Halothane exerts a potent negative inotropic effect on the heart and mimics many of the cardiac effects of lowered extracellular CaCl2. Reduced slow inward Calcium2+ current and sarcoplasmic reticular effects on intracellular Calcium2+ are likely involved. The authors reported previously that halothane protects against hypoxic and ischemia reperfusion injury in isolated hearts. The aim of this isolated heart study was to compare protective effects of halothane and low CaCl sub 2 (0.5 mM) administered during 1 day of hypothermic perfusion on return of normothermic perfusion.

Methods: Guinea pig hearts (n = 66) were isolated and perfused at 37 degrees Celsius with a Krebs' solution, gassed with 96% Oxygen2, 4% CO2, and containing 2.5 mM Calcium2+, and 4.5 mM Potassium sup +. Heart rate, isovolumetric left ventricular pressure, coronary flow, % Oxygen2extraction, Oxygen2consumption rate, and relative cardiac efficiency (EFF = heart rate *symbol* left ventricular pressure/Oxygen2consumption rate) were measured in five groups of hearts: time controls (no hypothermia); 1.5, and 3% halothane delivered by vaporizer; cold controls (hypothermia only); and 0.5 mM CaCl2. Halothane was administered, or CaCl2was decreased 0.5 h before hypothermia at 3.8 plus/minus 0.1 degree Celsius, during hypothermia for 22 h, and for 0.5 h after rewarming to 37.0 plus/minus 0.1 degree Celsius. Hearts were perfused at 25% of initial coronary flow during hypothermia.

Results: All groups had similar ventricular function and vasodilator responses before hypothermia. During normothermic reperfusion after hypothermia, both concentrations of halothane protected better than low CaCl2. Values, expressed as a percent of initial values in the five groups (time control, 3% halothane, 1.5% halothane, cold control, and 0.5 mM CaCl2, were respectively: 90 plus/minus 6, 54 plus/minus 6*, 48 plus/minus 5*, 27 plus/minus 8, 27 plus/minus 4% for left ventricular pressure; 84 plus/minus 5, 61 plus/minus 4*, 62 plus/minus 6*, 40 plus/minus 5, 34 plus/minus 5% for EFF; and 102 plus/minus 3, 63 plus/minus 3*, 66 plus/minus 3*, 55 plus/minus 2, 42 plus/minus 2% for coronary flow (*P < 0.05 halothane vs. 0.5 mM CaCl2). The coronary flow response to endothelium-dependent (acetylcholine) and endothelium-independent (nitroprusside) vasodilators was also greater after halothane than after 0.5 mM CaCl2.

Conclusions: Halothane administered during hypothermia restores left ventricular pressure, cardiac efficiency, basal coronary flow, and flow responses better than low CaCl2. Although halothane and low CaCl2both reduce intracellular Calcium2+, contractile force, and metabolic demand, the better protective effect of halothane is not likely simply due to a reduction in contractile function and metabolic rate before or initially after hypothermia because these were reduced much more by low CaCl2than by halothane.

LONG-TERM preservation of donor hearts will allow a greater and more easily attainable supply of hearts for transplantation. Previous studies from our laboratory indicate that long-term (24 h) ex vivo cardiac protection can be attained in guinea pig hearts by perfusing hearts with cold (3.8 degrees Celsius), normal ionic, oxygenated Krebs-Ringer solution containing reversible metabolic inhibitors and vasodilators. We have shown previously that halothane protects against cardiac damage after hypoxic perfusion, and after reperfusion subsequent to ischemia. The major purpose of this study was to examine if halothane is more or less effective than low perfusate solution CaCl2on preserving cardiac function after 1 day of preservation using low flow hypothermic perfusion. Halothane was chosen because it has a greater cardiac depressant effect in vitro than more recent volatile agents such as isoflurane or desflurane.

Halothane, as well as other volatile anesthetic agents, depresses contractile function by several interrelated mechanisms. Halothane attenuates slow Calcium2+ current through voltage-dependent, gated Calcium2+ channels, alters sarcoplasmic reticular (SR) handling of Calcium2+, and decreases Calcium2+ sensitivity of the myofibrillar apparatus. Lowering extracellular CaCl2also slows Calcium2+ current through voltage-dependent, gated Calcium2+ channels and reduces Calcium2+ induced Calcium2+ release from SR, which results in decreased cardiac work and metabolism. Lowering CaCl2also slows the rate of exchange of Calcium2+ via Sodium sup + and Potassium sup +-linked active transport pumps and reduces the degree of intracellular buffering and exchange with SR Calcium sup 2+ stores. These depressant effects of halothane and lowered CaCl2are generally applicable to vascular smooth muscle as well as to myocardial contractile factors but the vasodilatory effect may be partially counteracted by the decrease in myocardial work, which would tend to produce vasoconstriction via autoregulatory mechanisms.

