as much whole blood as possible from the cardiac graft with a double purpose. First, it serves as a way of euthanasia via exsanguination. Second, it minimizes the chances of coagulation within the cardiac graft during retrieval, cannulation, and transportation, as rat whole blood is known to have extremely short clothing times22,23. However, after hundreds of successful cardiac perfusions, it became apparent that the pressure applied to the rat organism during flushing is of utter importance, with the ideal flush pressure being around 10 mmHg. Higher portal vein flush pressures seem to result in damage to the vasculature of the cardiac graft, leading to increased vascular resistance (). Higher vascular resistance in effect results in target perfusion pressures being reached at lower flow rates. This imbalance between pressure and coronary flow is conveyed in the produced left ventricular pulse pressure (LVPP), resulting in significant variability.
The next instance of possible cardiac graft damage is during the connection of the graft to the system via the introduction of air bubbles into the coronaries. Air bubbles can be easily introduced by mishandling the cannulated heart (Figure 1B) or improper bubble removal from the perfusion system upstream of the bubble trap24. Due to the retrograde nature of this setup, any introduction of air will result in cardiac air embolism, leading to ischemic insults, fibrillation, and, very commonly, graft death. Finally, the last critical step to ensure protocol success occurs during the initiation of perfusion. Differently from the large majority of manuscripts that report utilizing Langendorff as a technique, the initiation of perfusion in this protocol is performed at relatively low flows (1 mL/min) with incremental increases (+0.2 mL/min), which warrant complete control over perfusion pressures5,8,15,19,20,21. This incremental increase in flow, and therefore pressure, is critical as abrupt changes in pressure irreversibly increase vascular resistance and alter the delicate flow/pressure balance.
High vascular resistance in pressure-controlled Langendorff perfusions is very consequential, as target perfusion pressures are reached at lower flows, and grafts result under-perfused. The large reliance on this perfect balance between flow and pressure is likely the largest limitation of this protocol, as any prior graft damage, intentional (i.e., extended cold preservation, warm ischemia insult, myocardial infarction, etc.) or unintentional, leads to increased vascular resistance. In effect, this protocol is particularly useful for research where the experiment starts after the initiation of perfusion (i.e., drug treatments, immunological response analysis, gene editing, normothermic graft preservation, etc.) but not prior. This limitation is a perfect example of one Langendorff not fitting all purposes and special care should be taken to tailor perfusion parameters to better meet experimental needs.
The authors have nothing to disclose.
This work was supported by generous funding to S.N.T. from the US National Institutes of Health (K99/R00 HL1431149; R01HL157803) and American Heart Association (18CDA34110049). We also gratefully acknowledge funding from the US National Institute of Health (R01DK134590; R24OD034189), National Science Foundation (EEC 1941543), Harvard Medical School Eleanor and Miles Shore Fellowship, Polsky Family Foundation, the Claflin Distinguished Scholar Award on behalf of the MGH Executive Committee on Research, and Shriners Children’s Boston (Grant #BOS-85115).
5-0 Suture | Fine Scientific Tools | 18020-50 | |
14 G Angiocath | Becton Dickinson | 381867 | |
16 G Angiocath | Becton Dickinson | 381957 | |
24 mm Heart Chamber adaptors | Radnoti | 140132 | |
Balloon Catheter | Radnoti | 170423 | |
BD Slip Tip Sterile Syringes- 10 mL | Fisher Scientific | 14-823-16E | |
BD Slip Tip Sterile Syringes- 1 mL | Fisher Scientific | 14-823-434 | |
BD Slip Tip Sterile Syringes- 50 mL | Fisher Scientific | 14-820-11 | |
Bovine Serum Albumin | Sigma | A7906 | |
Bubble Trap Compliance Chamber | Radnoti | 130149 | |
Calcium Chloride | Sigma | C7902 | |
Clamp Holder | United Scientic | RTCLMP1 | |
Dextran | Sigma | 31389 | |
DIN8 Extension Cable | Iworx | SKU C-DIN-EXT | |
Falcon High Clarity 50 mL conical tubes | Fisher Scientific | 14-432-22 | |
GSC Go Science Crazy Cast Iron Support Ring Stand | Fisher Scientific | S13748 | |
Heart Chamber | Radnoti | 140160 | |
Heated Water Circulator bath | Cole Parmer | N/A | |
Heparin sodium Injection | Medplus | G-0409-2720-0409-2721 | |
Hydrocortisone | Solu-Cortef | MGH Pharmacy | |
Insulin | Humulin R | MGH Pharmacy | |
Insvasive Fluid Filled Blood Pressure Sensor | Iworx | SKU BP-10x | |
Iworx Data Acquisition System | Iworx | IX-RA-834 | |
Krebs-Henseleit Buffer | Sigma | K3753 | |
Left Ventricular Pressure Balloon | Radnoti | 170404 | |
Masterflex L/S Easy-Load II Pump Head for Precision Tubing, PPS Housing, SS Rotor | VWR | MFLX77200-60 | |
Masterflex L/S Standard Digital Pump Systems | VWR | MFLX07551-30 | |
Membrane Oxygenating Chamber | Radnoti | 130144 | |
Penicillin-Streptomycin | ThermoFisher Scientific | 15140122 | |
Polyethylene Tubing | Fisher Scientific | 14-170-12H | |
Precision Pump Tubing-16 | VWR | MFLX96410-16 | |
Sodium Bicarobonate | Sigma | 5761 | |
Standard PHD ULTRA CP Syringe Pump | Harvard Aparatus | 88-3015 | |
Tygon Transfer Tubing | VWR | MFLX95702-03 |
.