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(Circulation. 1997;96:4372-4379.)
© 1997 American Heart Association, Inc.

Articles

Halothane Protects Cardiomyocytes Against Reoxygenation-Induced Hypercontracture

Berthold Siegmund, PhD; Wolfgang Schlack, MD; Yury V. Ladilov, PhD; Claudia Balser, MS; ; H. Michael Piper, MD, PhD

From the Physiologisches Institut, Justus-Liebig-Universität, Giessen, and Institut für Klinische Anaesthesiologie, Heinrich-Heine-Universität, Düsseldorf (W.S.), Germany.

Correspondence to H. Michael Piper, Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany. E-mail michael.piper{at}physiologie.med.uni-giessen.de

	Abstract

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Background Resupply of oxygen to the myocardium after extended periods of ischemia or hypoxia can rapidly aggravate the already existing injury by provoking hypercontracture of cardiomyocytes (acute reperfusion injury). Previous studies indicated that halothane can protect ischemic-reperfused myocardium. The aim of the present study was to analyze on the cellular level the mechanism by which halothane may protect against reoxygenation-induced hypercontracture.

Methods and Results To simulate ischemia-reperfusion, isolated adult rat cardiomyocytes were incubated at pH 6.4 under anoxia and reoxygenated at pH 7.4 in the presence or absence of 0.4 mmol/L halothane. Reoxygenation was started when intracellular Ca²⁺ (measured with fura 2) had increased to 10^-5 mol/L and pH_i (BCECF) had decreased to 6.5. Development of hypercontracture was determined microscopically. In the control group, reoxygenation provoked oscillations of cytosolic Ca²⁺ (72±9 per minute at fourth minute of reoxygenation) accompanied by development of hypercontracture (to 65±3% of end-ischemic cell length). When halothane was added on reoxygenation, Ca²⁺ oscillations were markedly reduced (4±2 per minute, P<.001) and hypercontracture was virtually abolished (90±4% of end-ischemic cell length, P<.001). Halothane did not influence the recovery of pH_i during reoxygenation. Similar effects on Ca²⁺ oscillations and hypercontracture were observed when ryanodine (3 µmol/L), an inhibitor of the sarcoplasmic reticulum Ca²⁺ release, or cyclopiazonic acid (10 µmol/L), an inhibitor of the sarcoplasmic reticulum Ca²⁺ pump, were applied instead of halothane.

Conclusions Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture by preventing oscillations of intracellular Ca²⁺ during the early phase of reoxygenation.

Key Words: reperfusion • prevention • ischemia • calcium • halothane

	Introduction

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In 1969, Spieckermann and colleagues¹ reported that inhalational anesthetics, in particular halothane, exert protective effects on the ischemic myocardium. Recent studies also discuss the cardioprotective effects of halothane during the process of myocardial reperfusion.^2–4 One of these studies,⁴ performed on the isolated perfused rat heart, found that halothane can be used to attenuate acute reperfusion injury when given on reoxygenation. This form of reperfusion injury ("oxygen paradox"⁵) is characterized by a rapid onset and extensive tissue destruction. The central aim of the present study has been to analyze the causal mechanism of a specific protective action of halothane on the reoxygenated cardiomyocyte on the cellular level.

The isolated ventricular cardiomyocyte model was used. In this model, we showed previously that acute reoxygenation injury is based on a sudden development of hypercontracture on reoxygenation^6–8 due to the reenergization of the cells at elevated cytosolic Ca²⁺ levels.^8–11 Ca²⁺ overload results from the preceding state of energy deprivation. Hypercontracture causes irreversible cytoskeletal alterations. In contrast to myocardial cells in tissue, which mutually exchange mechanical forces, isolated cardiomyocytes retain an intact sarcolemma when developing hypercontracture.⁸ Therefore, they represent an experimental model, in which the metabolic potential of the reoxygenated myocardial cell to restore a normal cation homeostasis can be investigated even if reoxygenation had induced hypercontracture.