We have reported that 1-day hypothermic perfusion of ex vivo guinea pig hearts with normal (low Potassium sup +) solution containing reversible metabolic inhibitors and/or vasolidators (such as, 2,3 butanedione monoxime, adenosine, nitrobenzylthioinosine, nitroprusside) is superior to a cold cardioplegic (high Potassium sup +) solution for restoring coronary flow and cardiac function. We postulated that halothane, like low CaCl2, might be a suitable cardioprotective agent because it depresses contractile function and metabolism by several mechanisms. In the current study, isolated hearts were treated before, during, and initially after hypothermia with either of two concentrations of halothane (1.5 and 3 vol%) or low perfusate CaCl2(0.5 mM) to compare their cardiac protective effects on normothermic reperfusion. Efficacy of these treatments on cardiac preservation was assessed by improvements in cardiac rate and rhythm, left ventricular (LV) systolic and diastolic pressure, basal coronary flow and vascular resistance, flow responses to endothelium dependent (acetylcholine) and independent (nitroprusside) vasodilators, percent oxygen extraction, myocardial oxygen consumption, and relative cardiac efficiency.

Preparation and Measurements

Approval from the institutional Animal Care Committee was obtained before the study was initiated. The investigation conformed with the Guide for the Care and Use of Laboratory Animals (NIH publication NO 85–23, revised 1985). Albino English short-haired guinea pigs (400–600 g) were injected intraperitoneally with 10 mg of ketamine and 1,000 units of heparin, and were decapitated when unresponsive to noxious stimulation. Isolation and preparation of hearts used in this study were done according to methods described in recent reports. The inferior and superior venae cavae were cut after thoracotomy and the aorta was cannulated distal to the aortic valve. Each heart was immediately perfused and then excised. Except during hypothermia, all hearts were perfused at a constant aortic root perfusion pressure of 55 mmHg. The perfusate, a modified Krebs-Ringer solution, was filtered (5 micro meter pore size) in-line filter disc (Cole-Parmer, Vernan Hills, IL) and had the following control composition in millimoles per liter: Sodium sup + 137, Potassium sup + 5, Magnesium sup 2+ 1.2, Calcium2+ 2.5, Chlorine sup - 134, HCO3sup - 15.5, H2PO4sup - 1.2, pyruvate 2, glucose 11.5, mannitol 16, glutamate 0.05, ethylene-diaminetetraacetic acid) 0.05, and insulin 5 units *symbol* L sup -1. Low CaCl2solution (0.5 mM) was prepared as described earlier except that the smaller amount of added CaCl2was substituted with an equimolar amount of NaCl. Left ventricular pressure was measured isovolumetrically with a transducer connected to a thin, saline-filled latex balloon that was inserted into the left ventricle through the mitral valve from a cut in the left atrium. Balloon volume was adjusted to maintain a diastolic LV pressure of 0 mmHg during the initial control period. Two pairs of bipolar electrodes (polytetrafluorethylene, coated silver, diameter 125 micro meter) were placed in each heart to monitor intracardiac electrograms from which spontaneous sinoatrial rate and atrioventricular (AV) conduction times were measured as noted previously. Coronary sinus effluent was collected by placing a cannula into the right ventricle through the pulmonic valve after ligating the vena cavae. Coronary inflow (aortic) was measured at constant temperature by an ultrasonic flowmeter. Coronary inflow and outflow (coronary sinus) Oxygen2tensions were measured continuously on-line (203B Oxygen2electrode, Instech, Plymouth Meeting, PA) and verified simultaneously off-line with an intermittently self calibrating gas analyzer system as described previously. Oxygen2consumption and % Oxygen2extraction were measured in all studies to assess the direct vasodilatory response of drugs apart from the response due to metabolic factors, e.g., a decrease in coronary flow and Oxygen2delivery secondary to decreased contractility and Oxygen2consumption. Use of this measurement assumes that local metabolites are produced in proportion to myocardial Oxygen2consumption, and that local metabolites are major factors regulating coronary flow. Percent oxygen extraction, Oxygen2consumption rate, and relative cardiac efficiency (heart rate *symbol* LV pressure per Oxygen2consumption rate) were calculated as reported previously. Coronary vascular resistance was calculated as perfusion pressure (constant during normothermia) divided by wet heart weight adjusted coronary flow (constant during hypothermia).

