Droperidol has recently been associated with cardiac arrhythmias and sudden cardiac death. Changes in action potential duration seem to be the cause of the arrhythmic behavior, which can lead to alterations in intracellular free Ca concentration ([Ca]i). Because [Ca]i and myofilament Ca sensitivity are key regulators of myocardial contractility, the authors' objective was to identify whether droperidol alters [Ca]i or myofilament Ca sensitivity in rat ventricular myocytes and to identify the cellular mechanisms responsible for these effects.
Freshly isolated rat ventricular myocytes were obtained from adult rat hearts. Myocyte shortening, [Ca]i, nitric oxide production, intracellular pH, and action potentials were monitored in cardiomyocytes exposed to droperidol. Langendorff perfused hearts were used to assess overall cardiac function.
Droperidol (0.03-1 mum) caused concentration-dependent decreases in peak [Ca]i and shortening. Droperidol inhibited 35 mm KCl-induced increase in [Ca]i, with little direct effect on sarcoplasmic reticulum Ca stores. Droperidol had no effect on action potential duration but caused a rightward shift in the concentration-response curve to extracellular Ca for shortening, with no concomitant effect on peak [Ca]i. Droperidol decreased pHi and increased nitric oxide production. Droperidol exerted a negative inotropic effect in Langendorff perfused hearts.
These data demonstrate that droperidol decreases cardiomyocyte function, which is mediated by a decrease in [Ca]i and a decrease in myofilament Ca sensitivity. The decrease in [Ca]i is mediated by decreased sarcolemmal Ca influx. The decrease in myofilament Ca sensitivity is likely mediated by a decrease in pHi and an increase in nitric oxide production.
DROPERIDOL is a butyrophenone derivative that was approved for clinical use in 1970 for the treatment of postoperative nausea and vomiting. In 2001, the US Food and Drug Administration released an alert on the use of droperidol because of its possible cardiotoxicity associated with ventricular arrhythmia (torsade de pointes) and sudden cardiac death. High concentrations of droperidol have been shown to cause a concentration-dependent prolongation in the QT interval in surgical patients, suggesting that droperidol may lead to polymorphic ventricular arrhythmias.1Similarly, large concentrations of droperidol used to treat psychiatric patients have been associated with significant QT prolongation, contributing to serious cardiac arrhythmias and even death in some susceptible patients.2,3In addition, droperidol has been shown to decrease left ventricular performance in humans,4although the mechanisms by which droperidol exerts these effects have not been clearly identified. Despite the potential for these adverse events, many physicians question the validity of warnings that have been imposed regarding the safety of droperidol, alone or in combination with other antiemetic drugs (serotonin receptor antagonists), for the treatment of postoperative nausea and vomiting.5,6
Cardiac contractility is regulated by changes in intracellular Ca2+concentration ([Ca2+]i), myofilament Ca2+sensitivity, or both. We tested the hypothesis that droperidol exerts a direct negative inotropic effect on individual freshly isolated rat ventricular myocytes. We assessed the effects of droperidol on Ca2+influx via voltage-gated Ca2+channels, action potential duration, and Ca2+release from the sarcoplasmic reticulum (SR). We also investigated the effect of droperidol on myofilament Ca2+sensitivity and measured droperidol-induced changes in two important mechanisms that regulate myofilament Ca2+sensitivity, intracellular pH (pHi) and nitric oxide production. Finally, we assessed the functional significance of our results in isolated myocytes by examining the effect of droperidol in a Langendorff perfused heart preparation.
Materials and Methods
All experimental procedures and protocols were approved by the Cleveland Clinic Institutional Animal Care and Use Committee (Cleveland, Ohio) and conformed to the guidelines for the care and use of laboratory animals.
