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CARDIOVASCULAR ANESTHESIA

The Impact of Isoflurane During Simulated Ischemia/Reoxygenation on Intracellular Calcium, Contractile Function, and Arrhythmia in Ventricular Myocytes

Martin Dworschak, MD^*,, Dirk Breukelmann, MD^*,, and James D. Hannon, MD^*

*Department of Anesthesia Research, Mayo Clinic, Rochester, Minnesota, Division of Cardiothoracic and Vascular Anaesthesia and Intensive Care, University Hospital Vienna, Austria, and Department of Anaesthesiology and Intensive Care, University of Münster, Germany

Address correspondence and reprint requests to Martin Dworschak, MD, Division of Cardiothoracic Anesthesia and Intensive Care, University Hospital Vienna, Waehringer Guertel 18–20, A-1090 Vienna, Austria. Address email to martin.dworschak{at}univie.ac.at

	Abstract

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Some of isoflurane’s cellular actions, such as interference with intracellular Ca²⁺ handling, inhibition of the respiratory chain, and the capability to produce oxygen radicals, could result in impaired cellular function during ischemia/reoxygenation (I/R). We investigated the effects of isoflurane applied during I/R on intracellular Ca²⁺, oxygen radical formation, arrhythmic events, and contractile function in rat cardiomyocytes. Single ventricular myocytes were subjected to 30 min of simulated ischemia followed by 30 min of reoxygenation. After baseline measurements, isoflurane-treated cells were exposed to 1 minimum alveolar concentration of isoflurane in air, whereas control cells were exposed to air only. Cytosolic Ca²⁺ overload was observed in the isoflurane group (P < 0.05). During ischemia, systolic cell shortening decreased in both groups. In the isoflurane group, arrhythmic events and hypercontracture occurred more often during I/R, and the recovery of contractility during reoxygenation was less marked (P < 0.05). Furthermore, increased oxygen radical generation was detected in isoflurane-treated myocytes during reoxygenation (P < 0.05). Isoflurane given during I/R in this study induced intracellular Ca²⁺ accumulation and impaired cell function. These potentially harmful effects were associated with a diminished Ca²⁺ clearance and an accelerated oxygen radical production.

IMPLICATIONS: Isoflurane, a volatile anesthetic, administered during ischemia/reoxygenation, aggravated functional impairment in heart muscle cells. These alterations were probably caused by interference with intracellular Ca²⁺ handling proteins and by generation of oxygen radicals. Cardioprotection by isoflurane when given before ischemia should be reevaluated in situations of continuing ischemia and subsequent reperfusion.

	Introduction

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Isoflurane is often used during cardiac surgery and serves as a drug for cardiac preconditioning (1). Therefore, it has to be applied as a pretreatment before a subsequent, potentially lethal, ischemic insult. However, 83% of patients with chronic stable angina pectoris have asymptomatic myocardial ischemia, and approximately 50% of patients scheduled for elective coronary artery bypass surgery exhibit electrocardiographic evidence of myocardial ischemia despite maximal medical therapy (2,3). Thus, in many clinical situations, isoflurane cannot be applied as a pretreatment but is given during recurrent ischemia and subsequent reperfusion. Kato and Foëx (4) in their detailed review on myocardial protection by anesthetics state that "it is still controversial whether anesthetics protect the heart when administered after the onset of ischemia."

The exact mechanism by which isoflurane exerts its preconditioning effect is still not completely understood, but generation of oxygen radicals plays an important role (5,6). Oxygen radicals, however, are generated in abundance during reperfusion. Furthermore, isoflurane has additional cellular actions. It interferes with intracellular Ca²⁺ handling (7–9) and inhibits the respiratory chain (10). All of these mechanisms could potentially result in impaired cellular function during ischemia/reperfusion.

As there are no data available on the cellular actions of isoflurane applied during continuing simulated ischemia/reoxygenation (I/R), we investigated whether isoflurane alters intracellular Ca²⁺ ([Ca²⁺]_i) and the incidence of arrhythmic events in isolated rat ventricular myocytes. We also studied its effects on cellular contractile function and on the time course of oxygen radical generation during simulated I/R.