In the present study, cardiomyocytes were subjected to anoxic conditions in media with pH 6.4 (simulated ischemia) until they developed severe cytosolic Ca²⁺ overload. This was followed by reoxygenation in media with pH 7.4 (simulated reperfusion) in the presence or absence of halothane. Previous studies^10,11 have shown that the early period of reoxygenation (first 10 minutes) when hypercontracture develops can be divided into two phases: In the first phase, lasting 2 minutes, cytosolic Ca²⁺ rapidly declines because it is sequestered into the sarcoplasmic reticulum (SR). During this period, the cytosolic pH is low (6.5). In the subsequent second phase, the cytosolic pH recovers to a normal value (pH 7.1), and a burst of spontaneous oscillations of cytosolic Ca²⁺ occurs. These oscillations are due to cycles of uptake and release of Ca²⁺ into and from the SR.⁹ The central hypothesis of the present study is that halothane, which is known to alter SR function,¹² inhibits the development of hypercontracture in reoxygenated myocardial cells by suppressing these SR-dependent Ca²⁺ oscillations. The effect of halothane was compared with the effects of agents that inhibit the SR function specifically, ie, ryanodine, which blocks SR Ca²⁺ release, and cyclopiazonic acid, which inhibits SR Ca²⁺ uptake.

	Methods

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Isolation of Cardiomyocytes
Ventricular heart muscle cells were isolated from 200- to 250-g adult male Wistar rats and plated in medium 199 with 4% FCS on glass coverslips that had been preincubated overnight with 4% FCS.^13,14 Four hours after plating, the coverslips were washed with medium 199. The wash removed broken cells, leaving a homogeneous population of rod-shaped quiescent cardiomyocytes (>95%) attached to the coverslip.

Ca²⁺, pH, and Cell Length Measurements
To measure cytosolic Ca²⁺ or H⁺ concentrations, cardiomyocytes were loaded at 37°C in 2.5 µmol/L fura 2 or 1.25 µmol/L BCECF as described previously.^9,11 The coverslip with the loaded cells was then introduced into a gas-tight, temperature-controlled (37°C), transparent perfusion chamber positioned in the light path of an inverted microscope (Diaphot TMD, Nikon). Alternating excitation of the fluorescent dye at wavelengths of 340 and 380 nm for fura 2 and 450 and 490 nm for BCECF was performed with an AR-Cation Measurement System adapted to the microscope (ISA). Emitted light (500 to 520 nm for fura 2 and 520 to 560 nm for BCECF) from the 10x10-µm area within a single fluorescent cell was collected by the photomultiplier of the system. The light signal was recorded and analyzed by an IBM PC/AT-based data analysis system (model DM3000CM, ISA). Simultaneously with the measurement of fluorescence, the microscopic image of the cell was recorded with a video camera, stored on tape, and printed with a video printer. Changes of the cell length were determined from these recordings. In the case of hypercontracted cells, the cell dimension along its previous longitudinal axis was determined.

Data were usually expressed in arbitrary units of fluorescence ratios of the emitted light of the two corresponding excitation wavelengths. To facilitate understanding, calibration protocols were performed to obtain numerical relationships between selected ratio values and ion concentrations. The fura 2 signal was calibrated according the method described by Li et al.¹⁵ For this purpose, the cells were exposed to 5 µmol/L ionomycin in modified Tyrode's solution (pH 7.4; for composition, see below) containing either 3 mmol/L Ca²⁺ or 5 mmol/L EGTA to obtain the maximum (R_max) and the minimum (R_min) ratios of fluorescence, respectively. The free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was calculated according to the equation [Ca²⁺]_i=K_dxßx(R-R_min)/(R_max-R), where ß represents the ratio of the 380-nm excitation signals of ionomycin-treated cells at 5 mmol/L EGTA and at 3 mmol/L Ca²⁺ and K_d, the dissociation constant of fura 2.¹⁶ The affinity constant of fura 2 to Ca²⁺ (K_d) inside the cell was determined in intact cardiomyocytes by a calibration curve. This in vivo K_d for fura 2 was found to be 312±9 nmol/L (mean±SEM, n=8) and was used for the transformation of fluorescence ratio values into Ca²⁺ concentrations. Calibration of the BCECF ratio signal was performed as described previously with 10 µg/mL nigericin, a K⁺/H⁺ ionophore, and incubation media with various pH values.¹⁷

Electrical Field Stimulation
Cardiomyocytes were electrically stimulated under normoxic conditions with two silver chloride electrodes immersed into the medium. Biphasic electrical stimuli composed of two equal but opposite rectangular 40-V stimuli, each of 3-ms duration, were applied with a frequency of 0.5 Hz.