Ventricular Myocyte Preparation
Ventricular myocytes were freshly isolated from adult male Sprague-Dawley rat hearts, as previously described.7Immediately after the animals were killed, the hearts were rapidly removed and perfused in a retrograde manner at a constant flow rate (8 ml/min) with oxygenated (95% O2–5% CO2) Krebs-Henseleit buffer (KHB; 37°C) containing the following: 118 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl2, 1.2 mm KH2PO4, 1.2 mm CaCl2, 37.5 mm NaHCO3, and 16.5 mm dextrose, at a pH of 7.35. After a 5-min equilibration period, the perfusion buffer was changed to a Ca2+-free KHB buffer containing collagenase type II (309 U/ml; Worthington Biochemical Corp., Freehold, NJ). After digestion with collagenase (20 min), the ventricles were minced and shaken in KHB, and the resulting cellular digest was washed, filtered, and resuspended in phosphate-free HEPES-buffered saline (HBS; 23°C) containing the following: 118 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl2, 1.25 mm CaCl2, 11.0 mm dextrose, 25.0 mm HEPES, and 5.0 mm pyruvate, at a pH of 7.35.
Measurement of [Ca2+]iand Shortening
Simultaneous measurement of [Ca2+]iand cell shortening was performed, as previously described by our laboratory.7Ventricular myocytes exhibiting a rod-shaped appearance with clear striations were chosen for study. Myocytes (0.5 × 106cells/ml) were incubated in HBS containing 2 μm fura-2/acetoxy methylester at room temperature for 15 min. Fura-2–loaded ventricular myocytes were placed in a temperature-regulated (30°C) chamber (Bioptechs, Inc., Butler, PA) mounted on the stage of an Olympus IX-70 inverted fluorescence microscope (Olympus America, Lake Success, NY). The cells were superfused continuously with HBS at a flow rate of 2 ml/min and field-stimulated via bipolar platinum electrodes at a frequency of 0.3 Hz with a 5-ms pulse using a Grass SD9 stimulator (Grass Instruments, West Warwick, RI).
Fluorescence measurements were performed on individual myocytes using a dual-wavelength spectrofluorometer (Deltascan RFK6002; Photon Technology International, Lawrenceville, NJ) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Because calibration procedures rely on a number of assumptions, the ratio of the light intensities at the two wavelengths was used to measure qualitative changes in [Ca2+]i. Just before data acquisition, background fluorescence was measured and automatically subtracted from the subsequent experimental measurement. The fluorescence sampling frequency was 100 Hz, and data were collected using software from Photon Technology International.
To simultaneously monitor cell shortening, the cells were also illuminated with red light. A dichroic mirror (600-nm cutoff) in the emission path deflected the cell image through a charge-coupled device video camera (Phillips VC 62505T; Marshall Electronics, Culver City, CA) into a video-edge detector (Crescent Electronics, Sandy, UT) with 16-ms resolution. The video-edge detector was calibrated using a stage micrometer so that cell lengths during shortening and relengthening could be measured.
Intracellular pH Measurements
Measurement of pHiwas performed, as previously described.8Ventricular myocytes were incubated in HBS containing 2 μm of the acetoxy methylester form of 2′,7′-bis(2-carboxy-ethyl)-5,6-carboxyfluorescein (BCECF/AM) at 23°C for 20 min. Similar to the procedure described for fura-2–loaded myocytes, BCECF-loaded ventricular myocytes were placed in a temperature-regulated (30°C) chamber mounted on the stage of an Olympus IX-70 inverted fluorescence microscope. The volume of the chamber was 1.5 ml. The cells were superfused continuously with HBS at a flow rate of 2 ml/min (30°C). Fluorescence measurements were performed on single ventricular myocytes as described above for [Ca2+]i; however, excitation wavelengths of 440 and 500 nm and an emission wavelength of 530 nm were used, as previously described.8The fluorescence sampling frequency was 10 Hz, and background fluorescence was determined as described above. To estimate the pHivalue from the ratio of 500/440 nm fluorescence, we used an in situ calibration procedure.8–10At the end of each experiment, the fluorescence ratio from each cell was calibrated in situ by exposing the cell to solutions of varying pH. Each solution contained 140 mm KCl, 1.0 mm MgCl2, 4.0 mm HEPES, 2.0 mm EGTA, 30 mm 2,3-butanedione monoxime, 50 μm BAPTA-AM, and 14 μm nigericin and was titrated to varying pH values (6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8) using KOH (1.0 N). The pHifor each cell was then determined from a linear regression of the fluorescence ratio versus the pH value of the calibration buffer. We previously determined that a linear relation exists between the 500/440-nm ratio and pHiin the physiologic range (pH 6.6–7.8).8
Analysis of [Ca2+]iand Shortening Data
The following variables were calculated for each individual contraction: resting [Ca2+]iand cell length, peak [Ca2+]iand cell length, change in [Ca2+]i(peak [Ca2+]iminus resting [Ca2+]i) and twitch amplitude, time to peak (Tp) for [Ca2+]iand shortening, and time to 50% (Tr) resting [Ca2+]iand relengthening. Variables from 10 contractions were averaged to obtain mean values at baseline and in response to the various interventions. Averaging the variables over time minimizes beat-to-beat variation.
