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Eur J Cardiothorac Surg 2005;28:435-442
© 2005 Elsevier Science NL
Original articles |
a Department of Cardiothoracic Surgery, Nagoya University Graduate School of Medicine, Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan
b Department of Cardiac Surgery, Aichi Medical University, Aichi, Japan
c Department of Cardiovascular Surgery, Gifu Prefectural Tajimi Hospital, Gifu, Japan
Received 2 March 2005; received in revised form 12 June 2005; accepted 15 June 2005.
* Corresponding author. Tel.: +81 52 744 2375; fax: +81 52 744 2383. (Email: yaraki{at}med.nagoya-u.ac.jp).
Abstract
Objective: It is widely accepted that both valve and cardiac functions are closely correlated. In order to investigate the relationship between the valve and cardiac functions, we developed a model of an isolated working heart with crystalloid perfusate in swine. We investigated the feasibility of this model to evaluate the precise left ventricular function using the pressure–volume relationship and metabolic measurement. Another objective was as a trial for the imaging and analysis of valvular interventions with a high-speed digital camera on this model. Methods: Six isolated working hearts were subjected in the pressure–volume study, and additional three hearts were used in the valve imaging study. Measurement of the pressure–volume relationship was undertaken in situ before the heart was removed, and on the working heart mode during the initial 30min as the baseline, and at every 60min. Lactate levels were measured at every stage in the working heart mode. Mitral valve interventions were performed in three hearts, and valve motions were observed by a high-speed digital camera via the left ventricle. Results: The end-systolic elastance maintained a baseline level (5.17±2.25) until 180min and decreased at 240min (3.97±1.97, NS) and 300min (2.85±1.29, P<0.01) of the working heart mode as compared with baseline. The Tau maintained a constant level until 180min and increased at 240min (62.2±13.3, NS) and 300min (85.5±43.4, P<0.05) as compared with baseline (50.3±13.6). The slope of the end-diastolic pressure–volume relationship gradually increased with no significance until 180min and increased significantly (0.147±0.066, P<0.01 vs 0.067±0.041 at baseline). Lactate increased accumulatively. The total heart energy was reduced from the initial phase of the working heart mode. The valves were well captured by the high-speed digital camera. Conclusions: The systolic and diastolic functions of an isolated heart were preserved at an acceptable level for 180min. The practical reliability of the swine working heart model was demonstrated. This model will be used reliably for the investigation of the interaction of valve and cardiac functions.
Key Words: Animal model • Cardiac function • Heart physiology • Heart valves • Valve imaging
1. Introduction
There is a mutual relationship between cardiac valves and cardiac functions. The cardiac function in patients with heart valve disease may be changed by means of valvular surgery. The post-operative cardiac function can be affected by the procedure of valvular surgery (replacement or plasty), and the types and sizes of artificial prosthesis (mechanical valve or bioprosthesis). Therefore, an investigation of the relationship between the valve and cardiac functions is important, and many types of clinical research has been performed [1–4]. However, it is also important to clarify these relationships through experimental research. The isolated working heart of a large animal with crystalloid perfusion facilitates the analysis of both relationships by investigating the cardiac function and visualizing the valvular motion. A high-speed digital video camera would possibly enable a detailed time-related analysis of valvular motion on working heart models in swine. We therefore needed to clarify the stability and durability of the left ventricular function of this model as the control study. Chinchoy et al. [5] proved the durability of the physiological condition for 4h with crystalloid perfusate. The objectives of this study are (1) to study left ventricular performance in detail based on the pressure–volume relationship to confirm the reliability of the crystalloid perfused working heart, and (2) to examine the feasibility of valvular analysis by using high-speed digital photography.
2. Material and methods
The research protocol was approved by the Nagoya University Laboratory Research Animal Care and Ethics Committee. The investigation conforms to the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health and revised in 1996.
2.1 Working heart apparatus
The working heart apparatus consisted of a main reservoir, a base reservoir, a preload column, an afterload column, a heat exchanger and a roller pump (Fig. 1
). Each reservoir or column was connected with a 10mm polyvinyl chloride tube (ID, 10mm). The main reservoir of 30l volume was placed 80cm higher and above the isolated heart. A preload column of 5l volume was placed at the same level as the left atrium of the isolated heart. An afterload column with a side branch placed 80cm above the aorta was set up as shown in the figure. The base reservoir was set below the heart and received coronary perfusate (coronary flow) and LV ejection volume through the afterload column (aortic flow).