Media
The perfusion chamber (1 mL filling volume) placed on the microscope stage was perfused at a flow rate of 0.5 mL/min with modified, glucose-free Tyrode's solution at 37°C containing (in mmol/L) NaCl 125.0, KCl 2.6, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.0, and HEPES 25.0; pH was 7.4 and 6.4, respectively. The medium was made anoxic by autoclaving as described previously¹⁸ and was equilibrated before and during use with 100% N₂. Normoxic medium was equilibrated with air. Halothane was administered during the reoxygenation period in a final concentration of 0.4 mmol/L (=1.5 times minimal alveolar concentration) prepared from a saturated solution in the modified Tyrode's solution.

Experimental Protocol
In the experiments for Ca²⁺ measurements, cardiomyocytes superfused with anoxic medium were allowed to accumulate Ca²⁺ until the saturation of the fura 2 ratio was reached. The anoxic superfusion was carried out in modified Tyrode's solution at pH 7.4 (anoxia) or at pH 6.4 (simulated ischemia). Cells were then reoxygenated in the absence (control) or presence of 0.4 mmol/L halothane, 3 µmol/L ryanodine, or 10 µmol/L cyclopiazonic acid in modified Tyrode's solution at pH 7.4.

Materials
Medium 199 was purchased from Boehringer, FCS from Gibco, acetoxymethyl esters of fura 2 and BCECF from Paesel and Lorey, ryanodine and cyclopiazonic acid from Sigma, and halothane from Hoechst. All other chemicals were from Merck and were of highest purity available.

Statistics
Data are given as mean±SEM from n individual cells investigated in separate experiments. Statistical comparisons were performed by Student's t test for unpaired samples and by ANOVA followed by the Tukey-Kramer post hoc test if appropriate. Differences with P<.05 are regarded as significant.

	Results

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Cytosolic pH Under Simulated Ischemia-Reperfusion
Variations in cytosolic pH (pH_i) were measured with the indicator BCECF. An intracellular pH of 7.18±0.05 (n=8) was observed in cells incubated in normoxic medium with pH 7.4. When anoxic incubations were carried out in medium at pH 6.4 (simulated ischemia), pH_i declined to 6.51±0.05 (n=8). When reoxygenation was carried out in medium at pH 7.4 (simulated reperfusion), the pH_i returned to the control value within 10 minutes. This pH_i recovery can be divided into two phases: The first phase, lasting 2 minutes, when pH_i is still acidotic, and the subsequent 6 minutes, when pH_i gradually recovers to the control value. The recovery of pH_i was not altered in any phase when reoxygenation was carried out in the presence of halothane, cyclopiazonic acid, or ryanodine (Fig 1).

View larger version (23K): [in this window] [in a new window]   Figure 1. Intracellular pH measured by BCECF method (500/460 nm) during reoxygenation under control conditions, in presence of 10 µmol/L cyclopiazonic acid (CPA), 3 µmol/L ryanodine, or 0.4 mmol/L halothane. Anoxic incubations were performed in media at pH 6.4, reoxygenation in media at pH 7.4. Inhibitors were applied from onset of reoxygenation. Data are mean±SEM, n=8 cells.

Ca²⁺ Overload and Cell Length
In oxygen-depleted and reoxygenated cardiomyocytes, the variations in [Ca²⁺]_i were monitored by the fura 2 ratio, ie, the ratio of the emitted light intensities at excitation wavelengths 340 nm and 380 nm (Fig 2). A fura 2 ratio of 1 found in cells under normoxic control conditions corresponded to a [Ca²⁺]_i of 80 nmol/L. In cardiomyocytes exposed to simulated ischemia, a small increase of the fura 2 ratio was observed some minutes before the onset of rigor shortening (after 30 minutes of anoxic incubation for the cell shown in Fig 2A). As described previously,^6,8 rigor shortening is a rapid process for the individual cell, completed within seconds. It reduced cell length by approximately one third. Only after rigor shortening had occurred did the fura 2 ratio rise progressively until severe cytosolic Ca²⁺ overload had developed. When saturation of the fura 2 signal (corresponding to pCa5) had been reached in cardiomyocytes under simulated ischemic conditions, the cells were reoxygenated in medium with pH 7.4, causing their shortening to 30% of the initial cell length. Simultaneously with the development of hypercontracture, the cells were able to reestablish a normal cytosolic Ca²⁺ control (Fig 2A). As shown in higher time resolution (Fig 2C), reoxygenation transiently induced spontaneous oscillations of the fura 2 signal, representing repetitive shifts of Ca²⁺ between cytosol and SR.⁹ Spontaneous Ca²⁺ oscillations were not observed during the anoxic period (Fig 2B).