	Methods

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All experimental procedures were reviewed and approved by the Animal Care and Use Committee of the Mayo Foundation. Protocols were completed in accordance with National Institutes of Health guidelines and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council).

Male Sprague-Dawley rats (n = 20) weighing 300–350 g were anesthetized with 100 mg/kg pentobarbital (Nembutal, Abbott Laboratories, North Chicago, IL) intraperitoneally and the hearts were rapidly excised. The hearts were perfused via aortic cannulation until clear of blood. The buffer contained the following (in mM): 75 NaCl, 2.4 KCl, 1 MgCl₂, 10 HEPES, 58 sucrose, 10 dextrose, 5 NaHCO₃, and 2.5 Glutamic acid (pH: 7.2) (11). Ventricular myocytes were dissociated as previously described (8). In brief, both ventricles were dissected and minced in a solution containing 0.6 mg/mL collagenase II and 1 mg/mL albumin (Sigma, St. Louis, MO) at 35°C. The cellular suspension was then centrifuged, the supernatant was discarded, and the cells were resuspended in Tyrode’s solution that was composed of the following (in mM): 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, and 5 HEPES (pH: 7.4).

After a resting period, aliquots of the cell suspension were loaded with the fluorescent Ca²⁺ indicator Fura-2 AM (5 µM) (Molecular Probes Inc., Eugene, OR). A different group of myocytes was loaded with 10 µM CM-H₂DCFDA (Molecular Probes Inc., Eugene, OR) to monitor the generation of intracellular reactive oxygen species, mainly H₂O₂, which is able to readily cross cell membranes, and the charged hydroxyl radical (12,13).

Aliquots of the cell suspension were introduced into a perfusion chamber on the stage of an inverted epifluorescence microscope (Model TE 300, Nikon Instruments Inc., Melville, NY). Ventricular myocytes were then superfused with Tyrode’s solution and field stimulated at 0.25 Hz with a stimulus duration of 5 ms and a voltage of approximately 20% above threshold (Grass S88 Stimulator, West Warwick, RI).

Contracting, rod-shaped ventricular myocytes with clear striations and no blebs were positioned in the light path of the microscope and alternately excited at 340 and 380 nm for Fura-2, and 490 nm for CM-H₂DCFDA respectively. Emitted light was collected at 520 nm through an oil immersion lens (S Fluor, Nikon Instruments Inc., Melville, NY) and detected by a photomultiplier (C&L Instruments, Inc., Hummelstown, PA). After subtracting autofluorescence, [Ca²⁺]_i was expressed as the ratio of the emitted light produced by Fura-2. Fura-2 ratios were normalized to the baseline value of each cell.

Electrical stimulation of myocytes results in systolic cell shortening and diastolic relengthening. Cell shortening is preceded by a characteristic cytosolic increase of [Ca²⁺]_i. After a systolic upstroke succeeding depolarization, a peak level is reached and an exponential decay of the transient to a resting diastolic level follows. Irregularities in these Fura-2 transients, i.e., stimulation-independent transients, and absence of transients after electrical stimulation were defined as arrhythmic events (14,15).

Microscopic images of the ventricular myocyte were recorded and stored for off-line determination of cell length. Hypercontraction was defined as cell length <55% of initial diastolic length. The degree of systolic cell shortening was assessed semiquantitatively at each time point for each individual myocyte. Cell shortening of a normal contracting ventricular myocyte is reported to be 10%–12% of its initial diastolic cell length (16). Accordingly, systolic cell shortening was determined as follows: a) very strong contraction (reduction of initial cell length >12%), b) normal or diminished contraction (reduction of cell length < 12%), and c) no contraction upon electrical stimulation.

Experiments were performed at room temperature (20°C–22°C), which was monitored in the superfusion buffer. The temperature was chosen to minimize the effect of cellular processes that transport negatively charged Ca²⁺ indicators from the cell. In addition, increased metabolism and faster intracellular Ca²⁺ accumulation at higher temperatures may result in earlier cell death and preclude observation of group differences that develop at a slower pace.