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The perfusate used was a modified Krebs Ringer Solution composed of the following ingredients (mmol/l): NaCl, 120.0; KCl, 4.0; MgSO4·7H2O, 1.3; NaH2PO4·2H2O, 1.2; CaCl2·2H20, 1.2; NaHCO3, 25.2; and Glucose 11.0. For each experiment 20l of perfusate was used. Perfusate was always circulating in the recirculation circuit by a roller pump at a 1l/min flow rate, and the fluid temperature of the main reservoir was maintained at 38°C. The isolated heart was then exposed to room temperature. Therefore, the intracardiac perfusion temperature settled within the physiological range (36–37°C) due to the natural drop in the room temperature (23–25°C). Oxygenation depends upon only the solubility of the gas, and the PO2 and PCO2 were maintained at 420–450 and at 35–45mmHg before the perfusate entered the heart.
2.2 Animal preparation and in situ pressure–volume measurement
Fig. 2
shows the experimental protocols. Six Landrace and Yorkshire swine weighing about 25–30kg were used for the pressure–volume study. The animals were anesthetized with intramuscular ketamine (7.5mg/kg) and atropine (0.5mg), intubated with a 5.5 Fr tube and mechanically ventilated. Anesthesia was maintained by inhalation of 0.02–0.04% Halothane. The right cervical vein was cannulated with an 8 Fr vessel catheter and lactate Ringer solution was administered to maintain volume. The right cervical artery was cannulated with a 14 G tube. After median sternotomy the heart was exposed.
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All cervical arterial branches were ligated. After the descending aorta was cross-clamped, the inferior vena cava and the main pulmonary artery were incised for intracardiac decompression. Through the 14G tube in the right cervical artery, 500ml of cold St Thomas solution (Miotecter, Kobayashi, Tokyo, Japan) was infused into the coronary artery. It was used for the cardioplegia, and was composed of the following ingredients (mmol/l): NaCl, 110; NaHCO3, 10; KCl, 16; MgCl2, 16; and CaCl2, 1.2. The heart and lung were excised and preserved in ice slush for 40min for valve surgery. Forty minutes is estimated to be sufficient to preserve the myocardium using St Thomas solution. All lung lobes were ligated so the stagnant blood in the lung would not mix with the crystalloid perfusate.
2.3 Experimental protocol of pressure–volume study in an isolated heart
After 40min of cardiac arrest, the aorta was connected to the main circuit of the perfusion system, and the heart was reperfused with the perfusate for the Langendorff mode. Langendorff perfusion was continued until the cardiac beat resumed and stabilized. A purse-string suture was placed on the left atrium, and the preload tube from the preload column was cannulated. After steady-state cardiac beats were confirmed, the perfusion mode was changed from Langendorff to the working heart mode. Preload volume was increased gradually, the left atrial pressure was maintained to 15cmH2O, and the heart began to eject.
A 7 Fr conductance catheter through the apex and the 2 Fr Millar catheter tip pressure transducer via the left atrium were positioned again in the left ventricle. The LV pressure–volume relationship was measured under the working heart mode. The ESPVR was assessed during abrupt preload reduction by a transient preload tube clamping during short periods (10–15s). Data were sampled in the working heart mode at the initial 30min and then every 60min until 300min. Data were acquired by a Leycom Sigma-5-DF signal conditioner processor (CardioDynamics BV, Leiden, Netherlands) and recorded on an IBM PC.
2.4 Data acquisition and analysis from Pressure–Volume relationship
The hemodynamic variables acquired from the pressure–volume study, which are heart rate (HR) and end-systolic pressure (Pes), were measured at in situ and every stage in the working heart mode. Secondly, the evaluation of LV performance was assessed with LV contractility, filling and relaxation, and energetics.
LV contractility to evaluate the systolic function was assessed by the parameter of the end-systolic elastance (Ees) as the load-independent index. The Ees was the biggest inclination of the ESPVR, which was plotted on the left upper corners of the pressure–volume loop (Fig. 3 , Appendix).
