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Doxorubicin alters Ca2+ transients but fails to change Ca2+ sensitivity of contractile proteins
ELSEVIER
ETAP
Environmental Toxicology and Pharmacology 1 (1996) 131-139
Doxorubicin alters
Ca 2+
transients but fails to change contractile proteins
Ca 2+
sensitivity of
Kyosuke Temma a,,, Akihito Chugun a, Tai Akera b, Hiroshi Kondo a, Nagomi Kurebayashi c Department of Veterinary Pharmacology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Higashi 23-bancho, Towada-shi. Aomori 034, Japan b MSD Research Laboratories-Japan, Minato-ku, Tokyo 107, Japan c Department of Pharmacology, School of Medicine, Juntendo University, Bunkyo-ku, Tokyo 113. Japan Received 15 August 1995; revised 31 August 1995; accepted 1 December 1995
Abstract Doxorubicin produced a transient increase and a subsequent decrease in the amplitude of twitch contraction in myocytes isolated from guinea-pig heart and loaded with fura-2. These changes were associated with an increase and a subsequent decrease, respectively, in the amplitude of Ca 2+ transients (peak minus diastolic C a 2+ concentrations). Doxorubicin increased the diastolic Ca 2+ concentration with a concomitant shortening of the diastolic myocyte length. The time to peak Ca 2+ transients and the time to peak twitch contraction increasedin parallel. Doxorubicin failed to affect the Ca 2+ concentration-contraction curve in skirined fibers obtained from atrial muscle. We conclude that biphasic inotropic effects of doxorubicin result from biphasic changes in Ca 2+ transients, and that doxorubicin fails to alter C a 2+ sensitivity of contractile proteins. These findings are consistent with the hypothesis that doxorubicin enhances Ca 2+ release and impairs Ca z+ uptake by the sarcoplasmic reticulum. Keywords: Doxorubicin; Ca 2+ transient; Ca 2 + sensitivity of contractile proteins; Myocyte; Skinned fiber
I. Introduction Doxorubicin, an efficacious anticancer drug, causes a dose-dependent and often lethal cardiotoxicity. The exact mechanism responsible for cardiotoxic actions of doxorubicin is unknown. Olson et al. (1974) by using an atomic absorption spectrophotometry and Combs et al. (1985) from the 45Ca2+ uptake studies showed that doxorubicin increases intracellular Ca 2+ concentration ([Ca 2+ ]i). These investigators postulated that an increase in [Ca 2+ ]i may be the cause of doxorubicin-induced cardiotoxicity. Doxorubicin restored the slow action potentials in isolated chickheart preparations exposed to tetrodotoxin (Azuma et al., 1981) suggesting that doxorubicin stimulates sarcolemmal C a 2+ channels. Moreover, doxorubicin elevates the resting tension in isolated chick heart preparations (Azuma et al., 1981) indicating that heart muscle cells exposed to doxorubicin are overloaded with Ca 2+. Kusuoka et al. (1991)
Abbreviations: IOC, index of contraction; ([Ca 2 + ]i, intracellular Ca 2+ concentration; MOPS, 3-(N-morphinolino)propanesulfonic acid * Corresponding author. Tel.: Japan-176-23-4371; Fax: Japan-176-238703. 1382-6689/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved 1-6
SSDI 1 3 8 2 - 6 6 8 9 ( 9 5 ) 0 0 0 2
showed that doxorubicin increases [Ca2+]i in ventricular muscle preparations obtained from ferret hearts. Several investigators, however, reported contradicting results. For example, Klugmann et al. (1981), Rabkin et al. (1983) and Sridhar et al. (1992) showed that verapamil failed to protect the animals from doxorubicin-induced cardiotoxicity, and instead increased the mortality. Consistent with these findings, Bernardini et al. (1986) and Jensen (1986) reported that doxorubicin inhibits Ca 2+ influx across the cell membrane. We recently observed that doxorubicin decreases, instead of increases, the amplitude of C a 2+ transients (peak minus diastolic [Ca 2+ ]i) in fura2-loaded myocytes isolated from guinea-pig heart (Jiang et al., 1994b). Pretreatment with verapamil failed to modify the effect of doxorubicin to depress C a 2+ transients indicating that doxocubicin-induced depression of myocardial functions is not secondary to the C a 2+ overload caused by enhanced Ca z+ influx. Many inotropic drugs alter the developed tension primarily by changing the amplitude of C a 2+ transients (Thompson et al., 1990). Several of these drugs, however, alter the affinity of cardiac contractile proteins for Ca 2+, thereby modifying the magnitude of the inotropic effects