View larger version (32K): [in this window] [in a new window]   Figure 2. Time course of fura 2 ratio (340/380 nm, indicating changes in cytosolic Ca2+ concentrations; continuous trace of original recording) and cell length () in a single cardiomyocyte during anoxia-reoxygenation (A). B, End of anoxic period at higher time resolution. C, Part of reoxygenation period showing reoxygenation-induced spontaneous Ca2+ oscillations. Experimental protocol was as specified for Fig 1. Cells were reoxygenated when fura 2 ratio had reached its level of saturation (ratio value, 12). A fura 2 ratio of 1 corresponds to 80 nmol/L Ca2+; ratio of 12, to 10-5 mol/L.

Reoxygenation in Presence of Halothane
The time course during reoxygenation of the fura 2 signal and simultaneous cell length changes in the presence or absence of halothane are shown in Fig 3. In the absence of halothane, the cells progressively developed hypercontracture, accompanied by the occurrence of spontaneous reoxygenation-induced Ca²⁺ oscillations. In the individual cell shown, the fura 2 ratio dropped from a value of 12 to 9 during the first minute of reoxygenation, when pH_i was still acidotic (see above). This was followed by a period of oscillations of the fura 2 signal between minutes 1 and 7. On average of all investigated cells, the oscillation frequency reached a maximum at minute 4 of reoxygenation and declined to zero at minute 10 (Fig 5A). Finally, the fura 2 ratio regained the initial control level. During this phase of Ca²⁺ oscillations, hypercontracture developed gradually (Fig 3A) and pH_i returned to the control value (see above).

View larger version (24K): [in this window] [in a new window]   Figure 3. Time course of fura 2 ratio (continuous trace of original recording) and cell length in percent of end-ischemic cell length () in single cardiomyocytes during reoxygenation. Experimental protocol was as specified for Fig 1. A, Reoxygenation in absence of inhibitors; B, reoxygenation in presence of 0.4 mmol/L halothane.

View larger version (27K): [in this window] [in a new window]   Figure 5. Oscillations of fura 2 ratio and hypercontracture in reoxygenated cardiomyocytes under control conditions, 0.4 mmol/L halothane, 3 µmol/L ryanodine, and 10 µmol/L cyclopiazonic acid (CPA). Experimental protocol was as specified for Fig 1. A, Frequency of reoxygenation-induced Ca2+ oscillations during reoxygenation; B, cell length in percent of end-ischemic length during reoxygenation. Data are mean±SEM, n=8 cells. *P<.05 vs end-ischemic values.

When halothane was present during reoxygenation, recovery of the fura 2 signal was also observed, but its course was altered. Halothane effectively suppressed the oscillations of the fura 2 signal at times later than 2 minutes of reoxygenation (Fig 3B, Fig 5A). Hypercontracture, developing simultaneously with the oscillatory shifts of Ca²⁺ in the absence of halothane, did not occur in presence of halothane (Figs 3B and 5B).

Reoxygenation in Presence of Inhibitors of SR Function
To verify the hypothesis that halothane protects against reoxygenation-induced hypercontracture after simulated ischemia by inhibition of the Ca²⁺ oscillations, two specific inhibitors of SR function were applied: ryanodine in a concentration (3 µmol/L) at which it selectively blocks the Ca²⁺ release channel of the SR, or cyclopiazonic acid (10 µmol/L), which inhibits the Ca²⁺ pump of the SR. These agents were added to the perfusion medium at the onset of reoxygenation. Representative examples of the fura 2 signal and cell shortening during reoxygenation in the presence of these agents are shown in Fig 4. All data of these experiments are compared with those obtained with halothane in Fig 5. Treatment of reoxygenated cardiomyocytes with ryanodine or cyclopiazonic acid had a pronounced effect on Ca²⁺ oscillations. In cells not treated with these inhibitors, spontaneous Ca²⁺ oscillations occurred with a frequency of 72±9 per minute (n=8) at minute 4 of reoxygenation; halothane reduced the frequency to 4±2 (n=8) (Fig 5A). The frequency of these oscillations gradually declined to zero during the first 10 minutes of reoxygenation. Treatment of the cells with ryanodine completely abolished these reoxygenation-induced Ca²⁺ oscillations after 4 minutes. Cyclopiazonic acid also reduced the frequency of the oscillations significantly (14±5 per minute at minute 4 of reoxygenation, n=8, P<.01 versus control) and truncated the time period in which they occurred (Fig 5A).

