Published: August 16th, 2016
Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.
Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions.
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).
The study of isolated organs permits control of physiologic conditions beyond what is possible in vivo. Ex vivo heart preparations were first described by Otto Langendorff,1 who described an isolated model with retrograde perfusion. Subsequently, others described the "working heart" model, in which the myocardium performs both pressure and volume work.2 Such preparations have been instrumental in elucidating mechanisms of myocardial action,3 myocardial metabolism,4-6 and effects of cardiotonic medications.7-9
The use of medications that enhance myocardial contractility is common in critically ill patients. However, few data are available comparing the relative effects of these medications on contractility and myocardial oxygen consumption, data which may be useful in the care of patients with clinical signs of heart failure of in the postoperative setting.10 However, because most cardiotonic medications affect not only the myocardium, but also arteriolar resistance, venous capacitance11, and a patient's metabolic rate,12 ex vivo isolated heart models remain the optimal means by which to study the effects of such medications on the myocardium proper.
We describe the use of an ex vivo model for the load-independent study of inotropic medications on myocardial function and oxygen consumption. Hearts from Sprague Dawley rats were cannulated using a left ventricular working heart model and perfused using a modified Krebs Henseleit perfusate. Aortic and left atrial pressures were controlled. Pressure-volume impedance catheters were placed into the left ventricle via apical puncture for the continuous monitoring of systolic and diastolic function. Oxygen consumption was continuously measured as the indexed difference in oxygen content between left atrial perfusate and the pulmonary artery effluent. Medications to be tested were infused into the left atrial block, and changes in cardiac performance and oxygen metabolism were measured and compared with an immediately preceding baseline.
This protocol is performed under a current protocol under the institution's animal care and use committee.
1. Preparation for the Study
2. Animal Preparation and Dissection
NOTE: For best results, ensure animal is between 300 and 500 g; we have found that an animal weight between 425 to 450 g is ideal for our system.
3. Aortic Cannulation
4. Pulmonary Vein Occlusion and Preparation of the Pulmonary Artery for Cannulation
NOTE: The purpose of this step is to create a closed left atrial system to ensure that all volume and pressure from the left atrial block is transmitted to the left heart structures. Failure to completely occlude the pulmonary veins could result in preload deficiency and may falsify results or create an unstable working heart preparation.
5. Left Atrial Cannulation
6. Pulmonary Artery Cannulation and Transition to Working Heart Mode
7. Insertion of the Left Ventricular Pressure Volume Catheter
NOTE: The PV catheter may be placed either retrograde (through the aortic valve) or via apical puncture. The benefit of retrograde is that position is more consistent and it obviates the need for apical puncture and the concomitant risks of coronary injury or loss of preload. However, retrograde placement can sometimes be very challenging, so we describe both techniques herein.
8. Infusion of Medication
9. Physiologic Manipulations
A schematic of a fully instrumented heart in retrograde perfusion (Figure 1A) and in left ventricular working heart (Figure 1B). Typical aortic, left atrial and left ventricular pressure and volume tracings are shown in Figure 2A - D. The typical end diastolic pressure is approximately 3 - 5 mmHg in this model, and the peak systolic pressure is approximately 100 mmHg. Figure 2E demonstrates the change in left atrial tracing when the LA cannula is moved away from the atrial septum during placement and positioning of the cannula. In these experiments, aortic pressure was set at 90 mmHg, and LA pressure was set to 10 mmHg.
To test the effects of catecholamines, each physiologic parameter (derived primarily from the pressure-volume catheter and associated software) was compared to the immediately preceding baseline period. In the example shown, dopamine was infused at 15 μg/kg/min into the left atrial block. Although the end diastolic pressure is identical in the two conditions (given the fixed atrial pressure in this model), the left ventricular end diastolic volume decreases by 2.5%, and the left ventricular end systolic volume decreases by 4.9%, yielding an increased stroke volume (Figure 3A). Compared with placebo infusions, the left ventricular stroke work, identified as the area within the pressure-volume curve, increased by 32% during treatment with dopamine (Figure 3B, P < 0.001, t test, n = 10 per group). This was associated with a greater increase in myocardial oxygen consumption relative to placebo infusions (Figure 3C). In this way, the relative potency and energy costs of different cardiotonic medications and doses can be compared to one another independent of their effects on loading conditions.