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LV energetics was assessed by PVA (pressure–volume area) and EW (external work)/PVA. EW can be approximated as the stroke work, which is the product of the stroke volume and the end-systolic pressure (Pes) [7]. PVA means total potential energy of the isolated heart and can be defined as the sum of EW and the potential energy. EW/PVA means the rate of EW, which is the efficiency of energy transfer from PVA to EW.
These indexes were automatically calculated by the Leycom Sigma-5-DF.
2.5 Biochemical assessment
To evaluate the ischemic anaerobic status, the lactate of the perfusate was measured at the initial 30min as the baseline and then every 60min after the working heart mode initiation.
2.6 Valve surgery and investigation using high-speed digital camera
The trial of valvular imaging was performed in the other three swine hearts (Fig. 2). Intracardiac interventions in the mitral position were performed during cardiac arrest. Suturing the plegets under the mitral leaflet for the markers, bioprosthetic valve replacement, and mechanical valve replacement were performed in every heart. In the steady state of the working heart mode, valvular imagings were performed using the high-speed digital video camera (FASTCAM-PCI, Photron, Inc., Tokyo, Japan). Photographs were recorded at 250 frames per second (fps).
2.7 Data analysis
Data are reported both in in situ and in the working heart mode. At first, in situ data were compared with the data of the initial phase of the working heart mode as a reference value. However, the load condition and experimental environment in in situ were quite different from the working heart mode. Therefore, the data from the initial measurement at 30min of the working heart mode were defined as baseline data because this shows the optimum cardiac function.
Data are presented as the mean value±SD. In situ data were compared with baseline data using the paired t-test to evaluate the difference between the in situ and working hearts. Data at every 60min in the working heart mode were compared with the baseline using repeated measures (ANOVA) with Dunnett's test for multiple comparisons. A P-value of <0.05 was considered statistically significant.
3. Results
3.1 Hemodynamic evaluation
Heart rate did not change significantly during the entire study period including the in situ and working heart modes, and a normal sinus rhythm was maintained (Table 1
). The Pes increased significantly at baseline as compared with in situ. The Pes gradually decreased as time progressed after the working heart mode. It decreased significantly after 180min following the beginning of the working heart mode.
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3.3 LV contractility
As shown in Fig. 4
a, Ees was significantly increased at baseline (5.17±2.25) as compared with that at the in situ mode (3.13±0.69). Ees was preserved without significant decline during the first 180min. It slightly decreased at 240min (3.97±1.97) and significantly at 300min (2.85±1.29, P<0.01) as compared with the baseline (5.17±2.25) of the working heart mode.
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3.5 Efficiency of energy transfer
The value of PVA and EW/PVA decreased at the baseline, but there were no significant differences between the in situ mode and the baseline. The PVA gradually and constantly decreased showing significant difference compared with the baseline (2684±849) after 60min of the working heart mode (2132±533, P<0.01) (Fig. 4d). The EW/PVA also decreased slightly since working heart initiation until 240min without significant difference, but decreased significantly at 300min (0.48±0.01, P<0.01) as compared with the baseline (0.63±0.11) (Fig. 4e).
3.6 Biochemical evaluation
The lactate levels of perfusate showed an accumulation trend (Fig. 5
) that increased linearly and significantly at 120min (5.72±3.09, P<0.05), 180min (7.02±3.69, P<0.01) and 240min (9.14±5.50, P<0.01) as compared with the baseline (2.38±0.97).