View larger version (23K): [in this window] [in a new window]   Figure 4. Time course of fura 2 ratio (continuous trace of original recording) and cell length in percent of end-ischemic cell length () in single cardiomyocytes during reoxygenation. Experimental protocol was as specified for Fig 1. A, Reoxygenation in presence of 10 µmol/L cyclopiazonic acid (CPA); B, reoxygenation in presence of 3 µmol/L ryanodine.

During simulated ischemia, cardiomyocytes underwent rigor shortening, which reduced the cell length by one third (Fig 2). In control cells, hypercontracture developed gradually after minute 3 of reoxygenation and hypercontracture, superimposing the preceding rigor shortening, so that the length of the cells was eventually reduced by two thirds of their initial control length. In Fig 5B, changes in cell length due to reoxygenation are expressed as relative changes compared with the preceding end-ischemic length 10 minutes after the onset of reoxygenation. Halothane significantly lessened the development of hypercontracture. Treatment of the cells with ryanodine or cyclopiazonic acid, which abolished the Ca²⁺ oscillations or reduced their frequency, also attenuated hypercontracture.

To estimate net changes in the cytosolic Ca²⁺ balance during reoxygenation, the integral of the fura 2 signal was determined. The integrals from the beginning of reoxygenation to complete recovery of the fura 2 signal are given in Table 1. These data indicate that the changes of the cytosolic Ca²⁺ balance were not altered in the presence of halothane or cyclopiazonic acid. It was reduced in the presence of ryanodine, possibly indicating a sequestration of Ca²⁺ within the SR.

View this table: [in this window] [in a new window]   Table 1. Influence of Halothane, Cyclopiazonic Acid, and Ryanodine Total Cellular Ca2+ During Reoxygenation

Reoxygenation After Anoxia in Medium at pH 7.4
In an additional set of experiments, cardiomyocytes were incubated under anoxic conditions in medium at pH 7.4. As reported previously,¹¹ intracellular pH remains close to the control level (pH_i 7.1) under these conditions of oxygen deprivation (simulated anoxic perfusion) and stays at that level when cells are subsequently reoxygenated in medium at pH 7.4. Anoxic incubation of cardiomyocytes in medium at pH 7.4 as well as in medium at pH 6.4 (Fig 2) caused a reduction of the initial cell length of one third as a result of rigor formation and a subsequent rise of the fura 2 ratio to the saturation level of fura 2 fluorescence, indicating a severe cytosolic Ca²⁺ overload. As under simulated ischemic conditions, the cells were allowed to develop a severe Ca²⁺ overload, indicated by the saturation of the fura 2 signal, and were then reoxygenated. Reoxygenation also provoked hypercontracture under these conditions of anoxia-reoxygenation, but hypercontracture developed more rapidly than in simulated ischemia-reoxygenation (Fig 6). When cells had been exposed to anoxia (pH 7.4), hypercontracture developed within the first minute of reoxygenation (Fig 6A), ie, during the initial fall of the Ca²⁺ signal. Hypercontracture was already maximal after 2 minutes of reoxygenation, ie, it occurred during the earliest phase of Ca²⁺ recovery. This is in contrast to the delayed development of hypercontracture after simulated ischemia, and hypercontracture occurred during the oscillatory movements of cytosolic Ca²⁺, ie, during the second phase of Ca²⁺ recovery (Fig 6B). After anoxia in medium with pH 7.4, the cytosolic Ca²⁺ concentration nevertheless also returned to control levels, as indicated by the fall of the fura 2 signal. This decline was again followed by a period of oscillations of the Ca²⁺ signal. When cardiomyocytes were reoxygenated under these conditions in the presence of halothane, hypercontracture was not prevented, although the reoxygenation-induced oscillations of the Ca²⁺ signal were suppressed (Fig 7).