Ischemia was simulated by decreasing extracellular pH to 6.3 and adding 10 mM deoxyglucose to competitively inhibit glycolysis (17). In addition, hypoxic conditions (PO₂ < 15 mm Hg) were achieved by vigorously bubbling the solution with N₂ and introducing N₂ under the hood of the superfusion chamber (18). Myocytes were exposed to 30 min of simulated ischemia followed by 30 min of reoxygenation with 10 mM glucose containing Tyrode’s solution and normal ambient PO₂ (pH: 7.4). I/R was done either in the presence of 1 MAC isoflurane in air (Iso; n = 30) or with air only (Air; n = 40). Concentrations of the volatile anesthetic in the perfusate (0.36 ± 0.09 mM, mean ± SD) were verified by gas chromatography (model 5880A; Hewlett-Packard, Palo Alto, CA) and PO₂ was measured in a gas analyzer (ABL; Radiometer, Westlake, OH). One MAC of isoflurane in the rat was based on the temperature-independent aqueous concentration of isoflurane calculated by Franks and Lieb (19). Time controls without simulated I/R were also performed. Furthermore, a group of myocytes was treated with 1 MAC of isoflurane without I/R to investigate I/R-independent effects of isoflurane in the setting we used.

Two-way factorial analysis of variance followed by appropriate post hoc tests (Tukey, Dunn’s, or Bonferroni test) were used to determine differences within and between groups during the experimental phases I/R. In the case of an unequal distribution of data, analysis of variance on ranks was calculated. Kruskal-Wallis statistics was used to analyze contractility data. Categorical data were analyzed with the ² test. SigmaStat 2.0 was the software we used and a P < 0.05 was considered statistically significant. Data are presented as means ± SEM.

	Results

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No change from baseline values of each variable was observed in time controls monitored over the course of the experiment without being subjected to simulated I/R (data not shown).

Peak and resting cytosolic [Ca²⁺]_i increased in both groups from the beginning of ischemia until the end of reoxygenation. This increase was more pronounced in the isoflurane group (P < 0.05; Fig. 1). More cells in the isoflurane group developed extremely high [Ca²⁺]_i values, which occurred before hypercontraction (Fig. 2). Isoflurane alone reversibly decreased peak and to a much lesser extent also resting [Ca²⁺]_i in myocytes that were not subjected to I/R (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 1. A, time course of normalized diastolic Fura-2 ratio. Resting diastolic myoplasmic [Ca2+]i increased over time in both groups. This increase, however, was much more pronounced and almost three times larger in the isoflurane group. B, time course of the percentage of nonarrhythmic cells. Arrhythmic events increased during simulated ischemia but showed a tendency towards restoration during reoxygenation in both groups. However, more myocytes in the isoflurane group became arrhythmic in the course of ischemia and remained arrhythmic during subsequent reoxygenation. The abbreviations on the y-axis indicate the phase (I = ischemia; R = reoxygenation) and the corresponding time in minutes. *P < 0.05 (Iso versus Air)

View larger version (30K): [in this window] [in a new window]   Figure 2. As can be seen from these original, unsmoothed Ca2+ transients, isoflurane during ischemia/reoxygenation (I/R) caused a more severe increase of resting cytoplasmic [Ca2+]i over time. It also induced a more frequent incidence of hypercontracting myocytes. Panel 3 in the lower row shows the myoplasmic Ca2+ concentration of a myocyte that hypercontracted (note the different scale). Additionally, the center panels depict the decreased rate of decay and the more depressed amplitude of the Ca2+ transient caused by ischemia, which used to be more pronounced in isoflurane-treated cells. Simulated I/R also resulted in an increase of arrhythmic events that is visible in both groups at the end of reoxygenation. Although the recording of the isoflurane-treated cell at the end of ischemia depicts regular transients, this myocyte was classified as being arrhythmic at this time point because it showed stimulation-independent transients immediately before this recording was done.