K. Temma et al. / Environmental Toxicology and Pharmacology I (1996) 131-139
132
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K. Temma et al. / Environmental Toxicology and Pharmacology I (1996) 131-139
(Konishi et al., 1984; Endoh and Blinks, 1988; Gwathmey and Hajjar, 1990; Ferroni et al., 1991; Bosnjak et al., 1992). In the present study, the effects of doxorubicin on Ca 2+ transients and on Ca 2+ sensitivity of contractile proteins were examined. In isolated heart muscle preparations, relatively high concentrations of doxorubicin ( 1 0 0 - 2 0 0 /xM) cause a transient positive and a subsequent negative inotropic effect (Hagane et al., 1988; T e m m a et al., 1994). The negative inotropic effect of high concentrations of doxorubicin observed in isolated heart muscle preparations in vitro is likely to be mediated by the same mechanism responsible for the cardiotoxicity observed in vivo after a prolonged exposure to a lower concentration of doxorubicin in plasma (Hagane et al., 1988; Olson and Mushlin, 1990; Agata et al., 1991; Temma et al., 1994). The results of the present study show that the positive and negative inotropic effects of doxorubicin are caused by an increase and a decrease in the amplitude of Ca 2+ transients, and that doxorubicin fails to alter Ca 2+ sensitivity of the contractile proteins.
2. M a t e r i a l s a n d m e t h o d s
2.1. Single cell preparations
Male Hartly guinea pigs, weighing approximately 400 g, were used in this study. Cardiac myocytes were obtained from ventricular muscle by using collagenase as previously described (Jiang et al., 1994b). The myocytes were loaded with fura-2 by incubating at 30°C for 30 min in a Hepes buffer solution containing 1-1.5 /zM fura2 / A M (Molecular Probes, Eugene, OR, USA) and bovine serum albumin (20 m g / m l ) . The myocytes were washed 3 times to remove extracellular fura 2 / A M , and stored at room temperature (18-23°C) up to 4 h before use. 2.2. Measurements o f [ C a 2 + ] i concentrations and cell motions
The methods used to record and analyze Ca 2+ transients in fura-2 loaded myocytes were similar to those previously described (Akera et al., 1990; Koyama et al., 1991; Jiang et al., 1994a, 1994b). In the present study, however, we used a CCD video camera with the picture elements vertically reduced to one fourth (slit-scan video camera; C-2400-77, Hamamatsu Photonics, K.K., Hamamatsu, Japan). This camera is capable of recording fluores-
133
cence images at 4 ms intervals. Myocytes were placed in an open chamber with a thin quartz-glass bottom (0.2 ml in volume) on the stage of an inverted epifluorescence microscope (Nikon TMD, Nihon Kogaku, Tokyo, Japan). Myocytes were superfused at a flow rate of 0.1 m l / m i n with a Krebs-Henseleit bicarbonate buffer solution containing (mM); 118 NaCI, 27.2 NaHCO 3, 4.8 KCI, 1.2 MgSO 4, 1.2 CaCI2, 1.0 KH2PO4, 11.1 glucose and 2.5 sodium pyruvate. Myocytes were electrically stimulated at 2 Hz at 25°C by means of a pair of platinum electrodes for electrical field stimulation. In this system, a microcomputer controls the following: an electrical stimulator (SEN-3301, Nihon Kohden Kogyo, Tokyo, Japan), the timer for the CCD slit-scan video camera and a filter controller. The filter controller was used to excite myocytes with the ultraviolet light of either 340 or 380 nm wavelength using a 100 W xenon arc lamp. Fluorescence images resulting from 340 nm excitation were recorded at 500 nm at 4 ms intervals for 533 ms (33 ms before to 500 ms after the electrical stimulation). A total of 133 images