View larger version (41K): [in this window] [in a new window]   Figure 6. Time course of fura 2 ratio (continuous trace of original recording) and cell length in percent of end-ischemic cell length () in single cardiomyocytes during reoxygenation. A, Changes during reoxygenation after anoxic incubation in medium at pH 7.4; B, changes after simulated ischemia (anoxic incubation at medium pH 6.4).

View larger version (15K): [in this window] [in a new window]   Figure 7. Oscillations of the fura 2 ratio and hypercontracture in reoxygenated cardiomyocytes. Anoxia and reoxygenation were carried out in media at pH 7.4. A, Frequency of reoxygenation-induced Ca2+ oscillations 5 minutes after onset of reoxygenation; B, cell length in percent of end-ischemic length after 10 minutes of reoxygenation. Data are mean±SEM, n=8 cells. *P<.001.

Effects of Halothane on Normoxic Cardiomyocytes
We investigated whether halothane can prevent hypercontracture in paced cardiomyocytes that had not been exposed to oxygen depletion. Removal of Na⁺ from the extracellular medium was used to induce hypercontracture (Na⁺ withdrawal contracture). This procedure represents an easy way to produce a manifest Ca²⁺ overload, because in the absence of exogenous Na⁺, the cells take up Ca²⁺ by the "reverse mode" of the Na⁺/Ca²⁺ exchanger. The extent of hypercontracture induced by Na⁺ withdrawal was identical in the presence or absence of 0.4 mmol/L halothane (Fig 8). In contrast to the inability of halothane to prevent hypercontracture under these conditions, it completely abolished the electrically paced contractions. This is a result of a rapid suppression of cytosolic Ca²⁺ transients, as shown in the inset of Fig 8.

View larger version (29K): [in this window] [in a new window]   Figure 8. Response of cardiomyocytes to Na+ withdrawal (hypercontracture) and to electrical field stimulation (Ca2+ transients, solid line, and paced contractions, ; see inset) under normoxic conditions in presence () or absence () of 0.4 mmol/L halothane. Halothane abolishes paced contractions concomitantly with suppression of Ca2+ transients. Na+-free conditions cause development of Na+ withdrawal contracture.26 Data are mean±SEM, n=8 cells.

Cellular State of Energy
The cellular state of energy was determined after anoxic conditions and subsequent reoxygenation in the presence of the agents investigated (Table 2). In anoxic/reoxygenated cells, ATP contents recovered to 32% of the control level within 15 minutes of reoxygenation. Creatine phosphate (CrP) recovered fully to the control level. Treatment of the cells with ryanodine, cyclopiazonic acid, or halothane did not affect ATP and CrP contents significantly.

View this table: [in this window] [in a new window]   Table 2. State of Energy of Cardiomyocytes

	Discussion

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In the present study, the effect of halothane on reoxygenation-induced hypercontracture was investigated in isolated cardiomyocytes exposed to simulated ischemia-reperfusion. We found that after simulated ischemia, hypercontracture develops in synchrony with the occurrence of SR-dependent oscillations of cytosolic Ca²⁺ concentrations. Ryanodine, which inhibits Ca²⁺ release, and cyclopiazonic acid, which inhibits SR Ca²⁺ uptake, suppress these oscillations and, at the same time, the development of hypercontracture. These results show that the oscillations of cytosolic Ca²⁺ represent the cause of hypercontracture. The temporary presence of halothane during reoxygenation also prevents reoxygenation-induced hypercontracture. This protective effect of halothane also seems to be due to its ability to abolish SR-dependent oscillations of cytosolic Ca²⁺.

In previous studies, the time course of changes in cytosolic Ca²⁺, Na⁺, and H⁺ in oxygen-deprived and reoxygenated cardiomyocytes was described in detail.^9–11 It was shown that reoxygenated cardiomyocytes, although jeopardized by the development of hypercontracture, can rapidly reverse their cytosolic Ca²⁺ overload when reoxygenated, because oxidative energy production is reactivated. The recovery of Ca²⁺ control can be divided into two phases: In the first phase, lasting 2 minutes, cytosolic Ca²⁺ rapidly declines because it is sequestered into the SR. In the subsequent second phase, Ca²⁺ cycles in an oscillatory manner between cytosol and SR.⁹ These oscillations continue for the time needed by the reenergized cell to extrude the excess load of Ca²⁺ across the sarcolemma, mainly via the Na⁺/Ca²⁺ exchanger.¹⁰ It should be mentioned that these oscillations of the fura 2 signal were observed only during the reoxygenation period. Higher time resolution of the fura 2 signal revealed that Ca²⁺ oscillations did not occur during the preceding period of simulated ischemia as was reported for chemical anoxia by prolonged metabolic inhibition due to cyanide exposure.^24,25