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Isolated heart preparations are widely used on small animals because of the simple handling and small scale systems. On large animals, however, there are many surgical factors involving intricate preparations and the usage of cardioplegia, and many methods and devices have been reported by investigators. A large animal heart is necessary for the investigation of physiological changes similar to the human heart. Recently, there was the report of isolated hearts using human donor hearts [9]; therefore, intracardiac anatomical imaging under the physiological cardiac cycle may well become attractive research. Intracardiac imaging would require a transparent crystalloid perfusate. However, it has been controversial whether or not the isolated heart of a large animal will continue to work with only the dissolved oxygen of a crystalloid solution in a working heart model. There are only a few methods invented for the isolated working heart in a large animal ever reported. Van Rijk-Zwikker and colleagues [10] described a method in which only the coronary arteries, selectively cannulated, were perfused with the animal's own blood, and the left ventricle was made to pump a clear saline solution. Portnoy and colleagues [11] or Sunagawa and co-investigators [8] reported methods using cross-circulated fresh blood of the supported animal. They used the blood perfusion for stability of the myocardial function. However, the presence of blood disturbs the visualization of intracardiac valves. Further, the methods reported by Van Rijk-Zwikker and colleagues [10] require an expensive system and a high level of proficiency as in selective coronary perfusion. Our preparation and apparatus are considered simple, inexpensive and, most importantly, reproducible. Recently, Chinchoy et al. [5] proved that the swine heart continued to work with only dissolved oxygen in a crystalloid solution for 240min in a report of the hemodynamic characteristics and a pressure study of isolated swine heart. Their working heart model uses four-chamber perfusion rather than our two-chamber perfusion. Our system is simpler and differs from their system with respect to the circuit formation, load setting, and amount of perfusate. We developed our own swine working heart model to study the relationship between cardiac and valvular functions. We measured the precise cardiac function of the isolated working heart by using the P–V study as the control study to confirm the reliability of our model.
Derived from the P–V loop, we acquired satisfactory results in contractility, the abilities of filling and relaxation, and energetics. The LV contractility, as shown by Ees, was preserved with no significant decline for 3h in the working heart mode. The Tau and the EDPVR slope show the abilities of relaxation and filling as the diastolic function. The Tau kept the same value for 180min and increased gradually after 240min. The EDPVR slope increased with no significance within 180min. Thus, LV diastolic function indicated acceptable preservation for 3h in our experiment.
The consequence of the energetic change was of much interest. We examined PVA and EW/PVA as an index of cardiac energy. PVA shows the total energetic that the myocardium has, and represents the sum of both potential energy and external work. In this study, PVA decreased significantly after 60min of the working heart mode. The reason for this is likely to be that the perfusate is crystalloid and does not contain oxygen transfer as does blood. The perfusate gradually becomes unpurified with metabolites, and the energy of the myocardium may gradually be exhausted. Additionally, as the result of the cumulative increase of lactate, oxygenation might be insufficient. However, the ratio of EW/PVA indicated a very low-pitched down slope without significance. This means that effective external work can be virtually maintained for 3h. If blood is used, a continuity of cardiac energy will be expected. However, 3-h duration is sufficient for intracardiac inspection and evaluation.
Regarding oxygenation, the O2 content and the coronary flow are the important factors deciding O2 delivery. O2 content is determined by the following formula:
Assuming the parameters of Hb, SaO2 and PaO2 of the blood perfused in situ heart as 10g/dl, 0.98, and 90mmHg, the O2 content of the in situ heart is calculated into 13.4ml. In the isolated heart, the parameter of PaO2 was measured as about 420mmHg, so the O2 content of the crystalloid perfusate was calculated into 1.3ml. The ratio of O2 content of the crystalloid perfusate to the in situ blood perfusate was only 10%.
The coronary flow was not measured at in situ because it is too difficult. Coronary flow is calculated as 80ml/100g of myocardium, and our swine heart weighed about 300g, so the coronary flow was calculated into 240ml/min. In the isolated heart, we measured the coronary flow as about 900ml/min. Therefore, the coronary flow of the isolated heart increased about four times compared with that of the in situ heart. The coronary sinus oxygen saturation (ScsO2) of the in situ heart [12] was reported as 0.42–0.50. Assuming its value at 0.50, the partial pressure of O2 (PcsO2) was about 28mmHg based on the oxyhemoglobin dissociation curve. In the isolated heart, the PcsO2 after the coronary purfusion was measured as about 150mmHg. Then the O2 consumption of the blood perfused in situ heart was calculated as follows:
The O2 consumption of the isolated heart was calculated as follows:
The O2 consumption ratio of the isolated heart to the in situ heart was about 0.47. This means the ability of the O2 delivery of the isolated heart decreased 53% compared with the in situ heart, and the isolated heart did not receive sufficient oxygenation. The O2 content in the isolated heart was very low, but the remarkable increase of coronary flow compensated for the decrease of the O2 content. If the O2 content were increased, it would be expected to prolong the stability of the preparation. But the perfusate was exposed to atmospheric pressure, and it is difficult to increase the solubility of the gas. This is the limitation of this system. It is, however, adequate to perform the valve study for 3h with acceptable hemodynamics.