Perfusate and bath temperature were maintained at 37.2 plus/minus 0.1 degrees Celsius before and after hypothermia using a thermostatically controlled water circulator. During the 22–23-h hypothermic perfusion period, perfusate and bath temperature were maintained at 3.8 plus/minus 0.1 degrees Celsius. A switch to hypoperfusion at 3.8 degrees Celsius was accomplished by use of a separate refrigerated jacket and perfusion circuit placed in parallel with the warm perfusion circuit. Normothermic perfusion for 3 h at 37.2 plus/minus 0.1 degrees Celsius after cold perfusion was reinstated by switching back to the warm circuit. Warm and cold perfusion circuits were temperature equilibrated in advance. Time to reach half the temperature decrease from 37.2 to 3.8 degrees Celsius was 5 min. Upon decreasing temperature, at 15 degrees Celsius, cardiac perfusion was switched from constant pressure to a low constant flow (1.7 ml *symbol* g sup -1 *symbol* min sup -1) of about one fourth the baseline normothermic flow during constant pressure perfusion. This technique of low constant flow perfusion was employed to ensure that all hypothermia groups were perfused equally during the reduced metabolic requirement afforded by hypothermia. Perfusion pressure was monitored throughout hypothermia during the period of constant flow. On raising temperature at 25 degrees Celsius after hypothermia, cardiac perfusion was returned to the constant pressure (55 mmHg) mode. Time to reach half the temperature rise from 3.8 degrees Celsius to 37.2 degrees Celsius was 3 min. Warm and cold perfusate solutions were equilibrated with a gas mixture of 96% Oxygen2+ 4% CO sub 2. For hearts in all groups during the initial normothermic period, mean coronary arterial (inflow) pH averaged 7.46 plus/minus 0.02 (SEM), pCO225 plus/minus 1 mmHg, and pO2580 plus/minus 8 mmHg; inflow samples, collected at 3.8 degrees Celsius during the hypothermic period were 37 degrees Celsius, and had values of 7.15 plus/minus 0.02, 47 plus/minus 2 mmHg, and 787 plus/minus 16 mmHg, respectively. The apparent acidity and hyperoxia measured at 37 degrees Celsius reflects the greater solubility of Oxygen2and CO2in the hypothermic solution. There were no significant differences for these values among the groups at each of the two temperatures.

Halothane was administered by an agent-specific vaporizer by switching to perfusate equilibrated with 1.5 or 3 vol % halothane. Samples were collected at an aortic inflow port during halothane delivery before, during, and after hypothermic perfusion for determination of halothane concentration by gas chromatography as described elsewhere. Halothane's effective vapor fraction (vol %) was calculated (Table 1) using an estimate for its partition coefficient at 4 degrees Celsius. .

Table 1. Perfusate Halothane (HAL) Concentration (mM) and Effective Vapor Concentration (vol %) before (37.2 degrees Celsius), during (3.8 degrees Celsius), and after (37.2 degrees Celsius) Hypothermia

Table 1. Perfusate Halothane (HAL) Concentration (mM) and Effective Vapor Concentration (vol %) before (37.2 degrees Celsius), during (3.8 degrees Celsius), and after (37.2 degrees Celsius) Hypothermia
Table 1. Perfusate Halothane (HAL) Concentration (mM) and Effective Vapor Concentration (vol %) before (37.2 degrees Celsius), during (3.8 degrees Celsius), and after (37.2 degrees Celsius) Hypothermia

Electrograms, heart rate, AV conduction time, outflow Oxygen sub 2 tension, perfusion pressure, coronary flow, and LV systolic and diastolic pressures were graphically printed on a fast-writing, thermal-array eight-channel recorder (Astro-Med(R) MT9500) and digitally recorded (MacLab 8, AD Instruments, Castle Hills, Australia) for later detailed analysis. Description and identification of dysrhythmias in this model have been described in detail previously. Calculated variables were computed using a software program (Microsoft Excel, Redmond, WA). Hearts were weighed immediately after each experiment (28 h for hypothermia groups and 6 h for the time-treatment control groups) and dehydrated hearts were weighed to calculate dry heart weight expressed as a percentage of wet heart weight.