It has been demonstrated before^9,21 that reoxygenated cardiomyocytes may develop hypercontracture because of the resupply of energy to the myofibrils at elevated cytosolic Ca²⁺ concentrations after reoxygenation. It is now shown that hypercontracture develops during different phases in the reoxygenated cardiomyocytes, depending on the initial pH_i: (1) When cells are reoxygenated after oxygen depletion in absence of a significant intracellular acidosis, hypercontracture follows instantaneously the onset of reoxygenation. Under these conditions, it is the still excessive initial cytosolic Ca²⁺ concentration that, in conjunction with the reenergization, causes the contractile activation leading to irreversible hypercontracture. (2) Under conditions of simulated ischemia-reperfusion, however, hypercontracture develops in a delayed manner, in synchrony with the occurrence of SR-dependent Ca²⁺ oscillations. Under these conditions, pH_i is sufficiently low during the first 2 minutes of reoxygenation to prevent development of hypercontracture.¹¹ This effect of low pH_i has been attributed to Ca²⁺ desensitization of the myofibrils. One may thus distinguish between phase 1 hypercontracture and phase 2 hypercontracture. Using these terms, we may say that cardiomyocytes under simulated ischemia-reperfusion are jeopardized by phase 2 hypercontracture during reoxygenation.

The present study shows that phase 2 hypercontracture can be prevented by a specific inhibitor of SR Ca²⁺ release, ryanodine, and of SR Ca²⁺ uptake, cyclopiazonic acid. Both agents abolish phase 2 oscillations because they interrupt the cycling of Ca²⁺ between cytosol and SR. Hypercontracture is not prevented by an influence on pH_i normalization after simulated ischemia by the investigated agents, because neither ryanodine nor cyclopiazonic acid prolonged intracellular acidosis. Recovery of pH_i was unaltered when these agents were present. The fact that these chemically and functionally different agents both inhibit the development of Ca²⁺ oscillations, and the concomitant development of hypercontracture, indicate that phase 2 hypercontracture is caused by the transiently high Ca²⁺ concentrations occurring during Ca²⁺ oscillations.

The cells were also protected against phase 2 hypercontracture when halothane was applied on reoxygenation after simulated ischemia. Again, halothane did not influence pH_i recovery. As in the presence of the specific inhibitors of SR functions, the phase 2 oscillations of Ca²⁺ were suppressed. This coincidence suggests a mode of action similar to that for the specific inhibitors, ie, interruption of Ca²⁺ cycling between SR and cytosol preventing high oscillatory peak concentrations of Ca²⁺. Halothane has indeed been shown to interfere with the ryanodine receptor.¹² The approach of the present study, however, does not allow us to directly identify the mode of action by which halothane suppresses SR-dependent oscillations in reoxygenated cardiomyocytes. Therefore, one has to consider that suppression of Ca²⁺ oscillations might also be due to a change in the total cellular Ca²⁺, ie, to changes by an accelerated extrusion of Ca²⁺ from the reoxygenated cells or a reduced further influx of Ca²⁺. Both effects might occur during the early phase of reoxygenation. If any of these objections should apply, a change in the integral of the fura 2 signal during the period of Ca²⁺ recovery in reoxygenation could be expected, because this represents a relative measure for the net cytosolic Ca²⁺ load during this period of time. The integral of the fura 2 ratio was not found to be different, however, in cardiomyocytes reoxygenated in the presence or absence of halothane. The results thus render it unlikely that halothane inhibits SR oscillations because of accelerated Ca²⁺ efflux from or decelerated Ca²⁺ influx into reoxygenated cardiomyocytes. The integral of the fura 2 ratio during reoxygenation in the presence of halothane is similar to that in the presence of cyclopiazonic acid but different from the one in the presence of ryanodine. The fura 2 integral is smaller in the presence of ryanodine. This may be because in its presence, ie, when the release channel is kept closed, the SR is available as a Ca²⁺ storage capacity to remove a part of the accumulated Ca²⁺ from the cytosol.