One major advantage of this experimental model is the simultaneous measurements of valve and cardiac functions, because both functions are closely linked to each other. Clinical investigation between both functions has been debated for a long time, but an experimental design to correlate both functions was not seen in the literature because of difficulties in measuring both parameters simultaneously. This model provides real-time information between valve and cardiac functions. In the future, we propose to evaluate the pressure–volume relationships concomitant with valvular investigations.
Another advantage of this model is the usefulness in analyzing valvular characteristics, for instance, the artificial valve or normal heart valve, because leaflet excursion can be visualized. Although clinical assessment of valve motion is now limited by using X-ray or echocardiography, these techniques provide no direct imaging, and leaflet motion is not clearly visualized. In research to analyze the movement of intracardiac parts, a method using radioactive markers was reported to measure the annular motion and subvalvular apparatus [13,14]. However, it is said to be difficult to evaluate leaflet motion within their study's limitations [13]. Van Rijk-Zwikker et al. [15] had succeeded in visualizing mitral valve motion, and reported the mitral apparatus in detail 15 years ago. The purpose of our experiment was similar to their work, and our future study will be the analysis of valvular movement using a high-speed digital camera along with the simultaneous monitoring of the cardiac function. This real-time visualization of leaflet excursion will provide precise information of valvular functions in the future study.
For the study of valve functions, we have made use of a high-speed digital camera. This system enables us to analyze 1000 frames per second. Therefore, it is possible to calculate the velocity of leaflet motion, the duration of the opening and closing of the leaflet, the timing of the opening and closing of the valve, and the change in the annular diameter. This can provide more precise analysis than echocardiography or cine-angiography, and is, moreover, a direct image compared with other imaging modalities. The high-speed digital camera can also provide not only images but also more detailed digitalized data. For instance, by means of an auto-chase program with the markers of Fig. 6a, we can calculate the change of annular diameter every 4ms (Fig. 7 ). This is the data that could be revised by two-dimensional analyses; however, three-dimensional revision is the key issue that must be solved in the future.
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5. Conclusion
The systolic and diastolic functions of the isolated working heart in swine indicated an acceptable preservation for 180min by this system. However, the total heart energy was reduced from the initial phase of the working heart mode. We acquired dynamic visualization of heart or artificial valves using a high-speed digital camera. This system is useful for study of the relationship between valve and cardiac functions.
6. Study limitation
The circulation of the working heart preparation was quite different from that of the in situ condition, and the comparison of physiological performances therefore appears difficult. The perfusate used is crystalloid and not blood, so the viscosity is quite different in the two conditions. The four-chamber study was reported for mimicking in situ physiological circulation [5]; however, it is not clear if there will be any alteration of the left ventricular function between the two- and four-chamber studies. So, we applied the two-chamber study of the left side because of the simple preparation. Further, it can be pointed out that the geometry of the left ventricle could vary in an isolated heart compared with the in situ condition because the heart hangs freely in the apparatus [9]. In our system, the position of the hearts were fixed similar to the in situ fashion, placed on their back as far as possible, and the precise left ventricular function of that position was clarified using the P–V study in this study. In a future study, the imaging of the left ventricular chamber would also be able to prove the alteration of the dimensions of structures caused by a change in the geometry of the left ventricle. Time-relating changes of phenomena can be measured by two-dimensional imaging. However, there is the unsolved problem that the measurement of dimensions needs to be a three-dimensional analysis.
Supplementary data
Appendix
Supplementary material
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejcts.2005.06.015
References
This article has been cited by other articles:
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  H. Hasegawa, Y. Araki, A. Usui, J. Yokote, S. Saito, H. Oshima, and Y. Ueda Mitral valve motion after performing an edge-to-edge repair in an isolated swine heart J. Thorac. Cardiovasc. Surg., September 1, 2008; 136(3): 590 - 596. [Abstract] [Full Text] [PDF]
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 S. Saito, Y. Araki, A. Usui, T. Akita, H. Oshima, J. Yokote, and Y. Ueda Mitral valve motion assessed by high-speed video camera in isolated swine heart. Eur. J. Cardiothorac. Surg., October 1, 2006; 30(4): 584 - 591. [Abstract] [Full Text] [PDF]
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