In all groups, peak coronary responsiveness was tested with adenosine to temporarily arrest hearts. A bolus of adenosine (0.2 mL of a 200 micro Meter solution) was injected directly into the aortic (coronary perfusion) cannula to assess this response. In all five groups, endothelium-dependent responses were tested with a 3-min infusion of 1 micro Meter acetylcholine hydrochloride (Ach), endothelium-independent responses were tested with a 3-min infusion of 100 micro Meter nitroprusside, and inotropic and chronotropic responses were tested with a 1-min infusion of 0.5 micro Meter epinephrine. Each drug was administered in identical fashion in all groups between 1 and 1.5 h before lowering perfusate CaCl2or administering either of two concentrations of halothane (Figure 1). These responses were again tested 4.5–5 hr later (warm group) or 26.5–27 h later during normothermic reperfusion after discontinuing halothane or after restoring CaCl2to normal. Maximal (m), steady-state (s), and ventricularly paced (p; 240 beats/min) flow responses to are displayed. The initial Achmresponse is a peak response occurring before the slowed atrial rate produces a relative vasoconstriction through autoregulatory mechanisms; Achsis the response during the slowed atrial rate (150 beats/min); Achp, is the response during pacing that most closely corresponds to the resting metabolic rate.


Hearts (n = 66) were divided randomly into 5 groups of approximately 13 hearts each:(1) time control (6 h normothermic perfusion);(2) 3.0% halothane;(3) 1.5% halothane;(4) cold treatment control; and (5) 0.5 mM CaCl2. Each group was perfused with the standard Krebs-Ringer solution containing 2.5 mM CaCl2, except for the low CaCl2group, which was perfused with a Krebs-Ringer solution containing 0.5 mM CaCl2. Halothane was administered, or CaCl2was lowered, 0.5 h before, during, and after (for 0.5 h after rewarming) 22 hours of hypothermic perfusion at 3.8 plus/minus 0.1 degrees Celsius. Hearts were perfused at the reduced constant flow rate during hypothermia while aortic perfusion perfusion pressure was allowed to vary. The four hypothermia ("cold") groups were perfused normothermically at constant perfusion pressure for 3 h, hypothermically at reduced constant coronary flow for 22 h, and normothermically at constant pressure for 3 h after hypothermia for a total of 28 h. The duration of normothermia in the time control group was 6 h, which is the same duration of normothermia as in the hypothermic groups. In preliminary experiments, perfusion of normothermic hearts with either 3% halothane or 0.5 mM CaCl2for 1 h had no lasting effect on any variable measured. Isolated guinea pig hearts perfused normothermically for 25 h, with or without drug protection, exhibit only very slow erratic atrial beating and no ventricular rhythm or contractile function; hearts stored hypothermically for 22 h without perfusion are contractured and exhibit no myocardial activity (unpublished results).

(F1-20) shows the time course of the protocol. All variables were measured during the last minute of (1) a 0.5-h initial control (C1) period (h 0.5);(2) during initial test infusions of adenosine, acetylcholine hydrochloride, nitroprusside, and epinephrine (drugs, D1);(3) during the prehypothermia period before giving halothane or lowering CaCl2;(4) during halothane or lowered CaCl2before the hypothermic period;(5) during halothane or lowered CaCl2after 22 h of hypothermic perfusion or during continued normothermia;(6) every subsequent 0.5-h period beginning after treatment (C2) in cold groups (h 26–28) or in the warm group (h 4–6); and (7) during repeat infusions of adenosine, acetylcholine hydrochloride, nitroprusside, and epinephrine (D2).

Because rewarming provoked ventricular dysrhythmias in a few hearts at approximately 25 degrees Celsius, each heart in the cold groups received one prophylactic bolus injection of 0.1 mL of 10 mg lidocaine hydrochloride during rewarming at 25 degrees Celsius to reduce the occurrence of such dysrhythmias. Only dysrhythmias occurring at 37 degrees Celsius were tabulated.

Statistical Analysis

All data are expressed as means plus/minus standard error of the means (SEM). Mean values were considered significant at P < 0.05. For data expressed over time (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7), the six groups were compared for variability at each time interval by two-way analysis of variance (CLR ANOVA, Clear Lake Research, Houston, TX). For each variable, each cold group was compared to the 6-h time control warm group (dagger); each halothane group was compared to the 0.5 mM CaCl2group (*) and to the cold treatment group (section); and 3% halothane group was compared to the 1.5% halothane group (double dagger). Not every statistical symbol is marked in the "time" F2-20, F3-20, F4-20, F5-20, F6-20, F7-20if there was no change from the previous time period. Group comparisons were always noted during halothane or lowered CaCl2, during adenosine or epinephrine, and at the final time control.

