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J Thorac Cardiovasc Surg 1995;110:239-247
© 1995 Mosby, Inc.
SURGERY FOR ACQUIRED HEART DISEASE |
New York, N.Y.
From the College of Physicians and Surgeons, Columbia University, New York, N.Y.
Received for publication May 17, 1994. Accepted for publication Oct. 18, 1994. Address for reprints: James P. Slater, MD, Department of Surgery, Columbia Presbyterian Medical Center, Box 188, 622 West 168th St., New York, NY 10032.
Abstract
The purpose of this paper is to describe an isolated heart model that uses human hearts to study cardiomyopathy. Techniques of organ preparation and perfusion are described that result in successful restoration of explanted human hearts to a beating condition. Native hearts of transplant recipients were placed on an isolated perfusion circuit immediately after explant. After appropriate organ and circuit preparation these hearts were restored to a functional state. Studies were done to assess the stability over time and response to inotropic stimulation. Six of seven hearts were returned to a functional condition. Left ventricular pressure generation ranged from 56 to 118 mm Hg (mean 84.8±34.11) at physiologic loading conditions. Hearts remained functional from 67 to 271 minutes (mean 152±71.7) and retained up to 70% of functional capacity after 120 minutes. Hearts performed in isovolumic and ejecting modes. Hearts had a marked response to inotrope administration not previously described. We conclude that human hearts can be reproducibly restored to a functional condition after explant from transplant recipients and can be maintained in a beating state with reasonably stable pressure generation for an extended period of time, which makes this a useful model with which to study cardiac pathophysiologic conditions. These hearts demonstrate an appropriate response to inotropes not previously observed, most likely because of improved myocardial preservation and stringent control of perfusate chemical make-up. (J THORACCARDIOVASCSURG1995;110:239-47)
In 1895 Langendorff
1 first described the use of an isolated heart preparation as a model with which to study myocellular and myocardial function. With the use of animal hearts this model has yielded information that has become the cornerstone to understanding cardiac physiologic processes. 2, 3 A shortcoming common to all of these studies is the necessity of using animal-derived data to extrapolate to the human condition. This problem is compounded by the need to induce or otherwise experimentally create the heart failure condition in studies of pathophysiologic conditions.
In 1988 Burkhoff and co-workers 4 recognized these limitations and reported a new Langendorff model that made use of cardiac transplant recipients' native hearts. These hearts were restored to a beating condition by being placed on a cardiopulmonary bypass circuit immediately after explantation. This was the first reported use of the Langendorff model in which the mechanics of human hearts were studied. 5 Although not completely representative of the in vivo state, data from this model complement previous efforts to describe human cardiomyopathy. There are several limitations, however, with the model. As noted by the authors, 4 this preparation requires a mandatory period of ischemia, which may diminish cardiac function. No information is obtained from the hearts in vivo, which makes comparison between the in vivo and ex vivo states difficult. Finally, no comparison data from normal hearts are available. Several other limitations are apparent. Most notably, the perfused hearts beat for only a brief time and exhibited stable cardiac function over an even shorter period. In addition, no response to inotropic challenge was observed. Beyond listing the initial constituents of the perfusate no assessment of blood chemistry components was made throughout the experiment. Last, a univentricular rather than biventricular model was used.
The purpose of this paper is to describe an improved, biventricular, isolated human heart model for the study of cardiomyopathy. We present techniques of organ preparation and describe perfusion techniques that result in successful return of human hearts to a beating condition after explant. We assess the stability of cardiac function over time and test the responsiveness of the heart to inotropic drug administration.
METHODS
Clinical evaluation
All patients were participants in the cardiac transplant program at the Columbia-Presbyterian Medical Center and underwent standard evaluation before acceptance, including detailed demographic profile; echocardiography or gated blood pool scan, or both; and cardiac catheterization. Catheterization data from the right side of the heart were obtained from a Swan-Ganz catheter (Baxter Healthcare Corp., Edwards Division, Santa Ana, Calif.) placed at the time of transplantation. All information presented was obtained for clinical use. No additional information was obtained for this study.
This study was done with the approval of our Institutional Review Board.
Isolated heart preparation
Organ procurement and preparation.