were taken during one cycle of the Ca 2+ transient. Immediately after a set of fluorescence images were taken with 340 nm excitation, the process was repeated with 380 nm excitation. Fura-2 loading was minimized to reduce the chelation of intracellular Ca 2+. The intensity of the ultraviolet light was reduced using 30% and 50% neutral density filters to avoid damages to myocytes. The filters were placed before and after the 340 or 380 nm excitation filter. To obtain clear pictures, four fluorescence signals were averaged. The images were digitalized and stored in an Argus-50 system (Hamamatsu Photonics, Hamamatsu, Japan) for later analyses. The ratio of fluorescence intensities observed at 340 and 380 nm excitation ( 3 4 0 / 3 8 0 nm) was calculated at each pixel on the screen after subtracting the back ground fluorescence. The [Ca2+]i in myocyte was calculated as described in our previous reports (Akera et al., 1990; Jiang et al., 1994a, Jiang et al., 1994b). The calibration curves for Ca z+ concentrations were obtained using the 'activation solution' for skinned fibers (see below). Fura-2 pentapotassium salt (final concentration, 10 /xM) was added to the activation solution. The activation solution is a Ca2+-EGTA buffer solution containing 22 nM to 2.36 M Ca 2÷ (pCa from 7.66 to 5.63). Twitch contraction of myocyte was estimated from changes in myocyte length using the fluorescence ratio images on the monitor. The diastolic length of myocytes (immediately before the electrical stimulation, i.e., time
Fig. 1. Initiation, propagation and restoration of Ca2+ transients and the motion of myocytes before (A) and after doxorubicin treatment (B). Fura-2 loaded myocytes were stimulated at 2 Hz at 25°C. After a 30 rain equilibration period, fluorescence images were recorded at 500 nm with excitation wave lengths of 340 or 380 nm (A). Subsequently, 100 ~M doxorubicin (final concentration) was added to the medium. The fluorescenceimages were recorded 60 rain later (B). The number at the upper left comer of each panel represents the time in msec after electrical stimulation. Typical pictures from seven experiments.
K Temma et al. / Environmental Toxicology and Pharmacology I (1996) 131-139
134 [Ca2*l i (nM) 900
I' I
Length (ixm) '
'
'
'
i 1°2 I
~
~
0
100
~
~
200
300
loo 7oo 99 600
98
500
97 96
400 95 300
94
200
93 0
100
200
300
400
500
Time (msec)
400
500
Time (msec)
Fig. 2. The time course of Ca 2+ transients and twitch contractions. Fura-2 loaded myocytes were stimulated at 2 Hz at 25°C. After the myocytes were incubated with ([7, II) or without (©, O ) 100 /xM doxorubicin for 60 min, fluorescence images were recorded at 500 nm with excitation wave lengths of 340 or 380 nm. The values for [Ca2+ ]i and myocyte length were plotted against time after electrical stimulation. The left panel represents [Ca 2+ ]i, the right panel represents the length of the myocytes. Each point represents the mean of seven experiments.
recorder (model WI 840G; Nihon Kohden Kogyo, Tokyo, Japan). The Ca 2+ concentration-contraction curves for the skinned fiber were obtained using a series of Ca2+-EGTA buffer solutions (activation solutions) having the Ca 2÷ concentration of between 47 nM and 64.57/xM (pCa from 7.33 to 4.19). The solutions were prepared by mixing basic solutions A and B in varying portions. Solution A contained (mM); 10 EGTA, and solution B; 10 EGTA and 10 calcium methanesulfonate, respectively. In addition, both solutions contained (mM); 20 3-(N-morphinolino)propanesulfonic acid (MOPS), 4.3 Na2ATP and 5.0 magnesium methanesulfonate. The pH value was adjusted to 7.0 using KOH. Ionic strength was adjusted to 0.17 with potassium methanesulfonate. The skinned fiber was exposed to one of the activation solutions for 2 - 4 min, and then relaxed using the relaxation solution containing (mM); 20 MOPS, 2.0 EGTA, 4.3 Na2ATP, 112.7 potassium methanesulfonate and 5.0 magnesium methanesulfonate. The next activation of the skinned fiber was performed 4 - 5 min later.
