THE ISOLATED HEART PREPARATION

Transcription

1 Br. J. Anaesth. (1988), 60, 28S-34S THE ISOLATED HEART PREPARATION R. G. MERIN There are two basic reasons why the isolated heart has been used to study the effects of anaesthetics. In the first place, the use of an isolated heart allows precise control of the determinants of myocardial function: preload, afterload, heart rate and the perfusate. Second, the isolated heart allows investigation of cardiac metabolism to a much more controllable degree than does the intact animal. There have been two general methods for isolating the heart from the rest of the body. Perhaps the more physiological technique involves the heart-lung preparation as described by Knowlton and Starling [13]. A more modern adaptation has been the study of both animals and man during cardiopulmonary bypass. This topic is the subject of another paper in this issue and will not be considered further. The second, more simplistic, way of isolating the heart is merely to excise it from the body of the animal and then perfuse it, usually with a blood-free salt and nutrient solution. THE HEART-LUNG PREPARATION Among his may contributions to cardiovascular physiology and pharmacology, E. H. Starling [13] introduced the isolated heart lung preparation (fig. 1). The technique was certainly an invasive one! The heart was isolated in situ so that the inflow (vena cava) was supplied from a reservoir the height and volume of which could be adjusted. The outflow (aorta) was directed through one of the early Starling resistors so that the resistance to outflow could also be either maintained constant or adjusted as desired. In the more modern preparations either ventricular or atrial filling pressures are measured or controlled, the inflow is usually controlled by the height of the reservoir, the outflow is adjusted to the resistor, and cardiac output measured by either timed collections from ROBERT G. MERIN, M.D., Department of Anesthesiology, University of Texas Medical School at Houston, Houston, Texas, U.S.A. FIG. 1. Schematic diagram of Knowlton-Starling (1912) heart-lung preparation [13]. the resistor or, in some cases, flow probes on the aorta. The animal's lungs serve as the oxygenator and, in the case of inhalation anaesthetics, as the route of administration of the drug. For noninhalation drugs, appropriate concentrations are added to the venous reservoir. This preparation lends itself very well to the concepts of the Frank-Starling volume control of cardiac contractility and indeed, after the primitive experiments of Otto Frank using isolated frog hearts,

2 ISOLATED HEART PREPARATION was the major preparation in which this relationship was demonstrated. Although the heart-lung preparation does allow for the control of the determinants of cardiac function, responses to cardiodepressant drugs are exaggerated for two reasons. First, the body's normal reflex responses to cardiac depression are specifically and intentionally inhibited. Second, the preparation is traumatic and cardiac function is probably depressed to begin with. For example, in one of the most cited studies of the effect of anaesthetics on cardiac function, the heart-lung preparation was often supported by an infusion of adrenaline [21]! i Anaesthetic experiments using the heart lung preparation Among the earliest publications of the effect of anaesthetics on cardiac function using a heartlung preparation were those in 1949 of Moe and colleagues [16] and Woods and colleagues [27] who, respectively, showed dose-related depression of cardiac function, by cyclopropane and by the two ultra-short acting barbiturates, thiopentone and thiamylal. In the first comparative study, Fisher, Bennett and Allahwala [7] investigated the effects of chloroform, diethyl ether, cyclopropane and divinyl ether on the Starling heart-lung preparation, and reported that chloroform was the most depressant and that diethyl ether produced a dose-related depression, whereas cyclopropane and divinyl ether were reported to have less effect. On this side of the Atlantic, Prime and Gray [22] also compared ether and cyclopropane as well as investigating thiopentone and two new neuromuscular blocking drugs, gallamine and decamethonium. They observed marked depressant effects by thiopentone and cyclopropane (in only 3-5 % concentration) with a dose-dependent depression by diethyl ether similar to that reported by Fisher, Bennett and Allahwala [7]. No depression was seen with the neuromuscular blockers. Price and Helrich [21] also compared cyclopropane, diethyl ether and thiopentone, but further looked at the effect of hydrogen ion and nitrous oxide. They confirmed Prime and Gray's results [22] showing a dose-related depression in the ratio of cardiac output to right atrial pressure by ether, nitrous oxide, cyclopropane and thiopentone. They attempted to equate anaesthetic concentrations with this depression, and suggested that, for light surgical anaesthesia, cyclopropane, ether and