One must consider whether the differences in developing hypercontracture observed in the present study can be attributed to differences in ATP contents of cardiomyocytes after anoxia. Altschuld et al²⁷ observed that, in permeabilized cardiomyocytes incubated with Mg²⁺-ATP solutions in the absence of CrP, the apparent Ca²⁺ sensitivity of contractile activation may be increased when the ATP concentration is gradually lowered from a physiological level to one causing Ca²⁺-independent rigor shortening. Because CrP was absent in their study and permeabilized cells were used, a direct extrapolation of their observations to the state of energy in intact cells with high CrP contents is not possible. In the present study, reoxygenation was carried out with or without ryanodine, cyclopiazonic acid, or halothane. The presence of one of these agents caused a suppression of reoxygenation-induced Ca²⁺ oscillations and hypercontracture, but ATP and CrP contents did not differ significantly in their presence or absence. These findings render it unlikely that protection against hypercontracture is due to variations of the cellular state of energy.

Another possibility was also investigated to explain the protective action of halothane. It has been demonstrated before that agents that directly inhibit the contractile machinery prevent reoxygenation-induced hypercontracture.²² These agents also protect against phase 1 hypercontracture as found in cardiomyocytes reoxygenated after anoxia without significant changes of intracellular pH. Halothane did not prevent hypercontracture under such conditions, however, indicating a different mode of action. This point was further corroborated by investigation of the ability of halothane to interfere with other forms of Ca²⁺ overload–induced hypercontracture in cardiomyocytes. For this purpose, hypercontracture was induced in normoxic cardiomyocytes by Na⁺ withdrawal, a procedure that causes Ca²⁺ overload via Na⁺/Ca²⁺ exchange.²⁶ Halothane was also unable to protect against this type of hypercontracture. This finding is in agreement with the observation that halothane is unable to prevent hypercontracture in anoxia-reoxygenation without anoxic acidosis. Conversely, halothane inhibits Ca²⁺ transients and cell contractions of normoxic, electrically paced cardiomyocytes. This is consistent with its ability to protect against hypercontracture after simulated ischemia, when hypercontracture is elicited by SR-dependent Ca²⁺ oscillations.

We cannot infer from our results exactly how halothane interferes with intracellular Ca²⁺ handling, but with these alternative explanations ruled out, the most likely explanation remains that halothane prevents hypercontracture in the way the inhibitors of SR function do, namely, by suppressing the SR-dependent Ca²⁺ oscillations.

For ischemia-reperfusion of myocardium in vivo, failure of halothane to protect against reoxygenation-induced hypercontracture in cells entering reoxygenation with a normal cytosolic pH is probably irrelevant, because ischemia would always leave the cytosol acidotic. Simulated ischemia imitates the in vivo situation more closely, in that the cells are reoxygenated when the cytosolic pH is acidotic (pH_i 6.5). Under these circumstances, hypercontracture does not occur during the very initial period of reoxygenation (phase 1), because at this time, intracellular acidosis still exists and inhibits contractile activation.¹¹ The subsequent recovery of intracellular pH (phase 2), however, permits a delayed occurrence of hypercontracture. As shown in the present study, the Ca²⁺ oscillations are then the trigger for hypercontracture.

The findings of the present study are consistent with a previous one⁴ showing that in the isolated perfused rat heart, halothane protects against oxygen paradox injury when present during reperfusion. That previous study did not indicate the possible cause for this protective action of halothane, however. Protective properties of halothane for the myocardium during ischemia-reperfusion have been reported for a variety of different experimental models.^{1–4,28–33} In most of these studies, the protection can be attributed to direct or indirect effects of halothane exerted on the myocardium before or during ischemia. As an example of an indirect mode of action, halothane may reduce the sympathetic tone and thereby lower myocardial energy demand before ischemia. Because of this, myocardium may then tolerate ischemic malsupply for longer time. In some studies, possible protective effects of halothane against reperfusion injury have been discussed. Answers could not be provided, however, because the experimental designs did not allow differentiation between the actions of halothane in ischemia and reperfusion or between the possible target cell types. We can derive from the present study on isolated cardiomyocytes and the previous one on the anoxic-reoxygenated heart that halothane can indeed provide specific protection during the early, "vulnerable" phase of reperfusion and that protection against hypercontracture is due to a direct effect on the cardiomyocyte.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (grant Si 618/1) and the European Union (BIOMED-2 program).

Received May 12, 1997; revision received August 19, 1997; accepted September 1, 1997.

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