2.4. Chemicals and statistical analyses zero in Fig. 1B and Fig. 2, right panel) decreased with time in the presence of 100 /xM doxorubicin. To compensate the changes in diastolic myocyte length, changes in the myocyte length resulting from twitch contractions were expressed as percentages of the diastolic length observed before each contraction. The twitch contraction was expressed as the index of contraction (IOC) calculated as follow (Jiang et al., 1994a, Jiang et al., 1994b): IOC =
myocyte length at peak contraction ) 1diastolic myocyte length × 100
Collagenase (type A, isolated from Clostridium histolyticum) and doxorubicin (Adriacin) were purchased from Boehringer Mannheim (Mannheim, Germany) and Kyowa Hakko Kogyo (Tokyo, Japan), respectively. All other chemicals used were of reagent grade. Statistical analyses were performed with Student's t test. The criterion for statistical significance is a P value of less than 0.05.
3. Results
(1)
3.1. Ca 2 + transients and twitch contractions observed in myocytes
2.3. Skinned fiber preparations The skinned fibers (150-200 /xm in diameter; 2 - 3 mm in length) were prepared from guinea-pig hearts (male Hartly guinea pigs, weighing approximately 400 g) as previously described (Kitazawa, 1984) and treated with 1% ( v / v ) Triton X-100 for 30 min at 24°C to completely disrupt cellular membranes (Wendt and Stephenson, 1983; Kurebayashi and Ogawa, 1991). One end of the skinned fiber was fixed to the bottom of the chamber with a pin and the other end was mounted on a stainless steel hook that was connected to an isometric force displacement transducer (model TB-625T; Nihon Kohden Kogyo, Tokyo, Japan). The skinned fiber was stretched to a length corresponding to an average sarcomere length of approximately 2.4 /zm. The sarcomere length was monitored using a microscope. Changes in contraction were continuously recorded using a force-displacement transducer and a pen
Fura-2 loaded ventricular myocytes of guinea-pig hearts were superfused with a Krebs-Henseleit bicarbonate buffer solution at 25°C and stimulated at 2 Hz. After a 30 min equilibration, control fluorescence images were recorded. Under these experimental conditions, twitch contractions and Ca 2÷ transients were stable for next 120 min (Koyama et al., 1991; Jiang et al., 1994a, Jiang et al., 1994b). As shown in Fig. 1A, the Ca 2+ transients started 8-12 ms after the electrical stimulation. Initial increases in [Ca2+ ]i were observed at several sites of the myocyte, and rapidly spread to the entire cell volume 30-40 ms after the electrical stimulation (Fig. 1A). The left panel of Fig. 2 shows the time course of a Ca 2+ transient. The Ca 2+ transients reached the peak at 93 ms (the mean of seven myocytes) after the electrical stimulation, and then declined to the diastolic level (Fig. 2, left panel). The start and the peak of twitch contraction was delayed from those
135
K. Temma et al. / Envi ronmental Toxicology and Pharmacology 1 (1996) 131-139
Table I Effects of doxorubicin on Ca 2+ transients in myocytes isolated from guinea-pig hearts
Table 2 Effects of doxorubicin in Ca2+ transients and twitch contraction in myocytes isolated from guinea-pig hearts
Time of doxorubicin exposure (min)
Size of Ca2+ transients
Size of twitch contraction
Time to peak contraction
(%)
(%)
(ms)
100 115.1 ±7.0 97.7±9.2 74.8 ± 12.1 38.1 ± 10.2 a
100
0 8 30 60 90
Diastolic [Ca2+ ]i
Peak [Ca2+ ]i
Time to peak [Ca2÷ ]i
(nM)
(nM)
(ms)
Time of doxorubicin exposure (min)
259± 14 292± 11 346± 18 a
840±61 957±62 898 ±47 841 ±28 778±52
93±7 112±7 a 149_+ 12 a 174± 11 a 190±9 a
0 8 30 60 90
461 ±53
a
533 _+49 a
a
After myocytes were equilibrated at 25°C for 30 min at 2 Hz stimulation, fluorescence images were taken at time zero. Subsequently, doxorubicin (final concentration 100 p~M) was added to the incubation medium. Fluorescence images were recorded at 8, 30, 60 and 90 min later. Values are the mean ± SE. of seven experiments. " Significantly different from values observed at time zero (P < 0.05, paired t test).
o f the C a 2+ transients (Fig. 2). The cell started to shorten at 50 ms and reached the peak at 186 ms (Fig. 2, right panel). A t the peak o f twitch contraction, m y o c y t e s shortened to 92.6 + 1.3% ( n = 7) o f the diastolic length. A s s u g g e s t e d by m a n y investigators, d o x o r u b i c i n increases the diastolic [Ca2+]i ( O l s o n et al., 1974; C o m b s et al., 1985; A z u m a et al., 1981; K u s u o k a et al., 1991). A t 60 min after the addition o f 100 /.tM d o x o r u b i c i n (final concentration), the m y o c y t e s h o w n in Fig. 1B at t i m e zero was brighter than the s a m e m y o c y t e s h o w n in Fig. 1A. The diastolic [Ca2+]i i n c r e a s e d with t i m e in the presence of d o x o r u b i c i n (Table 1). T h e patterns o f the initiation, spreading and restoration o f the Ca 2+ transient were unc h a n g e d by d o x o r u b i c i n (Fig. 1B). D o x o r u b i c i n , h o w e v e r , p r o l o n g e d the time to p e a k Ca 2+ transient and d e l a y e d the decrease in [Ca 2+ ]i f o l l o w i n g the peak (Fig. 2, left panel). A s s o c i a t e d with these c h a n g e s in [Ca2+]i, the diastolic length o f the m y o c y t e s d e c r e a s e d and the time course of the twitch contraction was d e l a y e d in the presence o f 100 p,M d o x o r u b i c i n (Fig. 2, right panel). T h e s e effects o f d o x o r u b i c i n on [Ca2+]i and twitch contraction increased gradually o v e r a 90 m i n e x p e r i m e n t a l period with similar time courses (Tables 1 and 2). D o x o r u b i c i n m o n o t o n o u s l y increased the diastolic [Ca2+ ]i and the t i m e to p e a k C a 2+ "transients; h o w e v e r , the effect o f d o x o r u b i c i n on the p e a k Ca 2+ transient was biphasic (Table 1). T h e p e a k C a 2+ transient increased at 8 min and then decreased. T h e size o f the C a 2+ transients and the index o f contraction ( I O C ) were calculated to allow a direct c o m p a r i s o n b e t w e e n d o x o r u b i c i n - i n d u c e d changes in the a m p l i t u d e s o f C a 2+ transients and twitch contractions (Table 2). T h e size o f C a 2+ transients and I O C initially increased, and then gradually decreased. The t i m e course o f c h a n g e s in the size o f C a 2+ transients was a l m o s t identical to that o f c h a n g e s in the size o f twitch contractions. T h e plots o f the size o f C a 2+ transients against the I O C g a v e a straight line (Fig. 3). T h e r e was a
186_+2 194± 12 238± 16 a 245 ± 14 a 271±5 a
12.1 96.3±11.6 70.9 ± 6.5 a 48.9:t:10.3 a
110.1
4-
The size of the Ca 2+ transients was estimated as [Ca2+ ]i at the peak minus [Ca2+ ]i at diastolic state. The size of twitch contractions was estimated from the IOC values (see Eq. 1). These values are expressed as percentages of the control value observed at time zero. Values are the mean±S.E, of seven experiments, a Significantly different from the control values observed at time zero (P < 0.05, paired t test).
h i g h d e g r e e of correlation b e t w e e n the size o f C a 2+ transients and the size o f twitch contractions ( y = 0.809, 35 points, P < 0.001) 3.2. Ca 2 + s e n s i t i u i t y o f s k i n n e d f i b e r s
Sensitivity o f contractile proteins to C a 2+ was e x a m ined using skinned fibers isolated from left atrial m u s c l e o f g u i n e a - p i g heart and treated with Triton X-100. Atrial muscle, instead o f ventricular muscle, was used to obtain better skinned fiber preparations. T h e t o x i c o l o g i c a l effects
lOG (% of control) 160
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20
40
60
80
100
120
140
160
Size of Ca2" transient (% of control)
Fig. 3. The relationship between the size of Ca 2+ transients and twitch contractions observed in the presence of doxorubicin. Fura-2 loaded myocytes were stimulated at 2 Hz at 25°C. After a 30 min equilibration, myocytes were exposed to 100 p,M doxorubicin. The size of Ca2+ transients (peak [Ca2+ ]i minus diastolic [Ca2+ ]i) and IOC were calculated at 0, 8, 15, 30, 60 and 90 min later. Each value for the size of Ca2+ transient and IOC was expressed as a percentage of the values observed at time zero (immediately before the doxorubicin addition). Each point represents an individual time point observed in a total of seven preparations.