For coronary flow (Figure 8and Figure 10) and % Oxygen2extraction variables (Figure 9and Figure 11), the flow increase to vasodilators adenosine, acetylcholine hydrochloride, and nitroprusside was tested using one-way analysis of variance with repeated measures. The following comparisons were made: vasodilator responses to adenosine, acetylcholine hydrochloride, and nitroprusside (bullet) versus C1 (F8-20and F9-20, controls before hypothermia) and versus C2 (F10-20and F11-20, controls after hypothermia);(dagger) each cold group versus the time control group;(*) either halothane group versus 0.5 mM CaCl2;(section) either halothane group versus the cold control group; and 3% versus 1.5% halothane (double dagger). Individual data on 6 of 13 hearts in the time control group, 7 of 13 hearts in the low CaCl2group, 5 of 14 in the cold control group, and 0 of 26 in the halothane groups have been previously published. Individual historical data was included to minimize use of experimental animals. Replacement of older individual studies with newer studies ensured random experimental design. There was no statistical difference in grouped means with substitution of individual data within a given group. Fisher's least significant difference test was used to compare means. Software programs were run on compatible computers (Macintosh, Apple Computer, Cupertino, CA).

(T1-20) shows perfusate halothane concentration and calculated effective vapor concentration obtained before, during and after hypothermic perfusion at the two vaporizer settings. Halothane concentration was markedly higher during hypothermia and moderately higher after hypothermia for each of the two anesthetic concentrations delivered, but effective vapor concentration was similar at the two temperatures for a given vaporizer setting. Dry heart weight averaged 13.5 plus/minus 0.3% for all groups and was not significantly different among groups.

Continuous line plots (F2-20, F3-20, F4-20, F5-20, F6-20, F7-20) compare time-dependent changes in 6 measured or calculated variables among the five groups. These figures show that each group had similar control baseline values and flow responses to epinephrine and adenosine before hypothermia. Administration of 1.5% halothane, 3% halothane or low CaCl2similarly reduced heart rate before hypothermia (F2-20). Halothane decreased LV pressure (F3-20) in a concentration dependent manner; this effect was less with halothane than with low CaCl2. Both halothane fractions decreased % oxygen extraction less than low CaCl2(F4-20), and both halothane fractions decreased cardiac efficiency more than low CaCl2(F5-20). Neither fraction of halothane nor low CaCl2altered coronary flow (F6-20) or vascular resistance (F7-20) before hypothermia.

With the onset of hypothermia, heart rate decreased and AV time increased until AV dissociation occurred. Cold induced contractures, indicated by increased isovolumetric LV pressure, occurred between 25 and 20 degrees Celsius with low CaCl2(20 plus/minus 2 to 60 plus/minus 3 mmHg, P < 0.05) but did not occur in cold controls (97 plus/minus 6 to 90 plus/minus 5 mmHg), with 1.5% halothane (80 plus/minus 7 to 66 plus/minus 4 mmHg) or with 3% halothane (49 plus/minus 3 to 30 plus/minus 6 mmHg). At 20 degrees Celsius, diastolic pressure increased to about 9 plus/minus 2 mmHg in all groups.

Initially after hypothermia, with continued halothane or decreased CaCl2(at 25.5 h), all time-dependent variables (F2-20, F3-20, F4-20, F5-20, F6-20, F7-20) in the hypothermia groups were significantly decreased compared to the untreated normothermic time group (not all statistical notations are shown). Basal heart rate (F2-20) was similar in each hypothermia group during normothermic reperfusion compared to the time control group after washout of halothane or return to 2.5 mM CaCl2, and heart rate response to epinephrine were greater in the halothane groups than in the low CaCl2-treated group or the cold control group. The incidence of ventricular fibrillation was significant (P < 0.05) in cold control (36%) and low CaCl sub 2 (38%) groups. All hearts remained in, or had reverted to, sinus rhythm with boluses of lidocaine within 2 h of warm reperfusion. There were no significant ventricular dysrhythmias in either group treated with halothane.

Left ventricular developed pressure (F3-20) increased to a level intermediate to that of the time control and cold control groups in the halothane-treated groups after washout of halothane; the halothane off effect on LV pressure was not concentration dependent and there was no difference between the cold control and 0.5 mM CaCl2groups on LV pressure during reperfusion. Left ventricular diastolic pressure after hypothermia and discontinuation of halothane or low CaCl2was: cold control, 8 plus/minus 3 mmHg; low CaCl2, 13 plus/minus 5 mmHg; 1.5% halothane, 0 mmHg; and 3% halothane, 0 mmHg. The values for cold control and low CaCl2groups were similar and higher than those for the halothane groups (P < 0.05).