Only rod-shaped, quiescent cells with smooth striations were selected. The whole cell configuration was achieved with fire-polished and Sylgard-coated tipped glass pipettes (Corning G86165T-4; World Precision Instruments, Sarasota, FL) and a resistance of 2–3 mV when filled with pipette solution containing 135 mm KCl, 1 mm MgCl2, 10 mm EGTA, 10 mm HEPES, and 5 mm glucose, at a pH of 7.3. The bathing solution contained the following: 140 mm NaCl, 4 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 10 mm HEPES, and 10 mm glucose, at a pH of 7.3. Using the Axopatch 1C (Axon Instruments, Foster City, CA), series resistance was 4–8 mV and was 30–80% electronically compensated. Action potentials were evoked by repetitive square pulses of 3-ms duration at 1.5× stimulus threshold. Experiments were performed under constant flow conditions at 30°C using a temperature-controlled experimental chamber (Delta T Culture Dish; Bioptechs, Butler, PA). Myocytes were paced under current clamp conditions at a cycle duration of 1,000 ms. Runs of 12 steady state action potentials were recorded with filtering at 2 kHz and sampled at 10 kHz. Data were acquired using a Pentium computer that controlled data acquisition hardware and software (pClamp 6.03; Axon Instruments). Analysis of action potentials was performed by taking the average of each sweep of 12 stable and steady state records.
Measurement of Nitric Oxide
Nitric oxide production was assessed by measurement of nitrate/nitrite production using a colorimetric kit from Cayman Chemical (Ann Arbor, MI). Suspensions of cardiomyocytes were placed in wells and incubated in the presence or absence of droperidol (0.1, 0.3, 1 μm) at 30°C for 15 min. Total nitrite levels (after conversion of nitrate to nitrite) were determined with Griess reagent using a microplate reader (absorbance, 540 nm). Aliquots (150 μl) from each well (before and after addition of droperidol) were collected and mixed with an equal volume of Griess reagent (1% sulfanilic acid and 0.1% N -(1-napthyl) ethylenediamine dihydrochloride in 2% phosphoric acid). The mixture was incubated at 20°C for 10 min. Nitrite concentrations in the samples were determined based on standard calibration curves by using an aqueous solution of sodium nitrite. The background value from buffer alone was subtracted from the experimental value.
Isolated Perfused Langendorff Heart Preparation
Male Sprague-Dawley rats weighing 250–300 g, were given an intraperitoneal injection of heparin (200 U). After the animals were killed, hearts were excised rapidly and placed in ice-cold KHB before being mounted on a Langendorff apparatus for perfusion at 37°C with KHB at 330 beats/min and a constant pressure of 65– 70 mmHg. The buffer was equilibrated with 95% O2and 5% CO2and had the following composition: 118 mm NaCl, 4.7 mm KCl, 1.25 mm CaCl2, 1.2 mm KH2PO4, 1.2 mm MgSO4, 25 mm NaHCO3, and 11 mm dextrose. A balloon-tipped catheter was inserted through the left atrium into the left ventricle, and the left ventricular end-diastolic pressure in all hearts was adjusted to between 8 and 12 mmHg. Left ventricular developed pressure was monitored continuously throughout the experiment. Coronary blood flow was measured by timed collection of the effluent into a graduated cylinder.
All experimental protocols were performed at 30°C, with the exception of the perfused Langendorff heart experiments performed at 37°C.
Concentration-dependent Effects on [Ca2+]iand Myocyte Shortening.
A stock solution of droperidol was obtained by dissolving the drug in dimethyl sulfoxide. Baseline measurements were collected from individual myocytes for 1.5 min in the absence of any intervention. Myocytes were exposed to four concentrations of droperidol (0.03, 0.1, 0.3, and 1 μm) by exchanging the buffer in the dish with new buffer containing droperidol at the desired concentration. Data were acquired for 1.5 min after a 5-min equilibration period in the presence of droperidol. Summarized results for the concentration–response curves are expressed as percent of the control value. Dimethyl sulfoxide (0.05% vol/vol) alone has no effect on [Ca2+]ior shortening.7We verified that droperidol had no effect on fura-2 fluorescence at the concentrations tested. This was confirmed in separate cell-free experiments using fura-2 (pentapotassium salt) in buffers ranging from pCa (−log Ca2+concentration) 9 to pCa 5 in the presence or absence of droperidol (data not shown).
Effect on KCl-induced Increase in [Ca2+]i.
Droperidol was added to the superfusion medium for 15 min, and then KCl (35 mm) was applied to the cell. This concentration of KCl was chosen because it stimulates an increase in [Ca2+]iapproximately 50% of the maximum response. Peak [Ca2+]iinduced by KCl was compared with peak [Ca2+]ibefore addition of droperidol (1 μm) and is reported as percent change from control.
Effect on Action Potential Duration.
Action potentials were recorded before and after addition of droperidol (1 μm) in individual cardiomyocytes. Changes in action potential duration at 90% repolarization were determined.
Effect on SR Ca2+Stores.
Baseline values for [Ca2+]iwere measured in individual, field-stimulated myocytes for 1.5 min. Droperidol (0.1 and 1 μm) was then added to the superfusion buffer and allowed to perfuse the cell for 5 min. Field stimulation of the myocyte was then discontinued, and caffeine (20 mm) was applied to the cell 15 s later in the continued presence of droperidol. Peak [Ca2+]iinduced by caffeine was compared with peak [Ca2+]ibefore addition of droperidol and as a percent increase above the peak [Ca2+]iachieved during field stimulation (fractional release).
Effect on the Extracellular Ca2+–Shortening Relation.
Changes in the extracellular Ca2+concentration ([Ca2+]o)–shortening relation (indirect measurement of myofilament Ca2+sensitivity), were examined as previously described.10,11Baseline variables were collected from individual myocytes for 1.5 min. Concentration–response curves for [Ca2+]owere performed by exchanging the buffer in the dish with a new buffer containing the desired [Ca2+]o. Data were acquired for 1.5 min after establishment of a new steady state. Concentration–response curves for [Ca2+]owere then performed in the presence of droperidol (1 μm). Cells were allowed to stabilize for 5 min after addition of droperidol.
Effect on pHi.
Baseline pHiwas collected from individual myocytes for 1 min. Droperidol (0.03, 0.1, 0.3, and 1 μm) was added by exchanging the superfusion buffer in the dish with new buffer containing droperidol at the desired concentration. Each myocyte was exposed to only one concentration of droperidol. Results are expressed as the change in pHiover time with each concentration of droperidol.
Effect on Nitric Oxide Production.
Suspensions of ventricular myocyte were exposed to droperidol (0.1, 0.3, and 1 μm) for 15 min at 37°C in HBS buffer. Total nitrite/nitrate production was measured as an indicator of nitric oxide production. Results are expressed as percent change in total nitrate/nitrite from baseline.
Effect on Left Ventricular Developed Pressure.
Left ventricular pressure was continuously monitored in perfused Langendorff hearts paced at 330 beats/min at 37°C before (20 min) and during administration of droperidol (1 μm, 20 min). Results are expressed as percent change in pulse pressure (left ventricular end systolic pressure minus end diastolic pressure) from baseline.
Each experimental protocol was performed on multiple myocytes from the same heart and repeated in five hearts. Results obtained from myocytes in each heart were averaged so that all hearts were weighted equally. Comparison of several means was performed using repeated measures and two-way analysis of variance.12The Bonferroni post hoc test was used when significant differences among groups were detected. Differences were considered statistically significant at P < 0.05. All results are expressed as mean ± SD.
Droperidol, caffeine, and dimethyl sulfoxide were purchased from Sigma Chemical Co. (St. Louis, MO). Collagenase was purchased from Worthington Biochemical Corp. (Lakewood, NJ). BCECF/AM and fura-2/AM were purchased from Texas Fluorescence Labs (Austin, TX). The nitrate/nitrite colorimetric assay kit was obtained from Cayman Chemical.
Baseline Variables for [Ca2+]iand Shortening
Resting cell length was 125 ± 5 μm, and the baseline 340/380 ratio was 0.8 ± 0.1. Twitch height was 12 ± 1.5 μm (10.4 ± 1.6% of the resting cell length). The change in 340/380 ratio from baseline with shortening was 0.5 ± 0.1. Tp [Ca2+]iand shortening were 151 ± 22 and 177 ± 16 ms, respectively. Times to 50% recovery (Tr) for [Ca2+]iand shortening were 192 ± 19 and 228 ± 21 ms, respectively.
Effect on [Ca2+]iand Myocyte Shortening
A representative trace depicting the concentration-dependent effects of droperidol on [Ca2+]iand shortening in a single, field-stimulated ventricular myocyte is shown in figure 1A. Droperidol (1 μm) reduced peak [Ca2+]iand shortening by 22 ± 4 and 43 ± 5%, respectively. An increase in resting cell length of 2 ± 0.6 μm with no change in resting [Ca2+]iwas observed in most cells. The myocardial depressant effects of droperidol were reversible after washout. Summarized data for the concentration-dependent effects of droperidol on [Ca2+]iand shortening are also shown in figure 1B. Droperidol caused concentration-dependent decreases in [Ca2+]iand shortening. Figure 2represents an exploded overlay view of the individual [Ca2+] transient and shortening event before (A and B) and after being normalized to peak height (C and D) in the presence or absence of droperidol (1 μm) to illustrate changes in timing. Droperidol (1 μm) had no effect on Tp [Ca2+]i(98 ± 5% of control), Tp shortening (94 ± 6% of control), Tr [Ca2+]i(96 ± 8% of control), or Tr shortening (102 ± 3% of control). The continuous [Ca2+]i–shortening relations are also depicted as hysteresis loops in figure 2E. Droperidol caused a marked concentration-dependent downward shift in the continuous [Ca2+]i–shortening relation.
Effect on KCl-induced Increase in [Ca2+]i
Addition of KCl (35 mm) to quiescent myocytes resulted in a sustained increase in [Ca2+]i(fig. 3A). Pretreatment with droperidol (0.1 and 1 μm) attenuated the KCl induced-increase in [Ca2+]iby 24 ± 5 and 27 ± 5%, respectively (fig. 3B).
Effect on Action Potential Duration
Figure 4Ashows that exposure of the myocyte to droperidol (1 μm) had no effect on action potential duration compared with control myocytes not exposed to droperidol. Summarized data depicting the effect of droperidol on action potential duration at 90% repolarization are also shown in figure 4B.
Effect on Caffeine-induced Release of Ca2+from the SR
Although the peak [Ca2+]iachieved with caffeine was reduced by 22 ± 5% in the presence of droperidol (0.1 μm) compared with control, the fractional release of Ca2+from the SR was not different from that observed in the absence of droperidol (fig. 5A). Summarized data for the effects of droperidol on SR Ca2+content and fractional release are depicted in figure 5B. The decrease in SR Ca2+content is likely due to the inhibitory effect of droperidol on the L-type Ca2+channel, which reduces the driving force for refilling the SR.
Effect on the Concentration–Response Curve to [Ca2+]o
Figure 6demonstrates that increasing [Ca2+]ofrom 1 to 4 mm (control, without droperidol) resulted in a concentration-dependent increase in shortening (A) and a concomitant increase in peak [Ca2+]i(B). Droperidol (0.3 μm) caused a significant downward shift in the concentration–response curve to increasing [Ca2+]ofor shortening, with no concomitant effect on peak [Ca2+]i.
Effect on Intracellular pHi
Baseline pHiwas 7.11 ± 0.04. Summarized data depicting the effect of droperidol on pHiare shown in figure 7. Droperidol at 0.1, 0.3, and 1 μm caused concentration- and time-dependent decreases in pHi. The effects of droperidol on pHiwere reversible after washout with HBS (pHi= 7.08 ± 0.04). There was no significant change in extracellular pH in the presence of 1 μm droperidol (control: 7.35 ± 0.13, droperidol: 7.34 ± 0.11; not significant).
Effect on Nitric Oxide Production
Droperidol at concentrations of 0.1, 0.3, and 1 μm increased nitric oxide production by 22 ± 6, 53 ± 7, and 74 ± 10% compared with control. The nitric oxide donor, S -nitroso-N -acetylpenicillamine (10 μm), increased nitric oxide by 128 ± 12% compared with control.
Effect on Left Ventricular Developed Pressure
Addition of droperidol (0.1, 1 μm) to the perfusate resulted in concentration-dependent decreases in pulse pressure of 29 ± 6 and 43 ± 8%, respectively. There were no significant changes in coronary blood flow after perfusion with 1 μm droperidol (18 ± 1.4 ml/min before droperidol vs. 16 ± 1.8 ml/min after droperidol).
The major findings of this study are that droperidol causes a decrease in cardiomyocyte contractility via a decrease in [Ca2+]iand a decrease in myofilament Ca2+sensitivity. The decrease in [Ca2+]iis due to a decrease in sarcolemmal Ca2+influx, whereas the decrease in myofilament Ca2+sensitivity is likely mediated by a decrease in cardiomyocyte pHi, an increase in nitric oxide production, or both.
Effect on [Ca2+]iand Myocyte Shortening
There are no previous studies that have evaluated the effects of droperidol on ventricular cardiomyocyte [Ca2+]iand contractility. However, droperidol has been shown to slow pacemaker activity and depress maximum velocity of contraction in guinea pig ventricular muscle, although the cellular mechanisms for these effects were not investigated.13,14In the current study, we observed that clinically relevant concentrations of droperidol inhibit the peak [Ca2+]iachieved in response to field stimulation in a concentration-dependent manner. In addition, droperidol caused an increase in resting cell length in the absence of any change in diastolic [Ca2+]i, suggesting a decrease in myofilament Ca2+sensitivity. Taken together, these data suggest that droperidol exerts its effects on cellular mechanisms that regulate [Ca2+]iand myofilament Ca2+sensitivity. Because there were no changes in the timing variables of [Ca2+]i, which would have been reflected in the shortening and relengthening of the myocyte, it is unlikely that droperidol has any effect on the Na+–Ca2+exchanger or the SR Ca2+pump. Therefore, we investigated the effects of droperidol on Ca2+influx via voltage-gated Ca2+channels.
Effect on KCl-induced Increase in [Ca2+]i
The increase in cardiomyocyte [Ca2+]iafter addition of KCl is known to result from a depolarization-induced activation of the L-type Ca2+channel. We observed a decrease in the KCl-induced increase in [Ca2+]iin myocytes pretreated with droperidol. These findings suggest that droperidol may have a direct inhibitory effect on L-type Ca2+channels. Alternatively, a droperidol-induced activation of the transient outward K+current or the delayed rectifier K+current could abbreviate action potential duration and thereby reduce the time in which Ca2+enters the cell via L-type Ca2+channels. Therefore, we examined the effect of droperidol on action potential duration.
Effect on Action Potential Duration
Previous studies have demonstrated a prolongation in action potential duration and the presence of early afterdepolarizations with low concentrations of droperidol (10–300 nm), whereas a shortening of action potential duration was observed with high concentrations (10– 30 μm).13,15The prolongation in action potential duration was due to a droperidol-induced inhibition of the delayed rectifier K+current.15Droperidol has been reported to cause a concentration-dependent prolongation of the QT interval in humans1and subsequent torsade de pointes, which can progress to ventricular fibrillation and sudden cardiac death.2In the current study using rat ventricular myocytes, we did not observe a prolongation in action potential duration with any concentration of droperidol. This may be because rat ventricular myocytes lack a prominent delayed rectifier K+current and primarily rely on the transient outward K+current for repolarization. Therefore, we conclude that the inhibitory effect of droperidol on the increase in [Ca2+]iinduced by electrical field stimulation or KCl is not due to an indirect effect of droperidol on action potential duration.
Effect on Caffeine-induced Release of Ca2+from the SR
Ca2+-induced Ca2+release is the process by which influx of Ca2+through sarcolemmal L-type Ca2+channels triggers Ca2+release from the SR through an activation of the ryanodine receptor (Ca2+release channel) in cardiomyocytes.16Rapid application of caffeine to quiescent myocytes results in direct activation of the ryanodine receptor on the SR, triggering the release of Ca2+from the SR. The difference between the peak [Ca2+]iinduced by electrical stimulation and the caffeine-induced increase in [Ca2+]irepresents the fractional release of Ca2+from the SR and is used to assess whether interventions have a direct effect on SR Ca2+content or an indirect effect due to decreased sarcolemmal Ca2+influx. Our results indicate that although the peak increase in [Ca2+]iin response to caffeine is attenuated by droperidol, the fractional release of Ca2+is unaltered. This is because droperidol inhibits sarcolemmal Ca2+influx through the L-type Ca2+channels, resulting in an overall decrease in the size of the releasable pool of Ca2+in the SR. Therefore, it seems that droperidol does not exert a direct inhibitory effect on SR Ca2+release but does have an indirect effect on the size of the releasable pool of Ca2+.
Effect on the Concentration–Response Curve to [Ca2+]o
We hypothesized that a decrease in myofilament Ca2+sensitivity may also play a role in the inhibitory effects of droperidol on contractility, based on our observations of a droperidol-induced increase in resting cell length and a downward shift in the [Ca2+]i–shortening relation. Although changes in sensitivity can also be reflected in the timing variables of contraction, we did not observe a change in the timing variables. This indicates that droperidol likely exerts its effects at multiple sites of regulation for myofilament Ca2+sensitivity, which may offset or mask the effect of one another. Because alterations in myofilament Ca2+sensitivity can alter contractility,17we examined whether droperidol altered the concentration–response relation to [Ca2+]o. This protocol is an indirect assessment of myofilament Ca2+sensitivity that allows for a paired comparison of [Ca2+]iand contractile amplitude in the same cell in the presence or absence of droperidol over a range of values for [Ca2+]o. Droperidol caused a downward shift in the concentration–response curve to [Ca2+]ofor shortening, with no concomitant effect on [Ca2+]i. These data suggest that droperidol decreases the maximal response of the myofilament to Ca2+as [Ca2+]iincreases. Therefore, it seems that in addition to a decrease in sarcolemmal Ca2+influx, a droperidol-induced decrease in myofilament Ca2+sensitivity contributes to the inhibitory effect of droperidol on cardiomyocyte contractility. We next assessed potential cellular mechanisms that may be responsible for the decrease in myofilament Ca2+sensitivity.
Effect on pHi
One possible mechanism for a decrease in myofilament Ca2+sensitivity is intracellular acidification. It is well known that intracellular acidosis decreases the contractility of cardiac muscle,17although the mechanisms responsible for the decrease are complicated. Acidosis affects every step of the excitation–contraction coupling pathway, including the availability and delivery of Ca2+to the myofilaments, as well as the response of the myofilaments to Ca2+.18–21Droperidol decreased pHiin a time- and concentration-dependent manner in cardiomyocytes. These data suggest that droperidol may have an inhibitory effect on the Na+– H+exchanger, which would promote accumulation of H+in the cytoplasm, resulting in intracellular acidosis. Because these studies were conducted in a HBS buffer in the absence of carbon dioxide and bicarbonate, a possible interaction between droperidol and Na+– HCO3−symport or Cl−–HCO3−exchange is unlikely, because these transport systems are inactive in myocytes bathed in HCO3−-free solution.22,23The magnitude of the change in pHiin response to droperidol (0.1 μm) was similar in magnitude, although opposite in direction, to that previously reported by our laboratory for thiopental- or propofol-induced changes in pHiand myofilament Ca2+sensitivity.8,10,24Moreover, other studies have documented similar changes in pHifor phenylephrine- and endothelin-induced intracellular alkalosis, respectively, resulting in a positive inotropic response.25,26It is also possible that a droperidol-induced decrease in pHicontributes to the inhibitory effect of droperidol on the KCl-induced increase in [Ca2+]i. Further studies are required to confirm this possibility.
Effect on Nitric Oxide Production
An increase in cardiomyocyte nitric oxide has been shown to decrease myofilament Ca2+sensitivity, resulting from an alteration in troponin I phosphorylation27and/or an alteration in actin–myosin cross-bridge cycling by modulating critical thiols on the myosin head.28In addition, nitric oxide is known to activate the cyclic guanosine monophosphate signaling pathway, which has been shown to reduce the myofilament response to Ca2+in cardiac myocytes29but augment release of Ca2+from the SR by caffeine.30Droperidol caused a concentration-dependent increase in nitric oxide production in cardiomyocytes. The increase in nitric oxide production, as well as the decrease in pHi, are likely involved in the droperidol-induced decrease in myofilament Ca2+sensitivity. However, direct confirmation of this hypothesis and the relative roles of each will require additional studies.
Effect on Left Ventricular Developed Pressure
To identify whether the changes in cardiomyocyte function could be extrapolated to the working heart, we assessed the effect of droperidol on overall cardiac function in buffer-perfused Langendorff hearts. Only one study assessing the effects of droperidol on isolated hearts has been performed, although the focus was on action potential duration and cardiac repolarization with no assessment of inotropic status.15An in vivo study in humans demonstrated a droperidol-induced decrease in left ventricular performance,4whereas another in vivo study focused on prolongation of the QT interval.1In the current study, droperidol caused decreases in left ventricular developed pressure that correlated with changes seen at the level of the individual cardiomyocyte. These data indicate that droperidol, at clinically relevant concentrations, exerts a negative inotropic effect in isolated perfused hearts. However, the magnitude of the negative inotropic effect of droperidol (1 μm) observed in cardiomyocytes, where the experiments were performed at 30°C, was greater than that observed in the perfused Langendorff hearts, where the experiments were performed at 37°C. The negative inotropic effect of droperidol in the cardiomyocytes is likely greater because of a reduced diffusion gradient for drug interaction with the cardiomyocytes.
Limitations of the Study and Clinical Implications
All anesthetic agents bind to plasma proteins, reducing the concentration available to bind to tissues. In the clinical setting, the use of droperidol (0.125 mg/kg) is standard for the prevention of postoperative nausea and vomiting. The peak plasma concentration with this dose has been estimated at 2 μm.31Taking into account that 90% of droperidol is bound to protein, the free concentration of droperidol is approximately 0.2 μm.32However, the microkinetic behavior of droperidol within the vascular space has not been defined. In addition, small changes in the amount or binding capacity of proteins could result in significant increases in the free plasma concentrations of droperidol. Not only is there uncertainty in calculating the in vivo concentration of droperidol during normal circumstances, but the concentration in free plasma would certainly be higher when the concentration of protein serum is reduced (e.g. , hemodilution, liver disease, hypoproteinemia). Another potential limitation is the use of rat cardiomyocytes as a model for human cardiomyocytes, because species differences may exist. Action potentials in rodent cardiomyocytes are abbreviated compared with those recorded in human myocytes and are comprised primarily of the transient outward K+current, whereas in humans the delayed rectifier K+current predominates. This may explain our inability to observe a droperidol-induced prolongation in action potential duration. Moreover, this may also contribute to differences in the response of human and rat myocardium to anesthetic agents. It should be noted that this study deals only with intrinsic properties of the heart and that cardiac function also depends on preload, afterload, venous return, and heart rate, which are not factors in isolated cardiomyocytes. However, we have tried to overcome this limitation with the perfused Langendorff heart preparation. Despite these limitations, our results demonstrate that clinically relevant concentrations of droperidol decrease [Ca2+]iand myofilament Ca2+sensitivity in cardiomyocytes, resulting in a negative inotropic effect. Moreover, in addition to the α-adrenergic blocking effect of droperidol in the vasculature,33,34a decrease in the inotropic state of the heart will likely exacerbate the hypotensive effect of droperidol observed in the clinical setting.
Our results provide the first direct evidence that droperidol causes a negative inotropic effect in individual cardiomyocytes. This effect is mediated by both a decrease in [Ca2+]iand a decrease in myofilament Ca2+sensitivity. The decrease in [Ca2+]iis not due to an effect on action potential duration, but rather due to an effect on the L-type Ca2+channel to limit sarcolemmal Ca2+influx. The decrease in myofilament Ca2+sensitivity is likely mediated by a decrease in pHiand an increase in nitric oxide production.