During ischemia, systolic cell shortening decreased in both groups, whereas the number of arrhythmic cells concomitantly increased. Arrhythmic events occurred more often in the isoflurane group during ischemia as well as during reoxygenation (P < 0.05; Fig. 1 and 2). The observed recovery of contractility during reoxygenation was also delayed and less marked in the isoflurane group. Furthermore, the number of noncontracting myocytes was significantly larger during ischemia and remained larger during reoxygenation in isoflurane-treated cells (P < 0.05; Fig. 3). In addition, the percentage of cardiomyocytes that hypercontracted was larger in the isoflurane group (42% versus 16%; P < 0.05). Hypercontraction primarily occurred during reoxygenation. These myocytes eventually showed decreases of the 340 and 380 nm signal, indicating leakage of Fura-2 out of the cell through a disrupted sarcolemma. In contrast, this phenomenon was not obvious in myocytes that retained their rod-shaped appearance but would not contract upon electrical stimulation. Isoflurane alone also impeded systolic cell shortening yet without inducing arrhythmic events (data not shown).

View larger version (64K): [in this window] [in a new window]   Figure 3. Semiquantitative changes over time in systolic cell shortening in the control (A) and the isoflurane (B) groups. Ischemia severely diminished cellular contraction with a significantly larger percentage of noncontracting myocytes in the isoflurane group (P < 0.05). Reoxygenation induced a recovery again in both groups. However, the number of noncontracting cells remained larger in the isoflurane group throughout reoxygenation at each time point (P < 0.05). The abbreviations on the y-axis indicate the phase (I = ischemia; R = reoxygenation) and the corresponding time in minutes.

View larger version (25K): [in this window] [in a new window]   Figure 4. CM-H2DCFDA fluorescence intensity during ischemia-reoxygenation (I/R). Intensity initially decreased during simulated ischemia but showed a steep increase early on during reoxygenation in both groups. This increase occurred faster and was more pronounced in the isoflurane group. *P < 0.05 (Iso versus Air).

	Discussion

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Without I/R, isoflurane in this study caused reversible depressions in [Ca²⁺]_i, in the Ca²⁺ transient, and in the rate of decay of the Ca²⁺ transient. These results are in accordance with previously published data. The decreased decay of the Ca²⁺ transient is mainly attributable to isoflurane inhibiting the sequestration of Ca²⁺ by the sarcoplasmic reticulum (SR). This mechanism has already been described by Lopez and Kosk-Kosicka (9) in the isolated SR of rabbit skeletal muscle under nonischemic conditions. Similar alterations were demonstrable in a study by Jiang and Julian (20) in rat ventricular trabeculae. Decreased Ca²⁺ uptake in their investigation and in this study occurred despite a remarkable decrease in the amplitude of the Ca²⁺ transient, which indicates reduced Ca²⁺-induced Ca²⁺ release. Diminished SR Ca²⁺ release may result from depression of L- or T-type Ca²⁺ currents, i.e., Ca²⁺ influx (21) or from a depressed ryanodine channel, i.e., reduced Ca²⁺ release from the SR (20,22). Reduced Ca²⁺-induced Ca²⁺ release through both mechanisms has also been reported in isolated cardiac myocytes superfused in the presence of isoflurane (23). Thus, isoflurane acts on the function of various different Ca²⁺ handling proteins that manage Ca²⁺ homeostasis.

However, despite depressing Ca²⁺ influx and SR Ca²⁺ release, in combination with I/R Ca²⁺ accumulation occurred in myocytes treated with isoflurane. Thus, Ca²⁺ clearance seems to be affected to a greater extent by isoflurane during I/R. This can be explained either by a further diminished SR Ca²⁺ reuptake or by a decreased extrusion via the plasma membrane Ca²⁺ adenosine triphosphate (ATP)-ase (8). Because reuptake of Ca²⁺ by the sarcoplasmic Ca²⁺ ATPase (SERCA) requires ATP, sequestration is naturally impeded during ischemia. For halothane, decreased Ca²⁺ sequestration via SERCA has been found to be pH dependent with greater inhibition at lower pH values (24). Therefore, this action may be aggravated in situations, as during ischemia, where intracellular pH decreases. Increased [Ca²⁺]_i, apart from a very brief period at the onset of reoxygenation, was not accompanied by an increase of systolic cell shortening, which reflects diminished Ca²⁺ sensitivity (21). Cytoplasmic Ca²⁺ overload may furthermore give rise to arrhythmia and may explain the more frequent arrhythmic events that were detectable in myocytes of the isoflurane group. In 1997, Houltz et al. (25) reported impaired diastolic ventricular relaxation and increased left ventricle end-diastolic stiffness in coronary artery disease patients treated with isoflurane to control the stress response during sternotomy. This finding could, in part, be attributable to diminished cytosolic Ca²⁺ removal during episodes of myocardial ischemia in the presence of isoflurane.