Percent Oxygen2extraction (F4-20) was generally greater in all hypothermic groups than in the warm time control group and was similar after washout of halothane or return to 2.5 mM CaCl2. Relative cardiac efficiency (F5-20) was lower in all hypothermia groups compared to the normothermic group but was similarly higher in the two halothane-treated groups than in the two other hypothermia groups during reperfusion. Basal coronary flow (F6-20) and the response to adenosine were less in all hypothermia groups during reperfusion but generally were greater in the halothane-treated groups than in the other two hypothermia groups. Coronary vascular resistance (F7-20) increased 2–3-fold during hypothermia in all four hypothermic groups despite a reduction in coronary flow to about one fourth the normothermic flow. Vascular resistance was least during hypothermia in the presence of halothane or low CaCl2. During normothermic reperfusion vascular resistance was increased in all hypothermic groups but was less in the cold control and halothane groups than in the low CaCl2-treated group.

Bar (F8-20, F9-20, F10-20, F11-20) detail the differences in basal flow and % Oxygen2extraction and changes in flow and % Oxygen2extraction in response to infusion of endothelium-dependent and endothelium-independent vasodilators before (F8-20and F10-20) and after (F9-20and F11-20) hypothermic preservation. F8-20shows that each group had similar control baseline flow and flow responses to adenosine, acetylcholine, and nitroprusside before hypothermia. F10-20shows that steady-state % 0xygen2extraction was similarly reduced in each group by acetylcholine and nitroprusside before hypothermia. After hypothermia (F9-20) and 2 h later (i.e., at 26.5 h), during normothermic reperfusion, basal coronary flow was less in all hypothermia groups but was greater in the two halothane-treated groups than in the low CaCl2-treated group. The flow response to the three vasodilators was attenuated in all hypothermia groups but was greater in the halothane-treated groups than in the low CaCl2-treated group. There was no difference in basal flow or in flow responses to vasodilators at the two concentrations of halothane during normothermic reperfusion. Basal % Oxygen2extraction after hypothermia was similar to that measured before hypothermia (F11-20) but was greater in the low CaCl2-treated group than in the halothane-treated groups after hypothermia. Percent Oxygen2extraction decreased during infusion of Ach and nitroprusside in the time control group and during infusion of nitroprusside in the two halothane-treated groups. There was no significant change in % Oxygen2extraction by nitroprusside infusion in the cold control and low CaCl2-treated groups after hypothermia.

The current study demonstrates that halothane offers better cardiac protection than low-perfusate CaCl2when given before, during, and immediately after hypothermic perfusion as reflected by fewer ventricular dysrhythmias, better mechanical function, improved Oxygen2utilization, higher basal coronary flow, and greater flow responses to vasodilators after return to normal ionic, normothermic perfusate solution. The smaller fraction of halothane appears to afford as much protection as the larger fraction, because there were few significant differences between the halothane groups. This might indicate that the protective effect of halothane is not specific. A lower concentration of halothane may help to discern specificity.

Metabolic Protection by Halothane

Halothane was tested as a possible myocardial protective agent because in isolated hearts it acts as a myocardial depressant that produces a relative overperfusion of the myocardium and because it has been shown to improve function and reduce dysrhythmias on reperfusion after coronary occlusion and after hypoxic perfusion in isolated hearts. The mechanism of the beneficial effect of halothane on reducing cardiac dysfunction and protecting against ventricular dysrhythmias is unknown. The protective effect of halothane on normothermic reperfusion after long-term, hypothermic perfusion, as well as after global ischemia or hypoxia, could result simply from the reduced metabolic demand that accompanies the reduced contractile effort before, during, or immediately after the insult. Indeed, reducing metabolic demand during cardiac stress or applying preconditioning maneuvers before stress can reduce the degree of dysfunction, or "myocardial stunning," by reducing the extent or severity of ischemic damage. For example administration of adenosine or Potassium sup +ATP-sensitive channel openers before a long period of ischemia reduces the extent of infarction as well as the degree of reversible myocardial dysfunction on reperfusion. These drugs may mimic endogenous adenosine release and Potassium sup +ATPchannel opening during a very brief transient occlusion, which protects against stunning and infarction before a much longer period of occlusion. .

Like halothane, low CaCl2, Calcium2+ blockers, adenosine, and Potassium sup +ATPchannel agonists decrease myocardial contractile function and promote vasodilation, so cardiac protection by halothane could simply reflect induced hibernation. Recent evidence, however, indicates that volatile anesthetics may actually open Potassium sup +ATPchannels. Administration of a Potassium sup +ATPchannel antagonist may help to expose the possible role of volatile anesthetics on myocardial protection in the perihypothermic period. Because hypothermia itself abolished rhythm and contractile activity, it is not possible from this study to ascertain how halothane better protects the myocardium during hypothermia.

