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Ann Thorac Surg 1995;60:767-772
© 1995 The Society of Thoracic Surgeons
Max-Planck-Institute for Physiological and Clinical Research, Department of Experimental Cardiology, Bad Nauheim, Germany
Abstract
Background. Recently, our group showed that in human hearts proteins are extremely sensitive to ischemic injury. The purpose of this investigation was to evaluate the effects of ischemia on contractile and cytoskeletal proteins in rabbit and pig hearts and to compare these findings with those obtained in humans.
Methods. Rabbit hearts were arrested by perfusion with Euro-Collins solution at different temperatures. Hearts perfused with buffer served as controls. Tissue samples were incubated for varying time intervals and processed for immunohistochemistry and electron microscopy. Porcine hearts were treated in the same manner. Changes in the localization of myosin, desmin, and tropomyosin antibodies were evaluated and the degree of ischemic injury was determined by electron microscopy.
Results. Healthy animal hearts tolerate ischemia better than human hearts. Cardiac proteins are more sensitive to ischemia than the ultrastructural cellular organelles. Temperatures as low as 0°C produce more cell damage than 4°C and should therefore be avoided. The Euro-Collins solution protects the myocardium better than buffer.
Conclusions. We conclude that healthy animal hearts are more resistant to ischemia than diseased human hearts and that results from experimental studies should be interpreted with caution with regard to the human situation.
In a recent publication [1] our group described the elevated sensitivity of cardiac proteins to ischemic damage in the human heart. We investigated human myocardium removed at the time of transplantation operations and incubated at various temperatures for different time intervals. The methods of investigation were electron microscopy for determination of the degree of ischemic injury in the tissue and immunohistochemistry employing monoclonal antibodies against various contractile and cytoskeletal proteins. From this study it became evident that the proteins of the contractile machinery, ie, myosin and the thin filament complex, are highly sensitive to ischemia, that the cytoskeleton shows a moderate sensitivity, and that proteins such as vinculin, responsible for the structural integrity of the myocyte, are the most resistant to the effects of ischemia. In general, the various proteins of the cardiomyocyte proved to be less tolerant to ischemia than the subcellular organelles such as mitochondria and cell nuclei. These findings were completely new and only possible with the availability of the modern histologic techniques of immunohistochemistry. They clarified the problem why human hearts need a certain period of time to recover from ischemia in the postoperative phase even though the ultrastructural appearance of the myocardial tissue is almost normal.
These findings are also important for the evaluation of the degree of preservation of donor hearts intended for transplantation. Because this problem usually is studied in animal hearts preserved by various means it seemed important to us to investigate the changes in protein localization in healthy animal hearts and to compare these with the findings obtained in diseased human hearts. We report here that myocardium from rabbit and porcine hearts is much more tolerant to the effects of ischemia with regard to protein integrity but that the ultrastructural changes in both situations are nearly the same. Caution should be applied, therefore, when interpreting findings in animal hearts and transferring them to the human situation.
Material and Methods
The experimental protocol described in this study was approved by the Bioethical Committee of the District of Darmstadt, Germany. All animals were handled in accordance with the American Physiological Society guidelines for animal welfare.
Rabbits
Eighteen male rabbits weighing between 5.0 and 6.0 kg were divided into five groups with 3 animals in each. The rabbits were premedicated with 0.4 mg/kg piritramide intramuscularly and anesthetized with 10.0 mg/kg pentobarbital. The thorax was opened and the hearts were perfused with Euro-Collins at 0°C, 4°C, 17°C, 27°C, and 37°C. The 3 remaining animals were anesthetized as described above and the hearts were perfused with phosphate-buffered saline (PBS) buffer at 10°C. The perfusion with either solution was continued until cardiac arrest was achieved. The hearts were then excised and cut into small pieces.
One sample of each animal was fixed in glutaraldehyde for electron microscopy and another was frozen in liquid nitrogen for immunohistochemistry for control purposes. The remaining tissue was incubated in Euro-Collins solution at temperatures corresponding to that of the perfusion solution. The tissue perfused with PBS buffer, however, was incubated in PBS buffer at 0°C, 4°C, and 37°C. Time points for the removal of samples for immunohistochemistry and electron microscopy at different temperatures and solutions were as follows:
Removal from the Euro-Collins solution
Pigs
Landrace pigs with a bodyweight of 20.0 to 25.0 kg were sedated with azaperone, 2 mg/kg intramuscularly, and anesthetized with pentobarbital, 30 mg/kg intravenously. After thoracotomy the heart was removed. Left ventricular tissue samples were taken and immediately frozen for control purposes. The remaining tissue was incubated in phosphate buffer at 20°C and 40°C, and samples were removed and frozen at 10, 20, 60, 120, and 240 minutes.