nitrous oxide all produced 29S about a 50% depression in cardiac function, whereas thiopentone produced only a 30% depression. In a rather scantily documented report to the Council on Drugs considering the new anaesthetic, halothane, Burn and colleagues [2] also studied the effect of chloroform, ether, trichloroethylene and halothane on the Starling heart-lung preparation. They reported that halothane and chloroform produced a relatively equivalent dose-dependent depression of cardiac output, whereas both diethyl ether and trichloroethylene showed little effect at anaesthetizing concentrations. However, it is of some note that they administered the latter two anaesthetics for only 10 min, not realizing the importance of high blood solubility of these two agents as far as organ system effect was concerned. Finally, Flacke and Alper [8] reported that 0.25 and 0.5 % halothane had little effect on cardiac function, but 1 % produced a rapid and severe decrease in cardiac output, with simultaneous increases in right atrial and left atrial pressure. They also reported that reserpinized dogs responded in the same fashion and that an infusion of noradrenaline was able to reverse completely the effects of concentrations of halothane as great as 1.5%, but that higher concentrations, even in the presence of noradrenaline, resulted in depression of cardiac function. These experiments established the principle that all potent inhalation anaesthetics are direct dose-dependent cardiac depressants in the dog heart-lung preparation. The relatively minor effects of diethyl ether and cyclopropane in intact animals then suggest that either hormonal or central nervous system effects produced by these anaesthetics modify the direct cardiac depressant effects. EXCISED PERFUSED HEART Some of the early experimenters in cardiovascular physiology merely excised animal hearts, placed them in a dish containing saline and observed their beating response to various interventions. One of the first investigators to standardize an excised heart preparation, in 1895, was Langendorff [14] and, indeed, his preparation became a standard, recognized even to this day (fig. 2). Langendorff suspended the excised rat heart by its aortic cuff on a hollow metal tube connected via a stopcock and flexible tubing to a perfusion system which allowed control of perfusion

3 BRITISH JOURNAL OF ANAESTHESIA 30S FIG. 2. Copy of orginal engraving of the Langendorff (1895) preparation [14]. pressure and composition of perfusate. Function of this heart was measured by a hook in the apex connected to a lever system recording on a smoked drum which documented the displacement of the apex with each beat. A temperature-sustaining water jacket completed this relatively simple preparation. Major advantages of the Langendorff preparation were its simplicity and relative stability as long as the proper perfusate and perfusion pressure were maintained. However, this was a distinctly unphysiological preparation, for the heart in fact did not fuction as a pump, nor was there real preload. Subsequent modifications have introduced a fluid filled balloon to the left ventricle so that there was some sustained preload. In addition, this balloon allowed the measurement of developed pressure as well as the rate of pressure development (dp/ dr). Nevertheless, this was a relatively low work type preparation and, in particular, measuring ongoing metabolic performance of this heart was difficult. The major metabolic measurements that could be made used rapid "freeze clamping" of the heart for measurement of tissue metabolites, particularly the high energy phosphates. Seventy years ensued before different workers attempted to produce a more physiological isolated heart preparation. One of the more widely accepted of these has been that developed by Neeley and colleagues [17] whereby, not only was the heart suspended by the aorta, but the left atrium was cannulated also (fig. 3). Now, the heart actually pumped the perfusate using a "windkessel" type of aortic outflow resistance and cardiac output could be documented. In addition, it was possible to manipulate atrial and ventricular volumes and pressures and produce cardiac function curves similar to those obtained in the heart-lung preparation (fig. 4). Another modification also cannulated the pulmonary artery for measurement of oxygen consumption and ongoing metabolites without killing the heart by freeze clamping. However, this preparation was much more technically demanding and of limited stability (2 h), so that use of the Langendorff preparation continued even after development of this working heart preparation. However, even more than the heart-lung preparation, the isolated perfused heart was unphysiological. Obviously, only effects on the heart itself could be measured (the object of the preparation, after all!). Until the development, in recent years, of oxygenators for blood-perfused systems, blood-free perfusates were used. This limited the oxygen carrying capacity so that coronary blood flows were much greater than in the intact animal. However, these hearts were not ischaemic if properly prepared. The oxygen tension in the coronary effluent was consistently greater than 13 kpa; the hearts responded normally to changed