Perfusion Protection by Halothane

Related to the concept of myocardial stunning, is the concept of "microvascular stunning" based on the observation that ischemia also damages the endothelial lining so that production of intrinsic endothelium-dependent vasodilators like nitric oxide or prostaglandins is diminished and underlying vascular smooth muscle contracts so that perfusion is restricted as a result of vasoconstriction, obstruction, or both. Inadequate production of vasodilators, poor vascular smooth muscle responses to vasodilators, and production of vasoconstrictor factors would lead to inadequate supply of oxygen and nutrients to match metabolic demands. We tested responses to a maximal vasodilator agent (adenosine) and to endothelium-dependent (acetylcholine) and endothelium-independent (nitroprusside) agents to assess microvascular injury. Indeed, although responses to these agents were attenuated after hypothermia in all groups, the two halothane-treated groups exhibited better flow responses to these agents than the low CaCl2-treated group. It is unlikely that the nitric oxide synthase-guanylyl cyclase vasodilator system and the ion gradient stabilization systems are fully operational during hypothermia, but they may be differentially protected before hypothermia or differentially activated during rewarming in the presence of low CaCl2or halothane. Because the halothane groups tended to have lower vascular resistance during hypothermia and to have significantly better basal flow rates than the low CaCl2group after normothermic reperfusion, halothane may better protect against microvascular injury, and concurrently, myocardial injury.

The mechanism of halothane to improve perfusion flow after hypothermia also is unknown. A decrease in intracellular Calcium2+, whether by lowering CaCl2or by administering halothane, promotes vascular smooth muscle relaxation. One possibility is that halothane reduces L-type Calcium2+ channel opening time and so reduces accumulation of myoplasmic Calcium2+ during hypothermia. Another is that it opens Potassium sup +ATPchannels to promote vascular smooth muscle relaxation via membrane hyper-polarization during warm reperfusion. Use of a Potassium sup +ATPantagonist like glybenclamide may help to unravel the beneficial vascular effect of halothane over low CaCl2. Another possibility is that in addition to the direct vascular effect, low CaCl2decreases Calcium2+-dependent endothelial cell release of vasodilator substances whereas halothane has a lesser effect or no effect.

Halothane Versus Low CaCl sub 2 on Cardiac Protection

Putative protective effects of halothane were compared with lowered CaCl2because of halothane's well-known effects on altering Calcium sup 2+ handling. It is well known that both halothane and low extracellular CaCl2lower myoplasmic Calcium2+ as a result of reduced Calcium2+ influx and that this effect can decrease Calcium sup 2+ induced SR Calcium sup + release. A decrease in myoplasmic Calcium sup 2+ causes a decrease in myocyte contractile force. Most evidence indicates that volatile anesthetics alter availability of myoplasmic Calcium2+ for the contractile mechanism via several cellular and intracellular sites. Contributing mechanisms include (1) voltage-dependent depression of transsarcolemmal Calcium2+ current;(2) decreased net SR Calcium2+ uptake;(3) increased rate of Calcium sup 2+ leak from the SR during diastole, (4) decreased affinity of troponin C (TnC) for Calcium2+, and (5) decreased response of the myofilaments to a given level of occupancy of the Calcium2+ binding sites on troponin C. Halothane appears to have no effect on Calcium2+ affinity of troponin C. A possible mechanism for halothane's negative inotropic effect is that as Calcium2+ permeability of the SR is increased and SR Calcium2+ uptake is decreased, the amount of Calcium2+ available for SR release during systole decreases and intracellular Calcium2+ stores become progressively depleted by continued depression of the inward Calcium2+ current. These events further decrease Calcium2+-induced release of Calcium2+ from the SR during systole and result in a net loss of Calcium2+ from the cell because Sodium sup +/Calcium2+ exchange and Calcium2+ adenosine triphosphatase effectively pump out more of the available Calcium2+ during diastole. It is unknown from the current study to what extent changes in Calcium2+ flux, or changes in other transcellular ionic gradients, are differentially affected by low CaCl2or halothane. Interestingly, cold contractures did not occur with halothane between 25 and 20 degrees Celsius as they did with low CaCl2. This suggests that halothane can alter intracellular Calcium2+ concentration differently than low CaCl2, especially during mild hypothermia.