Immunohistochemistry
Cryostat sections 4 µm in thickness were fixed with 3% buffered paraformaldehyde at room temperature. They were first stained with hematoxylin and eosin to evaluate the state of tissue preservation and to select longitudinal sections. Those cut transversally or diagonally were reembedded to a longitudinal orientation.
Antibodies against myosin (kind gift of Dr R. Decker, North Western University, Chicago, IL) and tropomyosin (Sigma, Germany) were used to represent the contractile proteins and a desmin antibody detected one of the major proteins of the cytoskeleton (Sigma). The secondary detection system was a biotinylated donkey anti-mouse immunoglobulin (Amersham, England). The reaction was made visible with streptavidin coupled with the fluorochrome fluoroisothiocyanate (Amersham). Nuclei were stained with 0.0001% propidium iodide following the modified protocol from Jones and Kniss [2]. The sections were viewed in a Leitz Aristoplan or an Olympus Vanox T light microscope equipped with fluorescence filters and objectives, and micrographs were taken on Kodak professional 200 ASA color slide film. All micrographs presented here are reproductions from color slides.
The immunohistochemical results were assessed in several ways: (1) qualitative estimation of the localization of different antibodies. (2) determination of the time at which the first changes in localization were observed and the time when the changes were maximal, (3) measurements of the intensity of the fluorescent labeling using the photographic exposure time (in seconds) in the microscope. For these measurements glass slides were prepared with control (nonischemic) and ischemic tissue to standardize the exposure times measured, and all sections from the entire experiment were stained for one antibody at the same time. Care was also taken to avoid photobleaching and quenching. Ten randomly selected areas of each section were measured, resulting in a total of at least 100 areas per animal. Spot measurements were used.
Controls
Control tissue consisted of zero time nonischemic porcine and rabbit myocardium. Another control reaction involved omission of the first antibody to check for nonspecific staining by the detection system. All reactions on control and ischemic tissue were carried out simultaneously on the same slide to eliminate variations in labeling intensity.
Electron Microscopy
All samples, immersion fixed in 3% buffered glutaraldehyde, were postfixed in OsO4, rinsed in buffer, dehydrated in a graded series of alcohol and propylene oxide, and embedded in Epon. Ultrathin 50-nm sections were stained with uranyl acetate and lead citrate and viewed in a Philips EM 201 electron microscope.
All micrographs were evaluated for the degree of ischemic injury following a semiquantitative scoring system [3], and the data were compared with the results from immunohistochemistry.
Results
In rabbit and porcine myocardium the antibodies produced a cross striation pattern typical of the individual proteins labeled. These have been described previously [1].
In recent studies in ischemic human myocardium we observed that the cross striation was attenuated (recorded as beginning of the alterations) and later completely disappeared (recorded as fully developed disturbance of the antibody localization) [1]. The morphologic appearance of the beginning of the alterations was similar in rabbit, porcine, and human myocardium. However, the complete disappearance of the cross striations for the proteins investigated was typical for human myocardium only and never observed in rabbit and porcine cardiac tissue.
Rabbit Hearts
ELECTRON MICROSCOPY.
In Table 1
the degree of ultrastructural injury is listed in dependence of the duration of incubation and of the incubation temperature. It is obvious that the degree of injury increases with time of incubation, and temperature of incubation, and that injury is greater at any time point after cardiac arrest with PBS than with the Euro-Collins solution. Optimal tissue preservation is achieved at 4°C after perfusion with the Euro-Collins solution.
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. Figure 1 shows the normal localization of these three proteins, and Figure 2 shows examples of changes of the localization pattern after a longer period of ischemia. From Table 2 it becomes evident that the first signs of antibody dislocalization for myosin and tropomyosin occur as late as 2 hours after ischemia. The contractile proteins myosin and tropomyosin are affected by ischemia, but desmin is not. In contrast to the ultrastructural findings, the differences in the immunohistochemical appearance between the PBS and the Euro-Collins group are minimal.
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shows that the intensity of fluorescence is different at the end of the cardioplegic perfusion at different temperatures and that this is the decisive factor for tissue preservation (time 0 in the graph). During the incubation period itself changes in the intensity of the fluorescence were not observed. In addition, incubation at 4°C seems to provide the best cardiac protection, whereas 0°C certainly has the least beneficial effects.
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. In this table only the values obtained for 20°C are indicated because at 40°C the tissue deteriorated very quickly and was irreversibly altered at 20 minutes of incubation. Table 3 shows that the beginning of the alterations due to ischemia started to occur at 120 minutes for myosin and at 240 minutes for desmin, which is in accordance with the results obtained in rabbit hearts.
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The results of this study show that the temperature of the cardioplegic perfusion solution is a decisive factor for cardiac preservation, that a temperature of 4°C causes less damage than 0°C, and that the Euro-Collins solution should be preferred over a simple buffer. In our previous study [1] it was also shown that healthy animal hearts are significantly more resistant to the effects of ischemia than are diseased human hearts and that contractile proteins are more susceptible to the effects of ischemia than the components of the cytoskeleton.