4 ISOLATED HEART PREPARATION To reservoir for prelimi perfusion Oxygenating chamber and reservoir " Peristaltic pump FIG. 3. The Neely (1967) working rat heart preparation [17] myocardial oxygen demand situations; induced ischaemia produced functional and metabolic changes identical to those observed in intact animals; and finally, the high energy phosphates, the ultimate result of adequate oxygenation, were present in amounts similar to those seen in intact animal hearts [18]. Many manipulations can improve the quality of these hearts, including nutrient content of the perfusate, harvesting techniques, temperature, etc. Of course, the relevance of data in one species for results in another must be viewed with some caution, especially as regards clinical management. Nevertheless, the isolated perfused working heart can provide valuable insights to the cardiac pharmacology of anaesthetic drugs. Although there were a number of workers around the turn of the century who looked at the effect of diethyl ether and chloroform on various 31S excised hearts, most of these descriptions were sketchy and very difficult to interpret. For example, Sherrington and Sowton [26] reported experiments in the excised gorilla heart! According to their description, chloroform 100 mg litre"1 produced a "90% reduction of the beat," whereas with 750 mg litre"1 "the beat was extinguished temporarily." They also noted "resistance to chloroform has been somewhat greater in this heart than that shown previously in the cat's heart." Not even a kymograph tracing was presented. Anaesthetic experiments in excised perfused hearts Langendorff. In 1962, Asher and Fredrickson [1] compared the effects of chloroform and halothane in a Langendorff rabbit heart preparation. They reported that the two anaesthetics produced an equivalent dose-dependent depression in function of this isolated heart, confirming the sketchy observations of Burn and colleagues [2] in the heart-lung preparation. Paradise and Bibbins [19] compared the effects of diethyl ether, chloroform, halothane and methoxyflurane in a Langendorff rat heart preparation. Like Price and Helrich [21], they tried to equate anaesthetic concentrations with cardiovascular depression. They reported that diethyl ether and methoxyflurane produced the least cardiac depression compared with the concentrations necessary for anaesthesia, halothane was intermediate and chloroform was the most depressant to the function of the isolated perfused rat heart. Kissin's group at the University of Alabama [11] and Wechsler's cardiovasuclar surgery laboratory at Duke University [9, 10, 20] were looking at both metabolic and functional effects of anaesthetics using a LangendorfF preparation. Wechsler's group was primarily interested in the modifying effect of anaesthetics (and other drugs) on the ischaemic rat heart. In a series of investigations (only one of which [9] is published in other than abstract form), they showed that the three clinically available inhalation anaesthetics halothane [20], isoflurane [9] and enflurane [10] had little effect on the pre-ischaemic tissue content of high energy phosphates, the time required for ischaemic contracture to develop or the tissue content of high energy phosphates at the time of ischaemic contracture. In the isoflurane and enflurane experiments, the hearts were also reperfused after ischaemia and both anaesthetics increased the percent recovery of pre-ischaemia

5 BRITISH JOURNAL OF ANAESTHESIA 32 S Bubble trap II j i ifilter Coronaryoutflow I Mixing column leiectrodel Collection or waste FIG. 4. Schematic diagram of Ross (1972) working rat heart preparation [25]. developed pressure as well as tissue content of high energy phosphates. The same was also shown for the calcium channel blocker, nifedipine but, interestingly enough, there was no additive effect of the combination of nifedipine and enflurane [24]. Kissin's laboratory was also interested in the metabolic effect of anaesthetics, but they used a different approach. Using surface fluorimetry on a rat heart, they demonstrated that the three currently used inhalation anaesthetics and diethyl ether all produced an increase in myocardial reductive metabolism (NADH/NAD) [11]. At calculated equi-anaesthetic concentrations, ether produced the most increase in NADH, with enflurane intermediate and halothane or isoflurane producing the least. At anaesthetic concentrations producing a 50% decrease in cardiac function (dp/dr), ether and halothane produced the least increase in NADH, again with enflurane intermediate and isoflurane producing the most. In a companion study [12] in which a rabbit heart was used, the NADH production by halothane was shown to be a