Patients were brought to operation for the purpose of orthotopic cardiac transplantation. Patients were anesthetized, and cardiopulmonary bypass was established in standard fashion. Hearts were arrested with 10 ml/kg of 4:1 blood:crystalloid (KCl 120 mg/L, mannitol 12.5 gm/L, HCO3 mEq/L) cardioplegia solution administered through an aortic cannula after application of the aortic crossclamp. Hearts were explanted leaving the majority of atrial tissue and ascending aorta in situ. The hearts were cooled to 4º C by immediate immersion in an iced saline bath in the operating room to minimize warm ischemic time. All subsequent preparation was done with the heart in this bath.
A modification of techniques previously described was used to prepare the hearts. 4 In the initial five hearts chordae tendineae were left intact. In the last two hearts all chordae to valve leaflets were severed. Oblong-shaped brass rings were sutured to the mitral and triscupid anuli. Once positioned these rings blocked both the left and right ventricular (LV and RV) outflow tracts. These rings were fitted with cuffs containing set screws that positioned the heart on a volume servo system (discussed later). DeBakey coronary perfusion cannulas (C. R. Bard Inc., Tewksbury, Mass.) in 14F and 12F sizes were inserted into right and left coronary ostia, respectively. A Gundry RCSP catheter (DLP, Grand Rapids, Mich.) was inserted into the coronary sinus and the balloon inflated. The heart was then transported to the laboratory in the water bath.
Perfusion system.
The perfusion circuit was an open system similar to that previously described and illustrated in Fig. 1. 4 The system consisted of a COBE CML Ultra membrane oxygenator with a cardiotomy reservoir containing a 30 µm filter (COBE, Arvada, Colo.), a circulating water bath, a hollow fiber dialysis tubing cartridge (COBE), a roller head pump (Sarns, Ann Arbor, Mich.), an in-line pH and temperature meter (Cardiovascular Devices, Inc., Ann Arbor, Mich.), and 1/4 - and 3/16 -inch connective tubing. Total circuit volume was 600 ml.
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The system was primed with 750 ml Dianeal low calcium (25 mg/L) peritoneal dialysis solution with 1.5% dextrose (Baxter, Deerfield, Ill.) and debubbled. To the prime was added two units of recently outdated, type-specific, noncrossmatched packed red blood cells. The red blood cells were washed to remove excess potassium and citrate by mixing with 250 ml normal saline solution and centrifuging at 4400 rpm for 5 minutes followed by decanting. Two hundred units of heparin and 300 mg of calcium gluconate were added to each unit of packed cells. An additional 1000 units of heparin was added to the circuit. The prime was recirculated through the system against a heated water bath and the perfusate allowed to come to 37º C.
Before the heart was reperfused corrective measures were required to adjust potassium concentration, pH, and calcium concentration of the perfusate to make the solution physiologic. The perfusate was dialyzed against the same dialysis fluid used in the prime supplemented with 600 mg of calcium gluconate. Initially no potassium was added to the system and the perfusate was dialyzed to a potassium concentration of less than 1.5 mEq/ml. Once this low potassium concentration was achieved approximately 2 mEq of potassium was added directly to the prime and an additional 2 mEq/L of potassium was added to the dialysate. This resulted in a potassium concentration of 3.5 to 4.5 mEq/L in the perfusate, which was maintained throughout the experiment. Initial pH measurements were acidotic (pH 6.5) because of the composition of the dialysis fluid and stored blood. This was corrected and proper pH maintained by addition of approximately 50 mEq sodium bicarbonate directly to the perfusate. Similarly, calcium gluconate was added to the dialysate to maintain an ionized calcium level of approximately 0.9 mEq/L. Electrolyte and pH values were monitored throughout the experiment using a Nova Stat Profile 5 (Nova Biomedical, Waltham, Mass.) and the in-line pH monitor (CDI). ACT determinations just before reperfusion were higher than 200 seconds and remained at this level for the duration of the experiment.
The perfusate was oxygenated through the membrane oxygenator with an admixture of compressed air and carbogen (95% oxygen, 5% carbon dioxide2 ). The sweep gas and percent carbogen were adjusted to maintain a carbon dioxide tension of 30 to 34 torr. The hemoglobin oxygen saturation was 100% throughout the experiment as determined by periodic determinations of blood gas values.
The preparation described was done in the physiology laboratory before explant of the heart. When the heart was brought to the lab all parameters had been corrected and were within physiologic range. In this manner, the heart could be reperfused immediately on its arrival and ischemic time minimized.
Servo system.
After the surgical preparation was completed, the heart was attached to RV and LV volume servo-control systems. Details of their design have been reported previously. 6, 7 In brief, motor-driven piston pumps regulate the volume of a balloon placed within each ventricular chamber (see Fig. 1). The mitral and triscuspid rings serve to confine the balloon within the respective ventricular chamber. This system therefore provides the means to measure and control ventricular volume. In addition, a micromanometer sensor inside each balloon is used to measure intraventricular pressure. Both RV and LV volume servo systems are controlled by a computer that is programmed either to impose physiologic ejection and filling flow patterns or to constrain the ventricles to contract isovolumetrically as required by the protocol. Details of the design of this computer control system are similar to those described previously. 8, 9 Briefly, a digital computer was programmed with the differential equations describing the entire circulatory system with parameter values that can be adjusted from the computer keyboard. 9 The computer digitizes the instantaneous LV and RV pressures and calculates the appropriate instantaneous flow in or out of each ventricle for the specified simulated vascular properties. These flow signals are integrated and used as command signals for the RV and LV volume servo systems, respectively. The mode of contraction can be switched to isovolumic when desired, at which point there is independent control of the volume within each ventricle.
Reperfusion.
The heart was initially reperfused in retrograde fashion via the coronary sinus catheter to remove air from the coronary arteries. After a short time retrograde perfusion was stopped, the coronary sinus balloon was deflated and opened to air, and antegrade flow was initiated. Several hearts fibrillated initially on rewarming and required electric cardioversion with ventricular pacing. These hearts restarted after one 30-joule shock only. All hearts were paced via pacing wires inserted into the LV apex. Temperature was maintained between 35º C and 37º C by use of a circulating water bath and was monitored by both epicardial surface probe and the in-line temperature monitor (CDI).
Protocol.
The purpose of this experiment was to test the stability and responsiveness to inotropic stimulation of isolated myopathic human hearts and to demonstrate the potential usefulness of this model for the study of cardiac pathophysiologic conditions. To this end our experiments consisted of returning the hearts to a beating condition, showing acceptable pressure generation, demonstrating stability of function over time, and exhibiting an appropriate response to inotropic drug administration.
RESULTS
Seven human hearts were obtained from patients with end-stage congestive heart failure at the time of cardiac transplantation. Patient demographic data (age, sex, weight, and height) are presented in Table I along with pertinent transplant evaluation data including diagnosis, surgical history, and preoperative cardiac assessment. In addition, postinduction, intraoperative hemodynamic data taken at the time of transplantation are presented in Table II.
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We demonstrate that it is possible to reproducibly revive excised human hearts on a perfusion circuit for the purpose of studying cardiac pathophysiologic conditions. Although laboratory experimentation necessitates imposition of artificial conditions, we believe that this model will be useful in future studies of human heart physiologic and pathophysiologic conditions.
Despite the large number of reported studies on isolated animal heart preparations we believe our model and methods to offer certain advantages. With this model there is no need to extrapolate from data derived from animal models of heart failure. In contrast to results in a previous experience with this model we were able to demonstrate prolonged, reasonably stable cardiac function in repeated trials. In addition, this preparation exhibited the appropriate response to pharmacologic challenges with both inotropes and myocardial depressants not previously shown. In comparison with the previous study of isolated human hearts these data indicate that the methods reported here result in more stable ventricular function, which may result from improved myocardial preservation. The ability to obtain data from both ventricles represents a technologic improvement over the previously used univentricular model.