Effects of Temperature
The effects of temperature on cardiac preservation have been studied extensively [4–7]. Nowadays, in routine heart operations the cardioplegic solutions are commonly used at a temperature of either 28°C or between 20° and 22°C [4, 7], but 10° to 15°C is also advocated [6]. Hypothermia is able to reduce significantly the metabolic rate and therefore oxygen consumption of the tissue and is therefore preferred over normothermia. There are, however, also several reports describing deleterious effects of low temperatures on the myocardial energy balance [8] and left ventricular compliance [5], and Sakai and Kuihara [9] report a relative hypoxia in hypothermia. Changes in pH [10], adverse effects on the adenosine triphosphatase system [11], and an increased Ca2+ overloading [12] have also been described. Therefore, several authors during recent years, have advocated the use of warm cardioplegia [13–15]. From the data presented here, however, it may be concluded that a reduction in the cardioplegic temperature preserves effectively the integrity of the cardiac proteins.
For the preservation of donor hearts, which often have to be transported over a longer distance, an ice-cold cardioplegic solution is preferred by cardiac surgeons, and several studies recommend the use of 0° to 5°C [16–20]. From the data of this study it becomes evident, however, that 0°C should be avoided and that 4°C is the preferable temperature. It also is evident that the cardioplegic perfusion temperature should be kept low and the hearts be preserved at the same low temperature to maintain structural integrity.
Contractile Versus Cytoskeletal Proteins
From our previous study in human hearts we know that all contractile proteins are less resistant to the effects of ischema than those of the cytoskeleton [1]. This finding was confirmed here. Therefore, in the present investigation only myosin and tropomyosin were used to determine the effects of ischema, and both again showed a similar sensitivity. Myosin localization can be used to estimate the degree of early injury of the myocardium, and the above statements on temperature dependence of tissue injury are mainly based on the changes in myosin as observed by immunocytochemistry (see Fig 3
).
Nishida and associates [21] found an early disappearance of actin labeling in ischemic rat myocardium, but Iwai and colleagues [22] reported a prolonged resistance of actin in canine myocardium, which is in accordance with our findings in both rabbit and porcine myocardium but not in human hearts.
Desmin belongs to the group of intermediate filaments and is one of the major components of the cytoskeleton of muscle cells [23]. Desmin was more resistant to the effects of ischemia than the contractile proteins, which confirms the findings in human hearts [1]. In contrast to the rapid deterioration of desmin localization in human myocardium, in rabbit hearts it was unchanged up to 24 hours of incubation, and in pig myocardium it showed slight changes starting at 240 minutes.
Healthy Animal Versus Diseased Human Hearts
In the present study it was shown that the contractile and cytoskeletal proteins of myocardium from healthy animals show a tolerance to the effects of ischemia that is significantly higher than that of diseased human myocardium. No differences were found in the tolerance of the subcellular organelles as observed in the electron microscope. To exclude the possibility that this rather unexpected finding was caused by the binding properties of one particular antibody, several antibodies against different epitopes of the same molecule were tested. All these antibodies were also used in the human heart. In addition, two different animal species, rabbits and pigs, were investigated. Identical results were obtained in both species and with the different antibodies. We therefore concluded that healthy animal hearts are more resistant to the effects of ischemia than diseased human hearts.
This interesting finding is difficult to explain. We believe that in the human heart the accelerated breakdown of cardiac proteins during ischemic conditions may have been caused by the presence of an increased amount of intracellular proteases that are absent in the animal hearts. It is possible that an increased depolymerization of filaments occurs during ischemia, which would explain the deranged cross striation pattern in the presence of a persistant, though reduced, fluorescence intensity. This hypothesis, however, will be tested in later studies.
In the situation of regional ischemia with stenosis or occlusion of one coronary artery, the degree of collateral circulation plays a major role in determining the tolerance to ischemia [24, 25], but in global ischemia it is unimportant. The same applies to the differences in oxygen demand that greatly influence infarct size but have little influence on the ischemic tolerance in global ischemia. These two factors, therefore cannot explain the differences in normal animal and diseased human hearts described here. The fact that a preexisting disease process has damaged the myocardium preoperatively, as described by our group [26], seems to determine solely the ischemic tolerance when the entire heart is made ischemic as in a cardiac operation.
Acknowledgments
Supported by a grant from the Minna-James-Heineman-Stiftung, München, Germany.
Footnotes
Presented at the International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, Sep 25-28, 1994.
Address reprint requests to Dr Sprengel, Department of Experimental Cardiology, Max-Planck-Institute, Benekestrasse 2, D-61231 Bad Nauheim, Germany.
References
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