fraction of that produced by ischaemia and, when halothane was administered to the ischaemic heart, there was slightly less NADH production for an equivalent degree of ischaemia, suggesting that halothane at least did not aggravate the ischaemic production of NADH. The other thrust of the Kissin group in using the Langendorff rat heart was to look at drug interactions. Marshall and colleagues [15] showed that the interaction of halothane and nifedipine on the function of the isolated rat heart was mostly additive, and Reves and co-workers [24] indicated that the interaction of diazepam and fentanyl was also additive. Working heart. In their 1982 abstract [20], Wechsler's group also looked at the effect of halothane on a working rat heart preparation. They showed that 1.5% halothane produced a significant decrease in cardiac output and the product of cardiac output times mean arterial pressure at 5, 10, and 15mmHg left atrial pressure, thus confirming previous observations in both heart-lung preparations and intact animals concerning the negative inotropic effect of halothane. However, most of the anaesthetic experiments using the excised perfused working rat heart have come from our laboratory at the University of Texas Medical School in Houston, headed by Dr Leslie Cronau. In continuing experiments so far published only in abstract form [3-6], we have shown that halothane produces a dose-related depression in cardiac function while also decreasing glycolytic flux [5], When insulin was added to the glucose in the perfusate, essentially the same functional depression was seen, but glycolysis was somewhat less depressed [4]. Bupivacaine in equi-anaesthetic concentrations produced significantly more depression of the

6 ISOLATED HEART PREPARATION 33S TABLE I. Effects of halothane (H) and isoflurane (I) on working rat heart (mean values±sd). *P < 0.05 v. control; +P < 0.05 v. 0.8% halothane or 1.2% isoflurane Control 0.8% H 1.2% I 1.6% H 2.4% I MAP (mm Hg) Cardiac output (ml min" 1 ) Heart rate (beat min" 1 ) Coronary flow (ml min" 1 ) MAP x CO O 2 consumption (mol min" 1 g) Glucose uptake (mol min" 1 g) Tissue glycogen (mol g" 1 ) Lactate production (mol min" 1 g) 81.9 ± ± ± ± ± ± ± * * ± * * * ± * ± ± ± ± ± ± *t ± *t ± ± *f *t ±8 3.6*+ ± * * ± ± * t ± ± ± ±5 31.2*+ ±14 5.8* * * ± ±0.1 isolated working rat heart than did lignocaine [3]. However, lignocaine appeared to impair glycolytic flux more than bupivacaine. Finally, we have been investigating the comparative effects of halothane and isoflurane on function and metabolism in the isolated working perfused rat heart [6]. At equianaesthetic concentrations, halothane produced significantly more depression of cardiovascular function (mean aortic pressure and cardiac output) (table I). This effect was also accompanied by a smaller decrease in coronary blood flow. Similarly, myocardial oxygen consumption was unaffected by MAC concentrations of isoflurane, compared with halothane (table I). The effects on metabolism were more complex, but halothane also depressed glycolysis to a greater extent than did isoflurane. On analysis of the results, it appears that there is no qualitative difference between the effects of the anaesthetics on glycolysis in the working rat heart, but rather that the effects depend on the functional effects, particularly oxygen consumption. At equivalent myocardial oxygen consumptions, the effects of isoflurane and halothane appear to be similar. Thus although these experiments demonstrate that halothane produces more functional depression of the isolated working rat heart than does isoflurane at equi-anaesthetic concentrations, the mechanism of this difference does not appear to be related to the effect on glucose metabolism. SUMMARY The major advantage of the isolated heart over isolated cardiac muscle for studying the effect of anaesthetics relates to the maintenance of the anatomy and function of the heart as a pump and the use of the native coronary circulation for cardiac nutrition and oxygenation. For the latter function, perhaps the blood perfused heart-lung preparation is more physiological but less controllable, particularly for metabolic studies. However, both preparations are predominantly useful for evaluating mechanisms and comparative biochemical pharmacology, rather than being relevant for clinical management. REFERENCES 1. Asher M, Frederickson EL. Halothane versus chloroform dose response using the isolated rabbit heart. Anesthesia and Analgesia, 1962; 41, Burn JH, Epstein HG, Feigan CA, Pawn WDM. Some pharmacologic actions of fluothane. British Medical Journal 1957; 2: Cronau LH, Merin RG, Abouleish E, Steenberg ML, Melgarejo A. Comparative effect of lidocaine and bupivacaine on glucose uptake and lactate production in the perfused working rat heart. Federation Proceedings 1986; 45: Cronau LH, Merin RG, de Jong JW. Interaction of insulin and halothane on glucose utilization by the perfused working rat heart. Federation Proceedings 1985; 44: 680.