K. Temma et al. / Environmental Toxicology and Pharmacology 1 (1996) 131-139
136 Tension (%) 120 j
I
I
1
T 10
Ca 2+ activation of the contraction. The value observed in the presence of 100 /xM doxorubicin was 1.25 _ 0.11 ~ M (n = 5) which was not significantly different from the value for the second curve observed in the absence of doxorubicin. These results indicate that Ca 2+ sensitivity of the skinned fibers is not altered by a 60 min exposure to 100 /xM doxorubicin.
80 60 40 20 0 0.1
4. Discussion 100
Ca 2+ concentration fitJM)
Fig. 4. Effects of doxorubicin on the Ca 2+ concentration-response curves in skinned fibers. Skinned fibers obtained from atrial muscle guinea-pig hearts were incubated with 1 ( v / v ) % Triton X-100 for 30 min to disrupt intracellular membranes. Subsequently, a Ca 2+ concentration-contraction curve was generated for control preparations (O). After a 60 min incubation in the absence ([]), or presence of 100 mM doxorubicin (zx), a Ca 2+ concentration-contraction curve was generated again. Values observed at 64.59/~M Ca 2+ in the first curve were set at 100%. Values at each point were calculated for each preparation and then averaged.
of doxorubicin and the effects of doxorubicin on isolated heart muscle preparations are similar in atrial and ventricular muscle (Olson and Mushlin, 1990; Boucek et al., 1993; Temma et al., 1994) justifying the use of atrial muscle in this study. A high concentration of caffeine (50 mM) failed to cause a contraction in these fibers (data not shown) indicating that all cellular membranes, such as sarcoplasmic reticulum, were completely disrupted. Contraction of skinned fibers in the relaxing solution was stabilized within 10 min. At this point, the solution was switched to the activation solution containing an indicated concentration of Ca 2÷ ranging from 47 nM to 64.57/.tM to generate the Ca 2+ concentration-contraction curves. After the first Ca 2+ concentration-response curve was obtained, the skinned fiber was incubated for 60 rain in the presence or absence of 100/xM doxorubicin. Subsequently, the skinned fiber was exposed to the activation solution to produce the second Ca 2+ concentration-response curve. An exposure to the activation solutions produced a typical sigmoidal concentration-response curve in skinned fibers (Fig. 4). In the presence of a high concentration of Ca 2+, the force of contraction was 7.66 _ 0.71 mg (n = 9) in the first concentration-response curve. The maximal value of the twitch contraction for the second curve observed in the absence of doxorubicin was 78.1 ___5.6% (n = 4) of the corresponding value for the first curve. The EDs0 values for C a 2+ w e r e 1.34 _+ 0.03 /,tM (n = 9) and 1.33 + 0 . 0 9 / z M (n = 4) for the first and the second curve, respectively. The addition of 100 /xM doxorubicin failed to alter the maximal response of the second curve (81.8 _ 4.3%, n = 5) compared to the corresponding value of the second curve observed in a doxorubicin-free solution. Moreover, doxorubicin failed to alter the EDso values for
In an earlier study (Temma et al., 1994), we observed that in isolated atrial muscle preparations obtained from guinea-pig hearts and incubated at 36°C, 200 /zM doxorubicin caused a biphasic positive inotropic effect: specifically, an early and a late phase positive inotropic effect. However, the pattern of the inotropic effects was markedly influenced by the experimental conditions. At low temperature or at a high stimulation frequency, doxorubicin caused a gradual negative inotropic effect, instead of a positive inotropic effect, after the initial transient positive inotropic effect. It is postulated that the decrease in developed tension of heart muscle may be causally related to the doxorubicin-induced cardiotoxicity. In the present study, therefore, relatively low temperature and high stimulation frequency were chosen. Using myocytes at 24°C under 2 Hz stimulation, doxorubicin caused a near identical pattern of inotropic effects with that observed at a low temperature and 2 Hz stimulation in isolated left atrial muscle preparations. In these myocytes, size of peak Ca 2+ transients also increased transiently and then