On the basis of metabolic depression it was expected that low CaCl2would be more protective than halothane during reperfusion after hypothermia. If the greater improvement in cardiac function on normothermic reperfusion by halothane was associated with a greater depression of myocardial contractile function and metabolism induced by halothane than by low CaCl2before, and presumably during hypothermia, depression of the metabolic rate could be inferred as the protective mechanism. Low CaCl2or halothane did not change flow before hypothermia, but since they both reduced % Oxygen2extraction, metabolic regulation of flow was attenuated. The greater reduction in % Oxygen2extraction by low CaCl2is indicative of a greater perfusion relative to the metabolic rate. Although the low CaCl2solution depressed contractile function and reduced % Oxygen2extraction much more than either concentration of halothane before, as well as after hypothermia, halothane protected hearts much better than did low CaCl2. Unless there is more or less buffering of Calcium2+ at the contraction apparatus by these maneuvers, one would expect that myoplasmic Calcium2+ was lower in the presence of low CaCl2than halothane. Overall, it seems unlikely that the cardiac protective effect of halothane is simply a result of a greater reduction in myoplasmic Calcium2+ by halothane than by low CaCl2.

It is interesting that the low CaCl2perfusate did not improve cardiac function and efficiency on normothermic reperfusion any better than the normal CaCl2perfusate. Although low CaCl2decreases cell membrane Calcium2+ influx, promotes smooth muscle relaxation, and greatly decreases cardiac work, reducing Calcium2+ from 5 to 0.2 mM was found to have little effect on reducing SR Calcium sup 2+ release because rapid cooling contractures to 5 degrees Celsius are similar at both CaCl2concentrations. Severe hypothermia is itself protective because it greatly decreases metabolic rate, but mild hypothermia produces a positive inotropic effect. We found that initial cooling from 37 degrees Celsius to 25 -20 degrees Celsius increases peak LV pressure about threefold in the presence of low CaCl2, whereas LV pressure continues to decrease with temperature in the presence of normal CaCl2or halothane. We reported previously that the decrease in contractile force in cardiac Purkinje fibers by halothane at 35 degrees Celsius is converted to a marked increase in force at 25 degrees Celsius, whereas Calcium2+ transients, an estimate of myoplasmic Calcium2+, are changed little. Moreover, the curve relating contractile force as a function of extracellular CaCl2is shifted steeper and to the left of the curve at 35 degrees Celsius. These findings suggested to us that the increase in contractile force by mild hypothermia is due to increased myofibrillar Calcium2+ sensitivity and that this effect can be attenuated by anesthetics which decrease myofibrillary Calcium2+ sensitivity. Although halothane was given in the presence of severe hypothermia in the current study, the effect of halothane to decrease Calcium2+ sensitivity during hypothermia might explain in part the beneficial effect of halothane on myocardial preservation. This might be tested by combining low CaCl2solution with halothane during hypothermia.

By exclusion, it is likely that other actions of halothane have a greater impact on furnishing cardiac protection during various cardiac stresses than a simple decrease in intracellular Calcium2+ triggered by a sarcolemmal effect to decrease influx of Calcium2+. Most likely, halothane's protective effect results from complex, interrelated mechanisms on ultracellular structures. Volatile anesthetics probably alter the function of multiple organelles within cardiac cells in a nonspecific but reversible fashion. Halothane may alter lipid and protein interactions within membrane structures, modulate free radical production, alter pump-regulated ion gradients, and produce other unknown effects. Thus, the function of receptor- and second-messenger-associated proteins of the sarcolemma and SR, as well as other enzymes and intracellular structures, may be altered in a way that preserves cardiac tissue during long-term hypothermia.

Moreover, it is likely that the protective mechanism afforded by halothane in this model of hypothermic preservation is different than the protective mechanism afforded during or after global coronary occlusion or hypoxic perfusion. The latter two insults produce ischemia because the flow or oxygen demand to maintain the rate of metabolism are not met. Halothane may be protective in those models simply because the metabolic rate is pharmacologically reduced in the face of continued metabolic demands. In the hypothermic preservation model presented here, flow and oxygen demand are more than met by the decreased metabolic demand of hypothermia. We have reported that addition of other metabolic depressant agents such as butanedione monoxime and adenosine to cardiac preservation solutions greatly improves function after normothermic reperfusion. Thus, these additives may preserve function not only because they simply depress metabolic activity, but also because they too initiate or enhance intrinsic protective mechanisms to reduce cellular metabolic rate during a myocardial stress.

Clinically, administration of halothane, and perhaps other volatile anesthetics, may help to preserve coronary vascular compliance, enhance utilization of oxygen, and improve cardiac function in the early posttransplant period after hypothermic perfusion. Although the mechanism of the potential protective effect is unknown, our comparison of effects with a low CaCl2perfusate suggests it is not likely owing to a simple reduction in cardiac function and metabolism resulting from a decrease in myoplasmic Calcium2+.

The authors thank James S. Heisner, for technical assistance, and Forrest Moore and David Chang, for helping with this study.

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