We believe that the success of this model is dependent on two key elements: expedient preparation of the organ and meticulous preparation and maintenance of the blood perfusate. The heart must be placed in a cold saline bath immediately on explant. Heart preparation and transport must take place with the heart submerged to ensure that the heart is kept cold up until the time of reperfusion. This is required to minimize warm ischemic time. The perfusion circuit must be set up and primed well in advance of organ availability. In our experience the circuit and perfusate require approximately 2 hours to set up and prime. Physiologic parameters are reached approximately 
hour before organ arrival. Frequent electrolyte and blood gas value determinations are essential to make the necessary adjustments to the perfusate. A stable circulating water bath is also required to maintain physiologic conditions.
Having established the feasibility of this model we believe that its use may provide an opportunity to study specific aspects of myocardial disease not previously done in human beings. Ventricular interdependence has been shown to exist; however, the degree to which RV function is dependent on LV function for pressure generation in the diseased human heart is unknown. 10 The load-dependent nature of ventricular performance may also be more accurately quantified in the diseased human heart. This model may be used to compare different cardiac preservation solutions under varying delivery techniques. Properties of myocardial metabolism are also easily studied in these hearts.
Analysis of the experimental data presented reveals isovolumic peak LV pressure generation between 15 and 70 mm Hg without inotropic support. In an isovolumic model this represents a decrease in contractility compared with in situ conditions even for end-stage congestive heart failure. For example, previous studies that used normal dog hearts showed peak systolic pressures in excess of 155 mm Hg. 11 Potential reasons for this relatively low pressure generation include ischemic insult during the period of nonperfusion, suboptimal perfusate composition, and loss of inotropic support. No similar studies have been done on human hearts without heart failure and data from normal control hearts might better place in perspective the performance of these diseased hearts. Potential sources for such organs might be patients who donate organs other than the heart in whom the heart has been judged unsuitable for clinical use.
Previous studies have suggested that disruption of the valvular apparatus results in impaired ventricular pressure generation. 12 More recently other investigators have shown little effect on ventricular function after the chordae are severed. 13 This experiment depends on accurate volume measurement and assumes that the intraventricular balloon will conform to the shape of the ventricular cavity. Intact anterior chordae tendineae act as a barrier to the balloon, preventing it from coming to rest along the septal wall of the ventricle. To avoid this problem we have elected to sever the chordae tendineae.
In conclusion, we have presented a unique model for the study of human cardiac pathophysiologic conditions. Explanted human hearts obtained at the time of cardiac transplant can be reproducibly restored to a functional condition on a perfusion circuit. The heart can be maintained in a beating state for an extended period. Pressure generation remains reasonably stable over this interval, which allows for accurate study of ventricular properties of the diseased heart. These hearts respond appropriately to inotrope administration by increasing pressure generation. The longer duration of viability and responsiveness to inotropic stimulation represent improvements over previously used techniques to study isolated hearts. These improvements are likely caused by better myocardial preservation and more stringent control of blood perfusate chemistry values.
Acknowledgments
We acknowledge the assistance of Linda Mongero, James Beck, Joe Sistino, and the entire Columbia-Presbyterian Perfusion staff for their assistance with this project.
References
This article has been cited by other articles:
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  A. J. Hill, T. G. Laske, J. A. Coles Jr, D. C. Sigg, N. D. Skadsberg, S. A. Vincent, C. L. Soule, W. J. Gallagher, and P. A. Iaizzo In Vitro Studies of Human Hearts Ann. Thorac. Surg., January 1, 2005; 79(1): 168 - 177. [Abstract] [Full Text] [PDF]
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E. Chinchoy, C. L. Soule, A. J. Houlton, W. J. Gallagher, M. A. Hjelle, T. G. Laske, J. Morissette, and P. A. Iaizzo Isolated four-chamber working swine heart model Ann. Thorac. Surg., November 1, 2000; 70(5): 1607 - 1614. [Abstract] [Full Text] [PDF]
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 J. P. Slater, E. C. Lipsitz, J. M. Chen, H. R. Levin, M. C. Oz, D. J. Goldstein, R. C. Ashton, and D. Burkhoff SYSTOLIC VENTRICULAR INTERACTION IN NORMAL AND DISEASED EXPLANTED HUMAN HEARTS J. Thorac. Cardiovasc. Surg., June 1, 1997; 113(6): 1091 - 1099. [Abstract] [Full Text]
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