decreased with the same time course as that of twitch contraction. The increase and subsequent decrease in the size of Ca R+ transients and twitch contraction showed a good linear correlation. In addition, at a higher temperature (34°C), the significant decrease in twitch contraction and size of Ca 2÷ transients observed at 24°C after the transient increase, was almost abolished (our recent observations, data not shown). At high Ca 2÷ (2.4 mM), the decreases in twitch contraction and the size of Ca 2+ transients were measurable. Further, doxorubicin caused a monotonous increase in the diastolic [Ca 2÷ ]i and the time to peak Ca z+ transients. These changes resulted in monophasic changes in the diastolic myocytes length and the time to peak twitch contraction. Results of the present study are consistent with that of earlier experiments using isolated left atrial muscle preparations of guinea-pig hearts (Temma et al., 1993, 1994), and clearly show that changes in the size of Ca 2÷ transients and in the diastolic [Ca 2÷ ]i are responsible for doxorubicin-induced changes in developed and resting tension, respectively. In the present study, myocytes obtained from guinea-pig heart were stimulated at 2 Hz at 25°C. E s t i m a t e d [Ca2+]i at diastolic state and the peak of Ca 2+ transients was 259 and 840 nM, respectively. The time to peak Ca 2÷ transient
K. Temma et al. / Environmental Toxicology and Pharmacology I (1996) 131-139
was 93.4 ms after the electrical stimulation and the peak twitch contraction was observed at 186 ms. These values appear reasonable for guinea-pig heart muscle preparations stimulated at 2 Hz at 25°C. Moreover, the distribution of [Ca2+]i at diastolic state and also at the peak of the Ca z+ transient was relatively uniform. These results would indicate that potential problems associated with the use of fura-2 for the estimation of [Ca2+]~ in isolated myocytes (Frampton et al., 1991) are minor in the present study. The Ca 2÷ concentration-contraction curves observed in the skinned fibers show a typical sigmoidal curve. Doxorubicin failed to alter the Ca 2÷ concentration-contraction relationships. Under the conditions studied, these results indicate that the inotropic effects of doxorubicin are not modified by changes in Ca 2+ sensitivity of the contractile proteins. Instead, changes in Ca 2+ transients are the cause of the positive and negative inotropic effects of doxorubicin. Recently, Boucek et al. (1993) reported the lack of doxorubicin effects on the concentration-response curves for Ca 2÷ in skinned fibers isolated from rabbit hearts with a lower concentration of doxorubicin and a shorter exposure time. Doxorubicin caused a significant prolongation of the time to peak Ca 2÷ transients confirming the results of our previous study (Jiang et al., 1994b). These results suggest that doxorubicin impairs the mechanism(s) that regulates [Ca2+]i in the cardiac muscle. These mechanisms include an influx or an effiux mechanism across the cell membrane, or an uptake or release mechanism across the sarcoplasmic reticulum membranes. Doxorubicin has been reported to decrease the Ca 2÷ influx across the sarcolemma (Bernardini et al., 1986; Jensen, 1986). Similar to doxorubicin, the Ca 2÷ channel blocker, verapamil, delayed the time course of Ca 2÷ transients in myocytes isolated from guinea-pig hearts (Jiang et al., 1994b). Verapamil, however, decreased the diastolic [CaZ+]i (Jiang et al., 1994b) whereas doxorubicin increased the d i a s t o l i c [Ca2+]i in the present study. These results, therefore, indicate that a decrease in the Ca 2+ influx is unlikely to be the cause of doxorubicin-induced prolongation of the time to peak Ca 2÷ transients. Doxorubicin has been reported to stimulate Ca 2÷ release from the sarcoplasmic reticulum resulting from its specific binding to the Ca 2+ release channels and ensuing increases in open probability of the channels (Kim et al., 1989; Holmberg and Williams, 1990; Ondrias et al., 1990; Pessah et ai., 1990). Boucek et al. (1993) reported a doxorubicin-induced increase in Ca 2+ release in sarcoplasmic reticulum and mitochondria membrane intactskinned fibers. These actions of doxorubicin may resemble actions of low concentrations of ryanodine (Meissner, 1986; Lattanzio et al., 1987). If doxorubicin causes a continuous leak release of Ca 2÷ from the sarcoplasmic reticulum, and when the Ca 2+ release exceeds the reserve capacity of the Ca 2÷ pump of the sarcoplasmic reticulum, the increase in the diastolic [Ca2+]i and the prolongation