Figure 1: Diagram of Flow in a Fully Instrumented Heart in Retrograde Perfusion and Working Heart Mode. (Panel A: Langendorff mode; Panel B: working heart mode. In retrograde mode, KHB is infused at a set perfusion pressure into the aortic root. This mode is utilized to recover the myocardium following ischemic time and during instrumentation. In working heart mode, perfusate flows through the left heart before perfusing the coronary circulation. In this mode, the myocardium must generate its own perfusion pressure. Please click here to view a larger version of this figure.
Figure 2: Representative Pressure and Volume Tracings Obtained during Baseline Measurements. (A) Aortic root pressure, (B) left atrial pressure, (C) left ventricular pressure and (D) left ventricular volume tracings during a baseline measurement are displayed. Stroke volume, stroke work, cardiac output, tau, and other parameters can be automatically calculated and displayed in real-time by the software. A blunted left atrial tracing (E) associated with a poor cardiac output in working heart mode can be a clue that the cannula is malpositioned in the left atrium. Note that the prominent v wave in the well-placed left atrial pressure tracing is common, likely due to a decreased left atrial compliance in the fully instrumented animal. Please click here to view a larger version of this figure.
Figure 3: Effect of Dopamine on the Pressure-Volume Curve. Dopamine infusion results in a leftward shift in the PV curve (A), including an increased stroke volume, decreased end systolic volume, compared with baseline measurements. Note that the shape of some components of these PV curves differ from those typically measured in vivo (see Figure 4) due to the absence of arterial and venous elastance. (B) Relative to an immediately preceding baseline, stroke work increased significantly more during infusions of dopamine than placebo (**, P = 0.0017, t-test), as did myocardial oxygen consumption (*, P = 0.013, t-test, C). Using this model, the average myocardial oxygen consumption at baseline was 0.22 ± 0.02 mmol O2/gram tissue/minute, using an estimated dissolved oxygen content of 165 µmol/L in saline at 40 °C. Such measurements can be used to compare the myocardial oxygen consumption of various medications. Please click here to view a larger version of this figure.
Figure 4: Analysis of Pressure Volume Loops. The Theoretical Pressure-Volume Loop Shown Describes the Normal Cardiac Cycle. Following aortic valve (AV) closure (1), isovolemic contraction occurs (1 - 2) as ventricular pressure decreases below atrial pressure. The duration of this phase is represented by Tau. The mitral valve (MV) then opens contemporaneously with atrial systole, filling the ventricle (2 - 3). Systole then commences with isovolemic contraction (3 - 4) until ventricular pressure exceeds diastolic arterial pressure, at which time the AV opens. Stroke volume is the difference between lines 1 - 2 and 3 - 4. Stroke work is the area within the 1 - 2 - 3 - 4 curve. Please click here to view a larger version of this figure.
This working heart model enables assessment of ventricular performance with full control of ventricular preload and afterload, oxygen tension of the perfusate, as well as heart rate. Among other factors, it permits assessment of the intrinsic myocardial effects of inotropic medications independent of afterload and preload, which ways that are not possible using an in vivo model. Because this model utilizes a crystalloid perfusate, it permits assessment the myocardium without interference from hemoglobin, simplifying spectroscopic analysis of myocardial energy states, for example.14 In this model, the right atrium is not cannulated as part of our instrumentation, though it is possible to do so. We intentionally chose not to do so in order to facilitate sampling of coronary sinus flow for the assessment of myocardial oxygen consumption. Importantly, though, the right heart still performs pressure and volume work in this model as it pumps the coronary sinus flow into the pulmonary artery cannula. Providing some right ventricular preload improves positioning of the ventricular septum and enhances left ventricular performance, and is an important component of this model.15
There are several experimental pitfalls to mention. The first is the initial retrograde cannulation, which should be performed expediently (i.e., in less than 2 min) to minimize the period of ischemia. The most important skill to master is the efficient isolation, preparation and handling of the ascending aorta. It is important that the aortic stump not be cut excessively short, leaving insufficient room for cannulation above the aortic valve. However, it is also important that the aortic stump not be too long, which can cause torqueing of the aorta around the cannula. It is also important that the aorta cannula and aortic root be appropriately size-matched. An excessively large aorta on a small cannula can also lead to torqueing of the aortic root on the cannula. The right subclavian artery typically takes off from the ascending aorta approximately 7 mm above the aortic valve. Identifying the brachiocephalic vessels (approximately 1 mm in diameter) during dissection and trimming of the aorta serve as important landmarks for the transverse aortic incision. Trimming the aorta just below the takeoff of the first brachiocephalic artery is advisable. Inclusion of this vessel in the trimmed aortic root typically leads to a leakage of KHB, and loss of aortic root pressure upon transitioning into working heart mode.
Another technically challenging aspect of cannulation is the left atrial cannulation. Although it is feasible to cannulate the left atrial appendage, we found that the cannula frequently gets stuck within the appendage, and does not pass easily into the body of the left atrium. Thus, we prefer to make the incision in the body of the left atrium, approximately 2 mm superior to the atrioventricular groove. It is important to position the left atrial cannula in the proper plane before insertion in order to avoid tearing the thin-walled atrium when securing the cannula.
We found that the ideal size of the left atrium incision was approximately 3 mm. Creating too small of an incision may also make the placement of the left atrial cannula more difficult, and may lead to tearing of the left atrium. We use a straight, 8 mm, beveled piece of oxygen-impermeable tubing (inner diameter 2.9 mm) on the left atrial block. We have found that using this, rather than a cannula with a beveled edge, leads to most consistent atrial cannulation and facilitates the process of securing the left atrial block. Regardless of the tubing used, it is important to ensure that the end of the tubing is not occluded by the atrial septum or the mitral valve (as depicted above, we found that the left atrial pressure tracing was helpful in this regard), as even subtle movement of the atrial cannula can significantly alter left ventricular preload and resulting hemodynamic measurements. For the same reason, it is important to ensure that the left atrium does not leak following after opening the left atrial block. It is important regardless of the type of tubing used to ensure that the tubing within this system is oxygen impermeable to ensure adequate oxygen delivery to the heart.
Another technically challenging aspect of the procedure was the placement of the pressure-volume (PV) catheter. We initially favored a retrograde placement of the catheter through the aortic block. Though technically feasible, we found it to be much simpler and expedient to place the PV catheter via transapical puncture. Care must be taken to monitor the position of the catheter throughout the duration of the experiment, as at times the catheter may move in or out of the left ventricle. This can be done by monitoring the pressure and volume tracings over time.
Finally, care should be taken to ensure that KHB solution is created fresh for each experiment. It is possible to weigh out the constituents of KHB and store them in conical tubes in powdered form ahead of time. On the day of experimentation, these may be mixed with sterile, filtered water, carbon dioxide/oxygen, and then calcium added to the mixture. It is also important to wash the system with enzyme active powdered detergent such as Tergazyme (or similar) and replace the perfusate filter regularly.
Several limitations of this experimental preparation should be noted. First, similar to all crystalloid-perfused Langendorff preparations, KHB and other asanguinous perfusates have a significantly diminished oxygen carrying capacity relative to blood. Although this is partially compensated for through coronary vasodilation and supraphysiologic coronary flow, the preparation is not entirely physiologic for this reason. Second, because of the nearly infinite compliance of the Windkessel chamber used in this instrument, the systolic and diastolic pressures are only minimally separated (see Figure 2A) and thus the coronary perfusion pressure is non-physiologic. This may be overcome in future models by incorporating an elastance component to the afterload block. Third, as with all isolated heart preparations, the heart undergoes a defined period (2 - 3 min) of warm ischemia which is likely to create myocardial injury or dysfunction. Minimizing this injury through practice of the technique is of utmost importance to representative results. Further, although necessary for animal welfare, inhaled anesthetics may serve as a myocardial suppressant early in the reperfusion process, though it is expected that this effect is quickly abolished as the heart is reperfused with KHB.
The working heart system described allows for a wide variety of physiologic investigations relevant to patient care, research and teaching. With a few additional modifications, the system can also be used to simulate important physiology relevant to congenital heart disease, including pulmonary hypertension and single ventricle physiology. Limitations include that it is an ex vivo preparation, that the heart is being perfused by a buffer instead of a higher-oxygen content blood.
The authors have nothing to disclose.
The equipment and experiments described here were funded by the Department of Cardiology, Boston Children's Hospital and by philanthropic donations from the Haseotes family. We are grateful to Drs. Frank McGowan and Huamei He for providing us with early experiences with this model, and to Lindsay Thomson for assistance with artwork.
|8.401 g/4 L
|0.744 g/4 L
|1.580 g/4 L
|0.578 g/4 L
|0.220 g/ 4 L
|27.584 g/4 L
|7.208 g/4 L
|Calcium chloride dihydrate
|1.470 g/4 L
|Biventricular working heart model
|Pressure volume catheter
|6 mm spacing catheter used
|LabChart Pro 8
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