7 34S 5. Cronau LH, Merin R, de Jong JW, Kuttesch J, Rogers K. Effect of halochane on glucose metabolism in the isolated perfused working rat heart. Federation Proceedings 1983; 42: Cronau LH, Merin RG, Steenberg M, Melgarejo A. Comparative effects of halothane and isoflurane on glycolytic flux in the isolated working rat heart. Anesthesiology 1986; 65: A Fisher CW, Bennett LL, Allahwala A. Effect of inhalated anesthetic agents on the myocardium of the dog. Anesthesiology 1951; 12: Flacke W, Alper MH. Actions of halothane and norepinephrine in the isolated mammalian heart. Anesthesiology 1961; 23: Freedman B, Christian C, Hamm D, Everson C, Wechsler A. Isoflurane and myocardial protection. Anesthesiology 1983; 59: A Freedman BM, Hamm D, Everson CT, Wechsler A, Christian CM. Enflurane enhances postischemic functional recovery in the isolated rat heart. Anesthesiology 1985; 62: Kissin I, Aultman DF, Smith LR. Effects of volatile anesthetics on myocardial oxidation reduction states assessed by NADH fluorimetry. Anesthesiology 1983; 59: Kissin I, Thomson CT, Smith LR. Effect of halothane on contractile function of the ischaemic heart. Journal of Cardiovascular Pharmacology 1983; 5: Knowlton FP, Starling EH. Influence of variations of temperature and blood pressure on performance of the isolated mammalian heart. Journal of Physiology {London) 1912; 44: Langendorff O. Utersuchungen am uberlebenden Saugertierhersen. Pflugers Archiv fur der Gesamten Physiologie 1895; 61: Marshall AG, Kissin I, Reves JG, Bradley EL, Blackstone EH. Interaction between the negative inotropic effects of halothane and nifedipine in the isolated rat heart. Journal of Cardiovascular Pharmacology 1983; 5: BRITISH JOURNAL OF ANAESTHESIA 16. Moe GK, Rennick BR, Freyburger WA, Malton SD. Effect of cycloproprane on cardiac work capacity. Journal of Pharmacology 1949; 10: Neely JR, Liebermeister H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat heart. American Journal of Physiology 1967; 212: Opie LH. Adequacy of oxygenation of the isolated perfused rat heart. Basic Research in Cardiology 1984; 79: Paradise RR, Bibbins F. Comparison of effects of equieffective concentrations of anesthetics on force of contraction of isolated perfused rat hearts. Anesthesiology 1969; 31: Peyton R, Christian C, Fagraeus L, Van Trigt P, Spray P, Pellom G, Pasque M, Wechsler A. Halothane and myocardial protection. Anesthesiology 1982; 57: A Price HL, Helrich M. Effect of cycloproprane, diethyl ether, nitrous oxide, thiopental and hydrogen ion on myocardial function of the dog heart lung preparation. Journal of Pharmacolgy 1955; 115: Prime FJ, Grey TC. Effect of certain anaesthetic and relaxant agents on circulatory dynamics. British Journal of Anaesthesia 1952; 24: Reves JG, Kissin I, Fournier SE, Smith LR. Additive negative inotropic effect of a combination of diazepam and fentanyl. Anesthesia and Analgesia 1984; 63: Richards S, Davis G, Wechsler A. Effect of chronic nifedipine administration in the rat heart exposed to enflurane. Anesthesiology 1984; 61: A Ross BD. Perfusion Techniques in Biochemistry. Oxford: Oxford University Press, 1972; Sherrington CS, Sowton SCM. On the relative effects of chloroform upon the heart and upon other muscular organs. British Medical Journal 1905; 2: Woods LA, Wyngaarden JB, Rennick B, Seevers MH. Cardiovascular toxicity of thiobarbiturates: Comparison of thiopental and Surital in dogs. Journal of Pharmacology 1949; 95: