J Mol
Cell
Cardiol
&l&m
24,
(1992)
619-629
Transients
in Isolated Cardiac Myocytes are Altered by l,l,l-Tricbloroetha
P. Hofhanu’,
M. Breitenstein2, and M. Tomason
2*3Centersfor Disease Control, National Institutefor Occupational Safety and Health, Cellular Toxicology Section, 4676 Columbia Parkway, Cincinnati OH 45226 USA, and ‘Institute of Industrial Toxicology, Martin Luther University, Hall, F&era1 Republic of Germany and ‘National Research Council Research Associate, National Zntitutefor Occupational Safety and Health, Cellular Toxicology Section, 4676 Columbia Parkway, Cincinnati OH, USA. (Received 16 September 1991, accepted 21 January 1992) P. HOFFMANN, M. BREITENSTEIN and M. TORAASON. Calcium Transients in Isolated Cardiac Myocytes are Altered by l,l,l-trichloroethane. Journal oj Molccalar aad Cellalar Cardioloo (1992) 24, 619-629. 1 , 1,l -Trichloroethane is a widely used solvent that is annually linked to several cases of sudden death following accidental exposure or abuse. Sudden death is believed to be due to ventricular fibrillation or myocardial depression. The purpose of this study was to investigate the mechanism of myocardial depression by assessing the influence of l,l, l-trichloroethane on intracellular Ca transients in single neonatal rat ventricular myocytes using spectrofluorometric analysis of fura-2-Ca binding. Cells were exposed to 1,l , l-trichloroeihane in Hanks’ balanced salt solution aliquoted as a 0.2% DMSO solution by a single pass suffusion in an environmentally controlled chamber. l,l, l-Trichloroethane (0.25 mM-8mM) reduced the height of electrically (1 Hz, 6OV, 10 ms) induced Ca transients concentration dependently and reversibly to a maximum of about 50% with no effect on diastolic Ca concentration. Video motion analysis revealed an inhibition of contractility in the same concentration range. 1 (1 ,l-Trichloroethane inhibited cytosolic Ca increase in response to KCl-induced (90 mM) depolarizations and further decreased the limited Ca transients in ryanodine (PI pretreated myocytes. Increased external Ca (5 mM) antagonized the effect of 0.5 mM l,l,l-trichloroethane on the Ca transients. 1 , 1,l -Trichloroethane reduced the caffeine (10 mM) releasable Ca pool in myocytes. These results show that 1,l ,l-trichloroethane inhibits Ca mobilization during excitation-contraction coupling in ventricular myocytes. An inhibitory action on the influx of extracellular Ca as well as on sarcoplasmic reticulum Ca release and sequestration is likely to be responsible for this action. KEY WORDS: chloroethane.
Myocytes;
Ca
transient;
Fura-2;
Myocardial
Introduction 1 , 1,l -Trichloroethane (TCE) was introduced in 1954 as a non-toxic solvent (Stewart and Andrews, 1966). It continues to be used extensively today in industry as a degreaser and as a vehicle in a variety of household and office products such as typing correction fluid (King et al., 1985). TCE’s apparent low toxicity is attributable to its relatively low blood/air partition coefficient and low rate of metabolism (Eben and Kimmerle, 1974; Kyrklund and Haglid, 1991). Despite being relatively non-toxic, accidental high-level exposures in the workplace and inhalation abuse by adoles3To whom correspondence should Mention of products or company Safety and Health. 0022s2828/92/060619
+ 11 $03.00/O
be addressed. names does not constitute
contraction;
Cardiac
arrhythmia;
l,l,
1 -tri-
cents have repeatedly linked TCE exposure with sudden death (Bass, 1970; Northfield, 1981; McCarthy and Jones, 1983; King et al., 1985; MacDougall et al., 1987). Sudden death is believed to be due to ventricular fibrillation, and experimental sensitization of the mammalian heart to epinephrine induced arrhythmias by TCE is well documented (Reinhardt et al., 1973; Kobayashi et al. 1982). Experiments with anesthetized dogs indicate that myocardial depression may also lead to sudden death during TCE exposure (Aviado and Belej, 1975; Herd et al., 1974; Kobayashi etal., 1987). However, a study with cultured cardiac
endorsement
by the National @ 1992
Institute Academic
for Occupational Press
Limited
620
P. Hofbnano
myocytes indicates that TCE directly effects the myocardium (Toraason et al., 1990). As a halogenated hydrocarbon, TCE has physicochemical properties similar to the volatile anesthetic halothane (2-bromo-2-chloro1 , 1,l -trifluoroethane). The negative inotropic action of halothane is primarily a consequence of a reduction in intracellular Ca availability. Halothane reduces the influx of extracellular Ca during systole (Bosnjak and Kampine, 1986; Lynch and Frazer, 1989). This decrease of Ca influx is likely to be responsible for a smaller release of Ca by the sarcoplasmic reticulum resulting in a diminished systolic cytosolic Ca concentration (Bosnjak and Kampine, 1986). Moreover, halothane also directly alters Ca release from the sarcoplasmic reticulum (Su and Kerrick, 1979; Wheeler et al., 1988; Katsuoka et al., 1989). Compared to halothane, little is known regarding the mechanism of the cardiodepressant and arrhythmogenic actions of TCE at the cellular level of the heart. Experiments with anesthetized dogs and isolated rat papillary muscles revealed that an increase in extracellular Ca concentration protected against the TCE induced decrease in myocardial contractility (Herd et al., 1974). These findings provide indirect evidence that cellular Ca dynamics during myocardial contraction-relaxation may be altered by TCE. The purpose of the present study was to examine the effects of TCE on intracellular Ca transients in electrically stimulated neonatal rat ventricular myocytes and to examine the role of the sarcolemma and the sarcoplasmic reticulum in the altered Ca dynamics. The results demonstrate that Ca transients are concentration-dependently and reversibly inhibited by TCE exposure and this effect appears to be mediated via actions on Ca fluxes at the sarcolemma and sarcoplasmic reticulum. Preliminary results from this study were published recently in abstract form (Hoffmann and Toraason, 1991).
et al. breeding colony maintained in the animal quarters of the National Institute for Occupational Safety and Health which is accredited by the American Association for Accreditation of Laboratory Animal Care. Ventricular myocytes were plated at a density of 2 x lo6 cells per 35mm well, in six well plastic trays (Falcon) containing 19mm round quartz cover slips.
Chemicals Ryanodine was purchased from Agrisystems International, Wind Gap PA USA. Hanks’ balanced salt solution was purchased from Gibco, Grand Island NY USA. New born calf serum was purchased from Hyclone, Logan UT USA. Dimethyl sulfoxide (DMSO) and TCE were purchased from Fisher Scientific, Fair Lawn NJ USA. All other chemicals and reagents were purchased from Sigma, St. Louis MO USA.
Measurement of cytosolicfiee calcium
Myocytes were loaded with furaby a 10 min incubation at 37% with 1 pM of the acetoxymethyl ester dissolved in DMSO added as 0.1% solution to culture medium. The low acetoxymethyl ester concentration and short loading time prevented significant intracellular compartmentation (e.g., mitochondria) of fura- as evidenced by a rapid loss ( >80%) of intracellular furafluorescence within 5 min of digitonin (10~~) treatment. Cover slips containing myocytes loaded with furawere transferred to a temperature controlled (32%) suffusion chamber on the stage of an inverted microscope (Nikon) which was coupled with a dual excitation spectrofluorometer (Deltascan, Photo Technology), South Brunswick NJ USA). Myocytes were suffused with Hanks’ balanced salt solution at 2 ml/min and were initially viewed by bright field microscopy to allow for optimal focus. Two platinum electrodes, 5mm apart, were positioned so that the myocyte of concern was Materials and Methods between them. Cells were paced at 1 Hz by Preparationof myocytes field stimulation with pulses from a Grass S88 Cardiac ventricular cells were isolated from stimulator at 60 V for 10 ms duration. Experitwo- to four-day-old Sprague-Dawley rats by ments were performed to verify that myocytes the method previously described (Toraason et stimulated using these conditions were on a al., 1990). Rat pups were obtained from a voltage-vs.-peak Ca plateau.
Tricbloroethane
The Deltascan system provided UV excitation via a 75-W xenon lamp. A reflective chopper rotating at 60 Hz allowed rapid alternation between the two excitation wavelengths of 340 nm and 380 nm. The UV illuminated field was adjusted with a diaphragm to excite a single cell and avoid signals from neighboring cells. To minimize photobleaching, a shutter was placed on the excitation side of the light path and opened only during data acquisition. Emission from the cell at the two excitation wavelengths was filtered at 510nm and collected by a photomultiplier tube. The data collection rate for all experiments was 20Hz. Cytosolic free Ca concentrations were calculated from the 340/380 fluorescence emission ratios of the Ca-bound and Ca-free forms of furaaccording to Grynkiewicz et al. (1985). Calibrations were carried out at the conclusion of experiments. Cells were exposed to 2 FM carbonyl cyanide m-chlorophenylhydrazone (CCCP, for deenergizing) and 10~~ ionomycin (to permeabilize the membrane) in 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid for lOmin, which gave the minimum fluorescence ratio (Rmin). Maximum fluorescence ratio (Rmax) was obtained by the exposure to 2pM CCCP and 10 mM ionomycin in 2.5 mM Ca. Intracellular Ca concentration was estimated by a derivation of the formula of Grynkiewicz et al. (1985):
where K, is the apparent dissociation constant for the Ca-fura-2-complex (224nM) and is the ratio of fluorescence 380,,/38O,i, yields of the Ca-free/Ca-bound indicator at 380nm. It must be noted that Ca values obtained using this method are only estimates. Potential sources of error using intracellular fluorescence probes have been described by Groden et al. (1991) and Martinez-Zaguilan et al. (1991). Control experiments indicated that TCE not quench or enhance (8mM) did fura-Z-fluorescence, nor did TCE modify autofluorescence of myocytes. Therefore, changes in fura-2-fluorescence transients of ventricular myocytes upon addition of TCE represent changes in cytoplasmic Ca concentrations.
and
Calcium
621
MyoGyte contractility Video motion analysis was used to measure contractility of isolated cardiac myocytes as previously described (Toraason et al., 1990). In brief, a single myocyte was captured with a video camera (Dage) and displayed on a video monitor. Pixel-to-pixel movement of the image along a single raster line on the monitor was measured using an image processor (model 604) and motion analyzer (model 633) with sync-stripper (model 302-2), all manufactured by Colorado Video (Boulder CO USA). The analog voltage fluctuation was fed through a DC driver amplifier (model 7DA, Grass Instruments, Quincy, MA) to a personal computer (IBM-AT) containing DASA 4600 data acquisition/signal analysis system (Gould Electronics, Cleveland OH USA).
Exposure to test solutions Prior to exposure to TCE, cells were allowed to equilibrate for 30min in the suffusion chamber. TCE was dissolved in DMSO, added to Hanks’ solution and vortexed in an air tight vial immediately prior to the start of the suffusion. The final concentration of DMSO in the suffusion medium was 0.2%. This concentration was without effect on Ca transients and contractility. All control experiments were performed with Hanks’ solution containing 0.2 % DMSO. Concentrationresponse data for transients and contractility were obtained by doubling the exposure concentrations at 5 min intervals.
Data a7iatysis Fluorescence data were stored continuously on a computer (Princeton Computer Products, Inc. Lawrenceville NJ USA) throughout each 10s run. For kinetic analyses, eight Ca transients obtained during steady state were averaged. The mean Ca transient had a low signal-to-noise ratio. The relaxation phase of the transient was fit to a polynomial equation using commercially available computer software (Tablecurve, version 2.12). Half-time for relaxation was obtained from look-up tables calculated by the software. The slow rate of data acqusition (20 Hz) was inade-
622
P. Ho-
quate for calculating + d[Ca];ldt and time to peak. Results are presented as mean f S.D. Statistical analyses were performed using Student’s t test and ANOVA. In the statistical analyses, PcO.05 was accepted to indicate a significant difference.
Results Inhibitory e$ect of TCE on the Ca transients and contractility Figure 1 shows typcial Ca transients measured in fura-2-loaded ventricular myocytes under control conditions and after treatment with 0.25 mM-8mMTCE. A sequential series of four transients elicited by electrical stimulation is shown at steady state. Under control conditions the peak transient drift over a period of 1 h was less than 10% (data not shown). TCE decreased Ca transients in a concentration-dependent fashion but had no effect on the basal (diastolic) level of cytoplasmic Ca. To ensure that there was a correlation between Ca transients and mechanical properties, myocyte movement was measured by
Control
et oz. means of video motion analysis as an index of contractility. Figure 2 shows a typical example of five experiments recorded from single myocytes electrically stimulated at 1 Hz. As with Ca transients, myocyte contractions were inhibited by TCE in a concentration-dependent fashion. Washout of TCE resulted in a restoration of normal contractions. Although measurements were performed in different myocytes, the concentrationand time-dependent reduction in motion (Fig. 2) is comparable to the reduction in transients (Fig. 1). Note that at 4mM and 8mM TCE, little motion was detectable, but small Ca transients were still observed in response to electrical stimulation. Figure 3 shows summary concentrationresponse data for Ca transient reductions during TCE exposure from six different cardiomyocyte preparations. The effects of TCE on the transients developed gradually, and stable conditions were observed after 5 min of exposure. The lowest concentration used in these experiments was 0.25 mM TCE, which caused a 12% reduction of peak Ca (PcO.05). The maximum effect of -50% inhibition was observed with 8 mMTCE. A
O-25m~
4 mM TCE
TCE
0.5
mkt TCE
6 mM TCE
I mM TCE
Washout
15 min
FIGURE 1. Ca transients in a single electrically stimulated (1 H z ) ventricular myocyte. Transients were measured in the fura-Z-loaded cell under control conditions using Hanks’ solution and after treatment with 0.25 mM-8 mM TCE. Concentration-response data were obtained by doubling the exposure concentration at 5min intervals. TCE was washed out with Hanks’ solution. Fura-P-fluorescence measurements were used to calculate cytosolic Ca concentrations as described in Materials and Methods.
Trichloroethane
0.25
Control
2 rnt.t I
TCE
and
Calcium
623
mM TCE
0.5 mM TCE
4 mht TCE
8 rnM TCE
I mM TCE
Washout
15 min
Is
FIGURE 2. Effect of TCE on myocyte contraction during used as described in Materials and Methods and contraction performed as described in Figure 1.
total inhibition of Ca transients was observed at this concentration in one cell. Arrhythmic events occurred with TCE concentrations higher than 2 mM. TCE did not cause overt damage to the ventricular myocytes during the 30min exposure period in the range of 0.25 mM-8 mM. This is supported by the morphological integrity of the cells as indicated by an absence of furaloss through leaky membranes and the reversibility of the effects on the Ca transients after washout of TCE. Note that after a 15min washout period the transients were increased by 13.5% compared to preexposure values. The variation within the group was very high and consequently the difference did not attain statistical significance. Nevertheless, a marked increase in peak transient height (15%) 29% and 35%) was observed in three out of six experiments. Following recovery of normal Ca transients, a second exposure of a cell to TCE resulted in the same reduction of Ca transients with subsequent recovery (data not shown). The fast data acquisition required to resolve the kinetics of the Ca transients leads to a considerable level of noise. By combining the signals from a series of eight consecutive Ca transients under steady state conditions, an
electrical amplitude
stimulation is expressed
0.25
0.5
at I Hz. Video motion analysis as voltage changes. Suffusion
I
2
4
8
0
was was
0
rnM TCE FIGURE 3. Concentration-response data for inhibition of electrically induced peak transients (1 Hz) in fura-Z-loaded ventricuIar myocytes. Average Ca transient peak height after TCE addition is expressed as a percentage of pre-exposure peak height measured in the same cell. Suffusion was performed as described in Figure 1. Each column represents the mean f S.D of values obtained from six separate myocyte preparations. The reduction of the peak transient height was significant at 0.25m~-8mM (PcO.05). The last two columns represent values after 5min and 15min washout periods with Hanks’ solution.
R Ho5uann et d.
624
- Control Control 0.25 4.0
LA.-.
_i.---~
0.2
0.0
mrd TCE mM TCE
/-.
0.4
0.6
-
-J
0.6
I .o
Time(s)
FIGURE 4. Average Ca transients in a single fura-2-loaded Fura-P-fluorescence signals from a series of eight consecutive conditions were averaged. Fluorescence measurements were in Materials and Methods.
ventricular myocyte before and after TCE exposure. electrically induced transients (1 Hz) under steady state used to calculate cytosolic Ca concentrations as described
average Ca transient with a reduced noise level but no loss of temporal resolution was obtained. Figure 4 illustrates typical averaged Ca transients measured in a single ventricular myocyte before and after TCE exposure. Under control conditions cytosolic Ca concentrations increased from basal levels of W100 nM to a peak of -820nM. Addition of 0.25mM and 4mM TCE did not influence basal Ca concentrations but reduced peak levels to 539 nM and 337 nM, respectively. TCE clearly reduced the slope of the rise of the cytosolic Ca
concentrations. Half-time not significantly diminished 0.25mM (212 f 23 ms) (198 f 22 ms) compared (225 f 42 ms, BO.05, n=
90
Efects
I 4
I
r---
on sarcolemtd
Ca jhces
The inhibitory action of TCE on Ca transients could be due to reduction of sarcolemmal Ca entry. This hypothesis was tested by depolarizing quiescent cells with high extracellular
90 mM KCI +5 /.LM Verapamil
mM KCI
of TCE
for relaxation was after addition of or 4mM TCE to control values 6).
90 f4
mtd KCI rntd TCE I
25s -
FIGURE 5. Effect of verapamil and TCE on cytosolic Ca concentration increases in ventricular by the addition of 90 mMKC1. Cytosolic Ca concentrations were expressed using fura-2-fluorescence KC1 was added at the points indicated to a non-pretreated cell suffused with Hanks’ solution (control) treatment with verapamil(5fiM) and TCE (4mM), respectively. The results were obtained in three preparations. Representative tracings from four control KCl-depolarizations without pretreatment, ations with verapamil pretreatment, and three depolarizations with TCE pretreatment are shown.
myocytes induced in quiescent cells. or five min after different myocyte three depolariz-
Ti-ichloroethane
Control
Control
(I.2
mM
Co)
625
and Calcium 0.5
mht
TCE ( 1.2 mu Co)
(5 mM Ca)
FIGURE 6. Elimination of the inhibitory action of 0.5 mM TCE on Ca transients in a single ventricular myocyte by an increase in the extracellufar Ca concentration from 1.2 mM to 5 mM. Cytosolic Ca concentrations were expressed using fura-2-fluorescence. Under control conditions the electrically stimulated cell (1 Hz) was suffused with Hanks’ solution (1.2 mM Ca). Addition of 0.5 mM TCE to Hanks’ solution reduced peak transients. After washout of TCE the same cell was suffused with Hanks’ solution containing 5 mM Ca and then reexposed to 0.5 mM TCE. The effect described was observed in four separate myocyte preparations.
KC1 (90mM without osmotic adjustment) to activate voltage-dependent slow Ca channels. Figure 5 shows that application to the cell of 90 mMKC1 induced a cluster of Ca oscillations. Within 5-6s the damped oscillations formed a plateau. In the presence of verapamil (5-20 PM) the KCl-depolarization-induced Ca increase returned to basal levels in about 25 s (Fig. 5). In a series of experiments using three separate TCE exposed (4 mM) cardiomyocyte preparations, the plateau level of cytosolic Ca after KCl-depolarization was reduced to 42 f 8% (PcO.05) of four control depolarizations without TCE exposure (Fig. 5). Figure 6 illustrates that the effects of 0.5 mM TCE can be abolished by increasing extracellular Ca concentration from 1.2 mM to 5 mM. Addition of 0.5 mM TCE reduced peak transients. After washout of TCE, the same cell was reexposed to 0.5 mM TCE in the presence of 5 mMCa. Increased external Ca (5 mM) completely eliminated the effects of 0.5mM
TCE on Ca transients. The same result was obtained in four separate experiments. It has been shown that ryanodine reduces the cytoplasmic Ca increase through inhibition of sarcoplasmic Ca release (Hilgeman et al., 1989). Pretreatment of the cells with 1 ELM ryanodine for 10min reduced the magnitude of electrically induced transients to 62 + 8 % of untreated cells (PcO.05, n = 8). TCE (4mM) caused further reductions (34 + 11% of untreated control, PcO.05) in the peak height after pretreatment with 1 PM ryanodine. The TCE effect on the Ca transients of ryanodine pretreated cells disappeared after washout of the TCE. Effects
of TCE
OR sarcophzsmic Ga fhxes
The addition of 1 mM caffeine to cardiomyocytes results in a rapid release of sarcoplasmic reticulum Ca which is then removed from the cytosol presumably by the action of sarcolemma1 and other pumping mechanisms. In
626
p. Hosnarul
IO mM Caffeine
et ul.
IO mM Caffeine I ELM Ryanadine pretreatment
after
IO mM Caffeine 4 mM TCE pretreatment
after
I
FIGURE 7. Effect of ryanodine and TCE on the caffeine induced increase in cytosolic Ca concentrations of ventricular myocytes. Ca concentrations were expressed using fura-2-fluorescence in quiescent cells, Caffeine (10 mM) was added at the points indicated to a non-pretreated cell suffused with Hanks’ solution (control), ten min after pretreatment of a cell with ryanodine (1 pm), or five min after pretreatment of a cell with TCE (4 mM). The results were obtained in three different myocyte preparations. Representative tracings from six control caffeine effects without pretreatment, three caffeine effects with ryanodine pretreatment, and six experiments with TCE pretreatment are shown. 4 mM TCE
reduced Ca transients. However, the immediate response of a quiescent ventricular myocyte to the introduction of 4rn~ TCE in the suffusate was a small brief (
Cell
Cardiol
&l&m
24,
(1992)
619-629
Transients
in Isolated Cardiac Myocytes are Altered by l,l,l-Tricbloroetha
P. Hofhanu’,
M. Breitenstein2, and M. Tomason
2*3Centersfor Disease Control, National Institutefor Occupational Safety and Health, Cellular Toxicology Section, 4676 Columbia Parkway, Cincinnati OH 45226 USA, and ‘Institute of Industrial Toxicology, Martin Luther University, Hall, F&era1 Republic of Germany and ‘National Research Council Research Associate, National Zntitutefor Occupational Safety and Health, Cellular Toxicology Section, 4676 Columbia Parkway, Cincinnati OH, USA. (Received 16 September 1991, accepted 21 January 1992) P. HOFFMANN, M. BREITENSTEIN and M. TORAASON. Calcium Transients in Isolated Cardiac Myocytes are Altered by l,l,l-trichloroethane. Journal oj Molccalar aad Cellalar Cardioloo (1992) 24, 619-629. 1 , 1,l -Trichloroethane is a widely used solvent that is annually linked to several cases of sudden death following accidental exposure or abuse. Sudden death is believed to be due to ventricular fibrillation or myocardial depression. The purpose of this study was to investigate the mechanism of myocardial depression by assessing the influence of l,l, l-trichloroethane on intracellular Ca transients in single neonatal rat ventricular myocytes using spectrofluorometric analysis of fura-2-Ca binding. Cells were exposed to 1,l , l-trichloroeihane in Hanks’ balanced salt solution aliquoted as a 0.2% DMSO solution by a single pass suffusion in an environmentally controlled chamber. l,l, l-Trichloroethane (0.25 mM-8mM) reduced the height of electrically (1 Hz, 6OV, 10 ms) induced Ca transients concentration dependently and reversibly to a maximum of about 50% with no effect on diastolic Ca concentration. Video motion analysis revealed an inhibition of contractility in the same concentration range. 1 (1 ,l-Trichloroethane inhibited cytosolic Ca increase in response to KCl-induced (90 mM) depolarizations and further decreased the limited Ca transients in ryanodine (PI pretreated myocytes. Increased external Ca (5 mM) antagonized the effect of 0.5 mM l,l,l-trichloroethane on the Ca transients. 1 , 1,l -Trichloroethane reduced the caffeine (10 mM) releasable Ca pool in myocytes. These results show that 1,l ,l-trichloroethane inhibits Ca mobilization during excitation-contraction coupling in ventricular myocytes. An inhibitory action on the influx of extracellular Ca as well as on sarcoplasmic reticulum Ca release and sequestration is likely to be responsible for this action. KEY WORDS: chloroethane.
Myocytes;
Ca
transient;
Fura-2;
Myocardial
Introduction 1 , 1,l -Trichloroethane (TCE) was introduced in 1954 as a non-toxic solvent (Stewart and Andrews, 1966). It continues to be used extensively today in industry as a degreaser and as a vehicle in a variety of household and office products such as typing correction fluid (King et al., 1985). TCE’s apparent low toxicity is attributable to its relatively low blood/air partition coefficient and low rate of metabolism (Eben and Kimmerle, 1974; Kyrklund and Haglid, 1991). Despite being relatively non-toxic, accidental high-level exposures in the workplace and inhalation abuse by adoles3To whom correspondence should Mention of products or company Safety and Health. 0022s2828/92/060619
+ 11 $03.00/O
be addressed. names does not constitute
contraction;
Cardiac
arrhythmia;
l,l,
1 -tri-
cents have repeatedly linked TCE exposure with sudden death (Bass, 1970; Northfield, 1981; McCarthy and Jones, 1983; King et al., 1985; MacDougall et al., 1987). Sudden death is believed to be due to ventricular fibrillation, and experimental sensitization of the mammalian heart to epinephrine induced arrhythmias by TCE is well documented (Reinhardt et al., 1973; Kobayashi et al. 1982). Experiments with anesthetized dogs indicate that myocardial depression may also lead to sudden death during TCE exposure (Aviado and Belej, 1975; Herd et al., 1974; Kobayashi etal., 1987). However, a study with cultured cardiac
endorsement
by the National @ 1992
Institute Academic
for Occupational Press
Limited
620
P. Hofbnano
myocytes indicates that TCE directly effects the myocardium (Toraason et al., 1990). As a halogenated hydrocarbon, TCE has physicochemical properties similar to the volatile anesthetic halothane (2-bromo-2-chloro1 , 1,l -trifluoroethane). The negative inotropic action of halothane is primarily a consequence of a reduction in intracellular Ca availability. Halothane reduces the influx of extracellular Ca during systole (Bosnjak and Kampine, 1986; Lynch and Frazer, 1989). This decrease of Ca influx is likely to be responsible for a smaller release of Ca by the sarcoplasmic reticulum resulting in a diminished systolic cytosolic Ca concentration (Bosnjak and Kampine, 1986). Moreover, halothane also directly alters Ca release from the sarcoplasmic reticulum (Su and Kerrick, 1979; Wheeler et al., 1988; Katsuoka et al., 1989). Compared to halothane, little is known regarding the mechanism of the cardiodepressant and arrhythmogenic actions of TCE at the cellular level of the heart. Experiments with anesthetized dogs and isolated rat papillary muscles revealed that an increase in extracellular Ca concentration protected against the TCE induced decrease in myocardial contractility (Herd et al., 1974). These findings provide indirect evidence that cellular Ca dynamics during myocardial contraction-relaxation may be altered by TCE. The purpose of the present study was to examine the effects of TCE on intracellular Ca transients in electrically stimulated neonatal rat ventricular myocytes and to examine the role of the sarcolemma and the sarcoplasmic reticulum in the altered Ca dynamics. The results demonstrate that Ca transients are concentration-dependently and reversibly inhibited by TCE exposure and this effect appears to be mediated via actions on Ca fluxes at the sarcolemma and sarcoplasmic reticulum. Preliminary results from this study were published recently in abstract form (Hoffmann and Toraason, 1991).
et al. breeding colony maintained in the animal quarters of the National Institute for Occupational Safety and Health which is accredited by the American Association for Accreditation of Laboratory Animal Care. Ventricular myocytes were plated at a density of 2 x lo6 cells per 35mm well, in six well plastic trays (Falcon) containing 19mm round quartz cover slips.
Chemicals Ryanodine was purchased from Agrisystems International, Wind Gap PA USA. Hanks’ balanced salt solution was purchased from Gibco, Grand Island NY USA. New born calf serum was purchased from Hyclone, Logan UT USA. Dimethyl sulfoxide (DMSO) and TCE were purchased from Fisher Scientific, Fair Lawn NJ USA. All other chemicals and reagents were purchased from Sigma, St. Louis MO USA.
Measurement of cytosolicfiee calcium
Myocytes were loaded with furaby a 10 min incubation at 37% with 1 pM of the acetoxymethyl ester dissolved in DMSO added as 0.1% solution to culture medium. The low acetoxymethyl ester concentration and short loading time prevented significant intracellular compartmentation (e.g., mitochondria) of fura- as evidenced by a rapid loss ( >80%) of intracellular furafluorescence within 5 min of digitonin (10~~) treatment. Cover slips containing myocytes loaded with furawere transferred to a temperature controlled (32%) suffusion chamber on the stage of an inverted microscope (Nikon) which was coupled with a dual excitation spectrofluorometer (Deltascan, Photo Technology), South Brunswick NJ USA). Myocytes were suffused with Hanks’ balanced salt solution at 2 ml/min and were initially viewed by bright field microscopy to allow for optimal focus. Two platinum electrodes, 5mm apart, were positioned so that the myocyte of concern was Materials and Methods between them. Cells were paced at 1 Hz by Preparationof myocytes field stimulation with pulses from a Grass S88 Cardiac ventricular cells were isolated from stimulator at 60 V for 10 ms duration. Experitwo- to four-day-old Sprague-Dawley rats by ments were performed to verify that myocytes the method previously described (Toraason et stimulated using these conditions were on a al., 1990). Rat pups were obtained from a voltage-vs.-peak Ca plateau.
Tricbloroethane
The Deltascan system provided UV excitation via a 75-W xenon lamp. A reflective chopper rotating at 60 Hz allowed rapid alternation between the two excitation wavelengths of 340 nm and 380 nm. The UV illuminated field was adjusted with a diaphragm to excite a single cell and avoid signals from neighboring cells. To minimize photobleaching, a shutter was placed on the excitation side of the light path and opened only during data acquisition. Emission from the cell at the two excitation wavelengths was filtered at 510nm and collected by a photomultiplier tube. The data collection rate for all experiments was 20Hz. Cytosolic free Ca concentrations were calculated from the 340/380 fluorescence emission ratios of the Ca-bound and Ca-free forms of furaaccording to Grynkiewicz et al. (1985). Calibrations were carried out at the conclusion of experiments. Cells were exposed to 2 FM carbonyl cyanide m-chlorophenylhydrazone (CCCP, for deenergizing) and 10~~ ionomycin (to permeabilize the membrane) in 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid for lOmin, which gave the minimum fluorescence ratio (Rmin). Maximum fluorescence ratio (Rmax) was obtained by the exposure to 2pM CCCP and 10 mM ionomycin in 2.5 mM Ca. Intracellular Ca concentration was estimated by a derivation of the formula of Grynkiewicz et al. (1985):
where K, is the apparent dissociation constant for the Ca-fura-2-complex (224nM) and is the ratio of fluorescence 380,,/38O,i, yields of the Ca-free/Ca-bound indicator at 380nm. It must be noted that Ca values obtained using this method are only estimates. Potential sources of error using intracellular fluorescence probes have been described by Groden et al. (1991) and Martinez-Zaguilan et al. (1991). Control experiments indicated that TCE not quench or enhance (8mM) did fura-Z-fluorescence, nor did TCE modify autofluorescence of myocytes. Therefore, changes in fura-2-fluorescence transients of ventricular myocytes upon addition of TCE represent changes in cytoplasmic Ca concentrations.
and
Calcium
621
MyoGyte contractility Video motion analysis was used to measure contractility of isolated cardiac myocytes as previously described (Toraason et al., 1990). In brief, a single myocyte was captured with a video camera (Dage) and displayed on a video monitor. Pixel-to-pixel movement of the image along a single raster line on the monitor was measured using an image processor (model 604) and motion analyzer (model 633) with sync-stripper (model 302-2), all manufactured by Colorado Video (Boulder CO USA). The analog voltage fluctuation was fed through a DC driver amplifier (model 7DA, Grass Instruments, Quincy, MA) to a personal computer (IBM-AT) containing DASA 4600 data acquisition/signal analysis system (Gould Electronics, Cleveland OH USA).
Exposure to test solutions Prior to exposure to TCE, cells were allowed to equilibrate for 30min in the suffusion chamber. TCE was dissolved in DMSO, added to Hanks’ solution and vortexed in an air tight vial immediately prior to the start of the suffusion. The final concentration of DMSO in the suffusion medium was 0.2%. This concentration was without effect on Ca transients and contractility. All control experiments were performed with Hanks’ solution containing 0.2 % DMSO. Concentrationresponse data for transients and contractility were obtained by doubling the exposure concentrations at 5 min intervals.
Data a7iatysis Fluorescence data were stored continuously on a computer (Princeton Computer Products, Inc. Lawrenceville NJ USA) throughout each 10s run. For kinetic analyses, eight Ca transients obtained during steady state were averaged. The mean Ca transient had a low signal-to-noise ratio. The relaxation phase of the transient was fit to a polynomial equation using commercially available computer software (Tablecurve, version 2.12). Half-time for relaxation was obtained from look-up tables calculated by the software. The slow rate of data acqusition (20 Hz) was inade-
622
P. Ho-
quate for calculating + d[Ca];ldt and time to peak. Results are presented as mean f S.D. Statistical analyses were performed using Student’s t test and ANOVA. In the statistical analyses, PcO.05 was accepted to indicate a significant difference.
Results Inhibitory e$ect of TCE on the Ca transients and contractility Figure 1 shows typcial Ca transients measured in fura-2-loaded ventricular myocytes under control conditions and after treatment with 0.25 mM-8mMTCE. A sequential series of four transients elicited by electrical stimulation is shown at steady state. Under control conditions the peak transient drift over a period of 1 h was less than 10% (data not shown). TCE decreased Ca transients in a concentration-dependent fashion but had no effect on the basal (diastolic) level of cytoplasmic Ca. To ensure that there was a correlation between Ca transients and mechanical properties, myocyte movement was measured by
Control
et oz. means of video motion analysis as an index of contractility. Figure 2 shows a typical example of five experiments recorded from single myocytes electrically stimulated at 1 Hz. As with Ca transients, myocyte contractions were inhibited by TCE in a concentration-dependent fashion. Washout of TCE resulted in a restoration of normal contractions. Although measurements were performed in different myocytes, the concentrationand time-dependent reduction in motion (Fig. 2) is comparable to the reduction in transients (Fig. 1). Note that at 4mM and 8mM TCE, little motion was detectable, but small Ca transients were still observed in response to electrical stimulation. Figure 3 shows summary concentrationresponse data for Ca transient reductions during TCE exposure from six different cardiomyocyte preparations. The effects of TCE on the transients developed gradually, and stable conditions were observed after 5 min of exposure. The lowest concentration used in these experiments was 0.25 mM TCE, which caused a 12% reduction of peak Ca (PcO.05). The maximum effect of -50% inhibition was observed with 8 mMTCE. A
O-25m~
4 mM TCE
TCE
0.5
mkt TCE
6 mM TCE
I mM TCE
Washout
15 min
FIGURE 1. Ca transients in a single electrically stimulated (1 H z ) ventricular myocyte. Transients were measured in the fura-Z-loaded cell under control conditions using Hanks’ solution and after treatment with 0.25 mM-8 mM TCE. Concentration-response data were obtained by doubling the exposure concentration at 5min intervals. TCE was washed out with Hanks’ solution. Fura-P-fluorescence measurements were used to calculate cytosolic Ca concentrations as described in Materials and Methods.
Trichloroethane
0.25
Control
2 rnt.t I
TCE
and
Calcium
623
mM TCE
0.5 mM TCE
4 mht TCE
8 rnM TCE
I mM TCE
Washout
15 min
Is
FIGURE 2. Effect of TCE on myocyte contraction during used as described in Materials and Methods and contraction performed as described in Figure 1.
total inhibition of Ca transients was observed at this concentration in one cell. Arrhythmic events occurred with TCE concentrations higher than 2 mM. TCE did not cause overt damage to the ventricular myocytes during the 30min exposure period in the range of 0.25 mM-8 mM. This is supported by the morphological integrity of the cells as indicated by an absence of furaloss through leaky membranes and the reversibility of the effects on the Ca transients after washout of TCE. Note that after a 15min washout period the transients were increased by 13.5% compared to preexposure values. The variation within the group was very high and consequently the difference did not attain statistical significance. Nevertheless, a marked increase in peak transient height (15%) 29% and 35%) was observed in three out of six experiments. Following recovery of normal Ca transients, a second exposure of a cell to TCE resulted in the same reduction of Ca transients with subsequent recovery (data not shown). The fast data acquisition required to resolve the kinetics of the Ca transients leads to a considerable level of noise. By combining the signals from a series of eight consecutive Ca transients under steady state conditions, an
electrical amplitude
stimulation is expressed
0.25
0.5
at I Hz. Video motion analysis as voltage changes. Suffusion
I
2
4
8
0
was was
0
rnM TCE FIGURE 3. Concentration-response data for inhibition of electrically induced peak transients (1 Hz) in fura-Z-loaded ventricuIar myocytes. Average Ca transient peak height after TCE addition is expressed as a percentage of pre-exposure peak height measured in the same cell. Suffusion was performed as described in Figure 1. Each column represents the mean f S.D of values obtained from six separate myocyte preparations. The reduction of the peak transient height was significant at 0.25m~-8mM (PcO.05). The last two columns represent values after 5min and 15min washout periods with Hanks’ solution.
R Ho5uann et d.
624
- Control Control 0.25 4.0
LA.-.
_i.---~
0.2
0.0
mrd TCE mM TCE
/-.
0.4
0.6
-
-J
0.6
I .o
Time(s)
FIGURE 4. Average Ca transients in a single fura-2-loaded Fura-P-fluorescence signals from a series of eight consecutive conditions were averaged. Fluorescence measurements were in Materials and Methods.
ventricular myocyte before and after TCE exposure. electrically induced transients (1 Hz) under steady state used to calculate cytosolic Ca concentrations as described
average Ca transient with a reduced noise level but no loss of temporal resolution was obtained. Figure 4 illustrates typical averaged Ca transients measured in a single ventricular myocyte before and after TCE exposure. Under control conditions cytosolic Ca concentrations increased from basal levels of W100 nM to a peak of -820nM. Addition of 0.25mM and 4mM TCE did not influence basal Ca concentrations but reduced peak levels to 539 nM and 337 nM, respectively. TCE clearly reduced the slope of the rise of the cytosolic Ca
concentrations. Half-time not significantly diminished 0.25mM (212 f 23 ms) (198 f 22 ms) compared (225 f 42 ms, BO.05, n=
90
Efects
I 4
I
r---
on sarcolemtd
Ca jhces
The inhibitory action of TCE on Ca transients could be due to reduction of sarcolemmal Ca entry. This hypothesis was tested by depolarizing quiescent cells with high extracellular
90 mM KCI +5 /.LM Verapamil
mM KCI
of TCE
for relaxation was after addition of or 4mM TCE to control values 6).
90 f4
mtd KCI rntd TCE I
25s -
FIGURE 5. Effect of verapamil and TCE on cytosolic Ca concentration increases in ventricular by the addition of 90 mMKC1. Cytosolic Ca concentrations were expressed using fura-2-fluorescence KC1 was added at the points indicated to a non-pretreated cell suffused with Hanks’ solution (control) treatment with verapamil(5fiM) and TCE (4mM), respectively. The results were obtained in three preparations. Representative tracings from four control KCl-depolarizations without pretreatment, ations with verapamil pretreatment, and three depolarizations with TCE pretreatment are shown.
myocytes induced in quiescent cells. or five min after different myocyte three depolariz-
Ti-ichloroethane
Control
Control
(I.2
mM
Co)
625
and Calcium 0.5
mht
TCE ( 1.2 mu Co)
(5 mM Ca)
FIGURE 6. Elimination of the inhibitory action of 0.5 mM TCE on Ca transients in a single ventricular myocyte by an increase in the extracellufar Ca concentration from 1.2 mM to 5 mM. Cytosolic Ca concentrations were expressed using fura-2-fluorescence. Under control conditions the electrically stimulated cell (1 Hz) was suffused with Hanks’ solution (1.2 mM Ca). Addition of 0.5 mM TCE to Hanks’ solution reduced peak transients. After washout of TCE the same cell was suffused with Hanks’ solution containing 5 mM Ca and then reexposed to 0.5 mM TCE. The effect described was observed in four separate myocyte preparations.
KC1 (90mM without osmotic adjustment) to activate voltage-dependent slow Ca channels. Figure 5 shows that application to the cell of 90 mMKC1 induced a cluster of Ca oscillations. Within 5-6s the damped oscillations formed a plateau. In the presence of verapamil (5-20 PM) the KCl-depolarization-induced Ca increase returned to basal levels in about 25 s (Fig. 5). In a series of experiments using three separate TCE exposed (4 mM) cardiomyocyte preparations, the plateau level of cytosolic Ca after KCl-depolarization was reduced to 42 f 8% (PcO.05) of four control depolarizations without TCE exposure (Fig. 5). Figure 6 illustrates that the effects of 0.5 mM TCE can be abolished by increasing extracellular Ca concentration from 1.2 mM to 5 mM. Addition of 0.5 mM TCE reduced peak transients. After washout of TCE, the same cell was reexposed to 0.5 mM TCE in the presence of 5 mMCa. Increased external Ca (5 mM) completely eliminated the effects of 0.5mM
TCE on Ca transients. The same result was obtained in four separate experiments. It has been shown that ryanodine reduces the cytoplasmic Ca increase through inhibition of sarcoplasmic Ca release (Hilgeman et al., 1989). Pretreatment of the cells with 1 ELM ryanodine for 10min reduced the magnitude of electrically induced transients to 62 + 8 % of untreated cells (PcO.05, n = 8). TCE (4mM) caused further reductions (34 + 11% of untreated control, PcO.05) in the peak height after pretreatment with 1 PM ryanodine. The TCE effect on the Ca transients of ryanodine pretreated cells disappeared after washout of the TCE. Effects
of TCE
OR sarcophzsmic Ga fhxes
The addition of 1 mM caffeine to cardiomyocytes results in a rapid release of sarcoplasmic reticulum Ca which is then removed from the cytosol presumably by the action of sarcolemma1 and other pumping mechanisms. In
626
p. Hosnarul
IO mM Caffeine
et ul.
IO mM Caffeine I ELM Ryanadine pretreatment
after
IO mM Caffeine 4 mM TCE pretreatment
after
I
FIGURE 7. Effect of ryanodine and TCE on the caffeine induced increase in cytosolic Ca concentrations of ventricular myocytes. Ca concentrations were expressed using fura-2-fluorescence in quiescent cells, Caffeine (10 mM) was added at the points indicated to a non-pretreated cell suffused with Hanks’ solution (control), ten min after pretreatment of a cell with ryanodine (1 pm), or five min after pretreatment of a cell with TCE (4 mM). The results were obtained in three different myocyte preparations. Representative tracings from six control caffeine effects without pretreatment, three caffeine effects with ryanodine pretreatment, and six experiments with TCE pretreatment are shown. 4 mM TCE
reduced Ca transients. However, the immediate response of a quiescent ventricular myocyte to the introduction of 4rn~ TCE in the suffusate was a small brief (