Excessive oxygen radical production during reperfusion can additionally amplify the functional damage caused by an increased cytoplasmic Ca²⁺ load (5,10). In this investigation, the generation of reactive oxygen species was accelerated in the isoflurane group during reoxygenation. Interestingly, CM-H₂DCFDA fluorescence decreased during simulated ischemia in both groups. This can either be explained by the minor quantities of oxygen radicals generated by our ischemia protocol or by the interchangeability of reactive oxygen species, which depends on the specific environment at the time radicals are generated. Additionally, because of the insufficient selectivity of CM-H₂DCFDA some radical species may not be detected by this fluorescent dye. CM-H₂DCFDA is more sensitive to H₂O₂ and to the hydroxyl radical (17). It is, however, less sensitive to the superoxide radical and other oxygen radical species that were detected during preconditioning with isoflurane (5,26). H₂O₂ has been shown to uncouple Ca²⁺ reuptake by SERCA from ATP hydrolysis and seems to be responsible for a depressed Ca²⁺ uptake in canine SR (27). Hydroxyl radicals that can evolve by the Fenton reaction from H₂O₂ and superoxide radicals have also been associated with diastolic dysfunction (28). In this setting, isoflurane induced a more rapid and a greater liberation of these oxygen species beginning with the onset of reoxygenation. This can be responsible for some of the impaired cellular functions we observed in the isoflurane group.

It is difficult to accurately simulate clinical myocardial ischemia and reperfusion in a cellular model with concomitant application of a drug. Although some blood flow may persist during myocardial ischemia, it is inadequate for cellular metabolism. In this regard, the model we used clearly has its limitations, as ventricular myocytes were continuously superfused. However, we applied a slightly modified model that was previously used to describe alterations in ventricular myocytes in the course of I/R (17). Although our results should be interpreted with caution, they do not reveal a cellular protective effect of isoflurane when given in the course of continuing I/R. Because we cannot answer the question of whether isoflurane would still exert beneficial effects on the heart in the whole organism, further controlled studies are warranted to address this clinically relevant issue. Protective effects could be conveyed through alterations in the systemic and the coronary circulation, via actions on the autonomic nervous system, and via modulation of the interaction between neutrophils and the coronary endothelium.

In conclusion, the application of 1 MAC isoflurane throughout simulated ischemia and subsequent reoxygenation results in cytosolic Ca²⁺ accumulation, enhanced liberation of reactive oxygen species during reoxygenation, diminished cell shortening, and an increased incidence of arrhythmic events in rat ventricular myocytes. These potentially harmful effects were associated with diminished cytosolic Ca²⁺ removal that has been observed with isoflurane and has previously been reported from our laboratory (7,8). Ca²⁺ accumulation with ensuing cellular dysfunction is likely to be the result of the interplay between direct effects of isoflurane on Ca²⁺ handling proteins and indirect actions via generation of oxygen radicals.

	Acknowledgments

 
Supported, in part, by the National Institutes of Health Grant No. GM57891 and by institutional funding from the Division of Cardiothoracic and Vascular Anesthesia and Intensive Care, University of Vienna, Austria.

	Footnotes

 
Presented, in part, at the 2003 IARS meeting in New Orleans and published in abstract form in the corresponding supplement issue of Anesthesia & Analgesia.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Dworschak, M. Articles by Hannon, J. D. Search for Related Content PubMed PubMed Citation Articles by Dworschak, M. Articles by Hannon, J. D. Related Collections Cardiovascular Mechanisms Pharmacology