137
of the time to peak C a 2+ transients would be anticipated. These results were observed in the present study. Liu and Vassalle (1991) recently suggested that 10-50 /zM doxorubicin impairs Ca 2+ uptake by the sarcoplasmic reticulum in myocytes isolated from ventricular muscle of guinea-pig hearts. Enhanced Ca 2+ release and inhibited Ca z+ uptake by the sarcoplasmic reticulum explain the observed increase in the diastolic [Ca2÷]i and the prolongation of the time to peak of Ca 2+ transients. In addition, the decrease in Ca 2+ uptake may also account for the slower decay of the C a 2+ transients and of the twitch contraction by doxorubicin. In this regard, the actions of doxorubicin resemble some actions of caffeine. Caffeine binds to the ryanodine receptors, increases the open probability of C a 2+ channels, and stimulates Ca 2+ release in membrane preparations obtained from cardiac or skeletal muscle sarcoplasmic reticulum (Salviati and Volpe, 1988; Kim et al., 1989; Ondrias et al., 1990; Pessah et al., 1990). Caffeine increased the diastolic [Ca2+]i (O'Nell and Eisner, 1990; Jiang et al., 1994b) and increased the time to p e a k C a 2+ transients (Konishi et al., 1984). These reports further support the concept that the cause of the prolongation of the time to peak Ca 2+ transients and the increase in the diastolic [Ca2+]i result from the impairment of C a 2+ uptake and release mechanisms in the sarcoplasmic reticulure. It should be noted that the prolongation of the time to peak Ca z+ transients and the increase in diastolic [Ca2+]i developed with almost the same time course in the present study. As described above, myocyte length shortened concomitant with an increase in the diastolic [Ca2+] r Although in isolated heart muscle preparations obtained from chicks, the increase in resting tension was noticed by Azuma et al. (1981), the increase in resting tension by doxorubicin was not observed in isolated left atrial muscle preparations of guinea-pig hearts (Temma et al., 1994). The cause of this discrepancy between myocytes and atrial muscle preparations is presently unknown. An initial increase and a subsequent decrease in the size of C a 2+ transients caused by doxorubicin may also result from the augmentation of Ca 2+ release from the sarcoplasmic reticulum. An enhancement of the Ca 2+ release is likely to initially increase the size of Ca 2+ transients. Subsequently when the Ca 2+ release begins to exceed the capacity of the C a 2+ pump, the amount of releasable Ca 2+ stored in the sarcoplasmic reticulum would decrease. This would result in a decreased size of Ca 2+ transients that are triggered by membrane excitation. In addition, it has been reported that release of C a 2+ from sarcoplasmic reticulum is decreased by an increase in diastolic Ca 2+ due to an inactivation of Ca 2+ release channels (Fabiato, 1983). It has been reported that Ca 2+ overloading of the cardiac cells causes a decrease in C a 2+ transients. Doxorubicin has been shown to cause an enhancement of slow channels, or a decrease in Na +, Ca 2÷ exchange reactions or Ca 2+ pump activities (Azuma et al., 1981; Caroni et al.,
138
K. Temma et al. / Environmental Toxicology and Pharmacology 1 (1996) 131-139
1981: H a r a d a et al., 1990). T h e r e f o r e , it is s u g g e s t e d that d o x o r u b i c i n m a y c a u s e C a 2+ o v e r l o a d i n g as a r e s u l t o f the a b o v e effects, a n d t h e r e b y c a u s e c a r d i o t o x i c i t y . H o w e v e r , d o x o r u b i c i n d i m i n i s h e d , i n s t e a d o f a u g m e n t e d , the oscillatory a f t e r c o n t r a c t i o n p r o d u c e d b y a toxic c o n c e n t r a t i o n o f o u a b a i n ( B i n a h et al., 1983). B e c a u s e , the o s c i l l a t o r y a f t e r c o n t r a c t i o n r e s u l t s f r o m C a 2÷ o v e r l o a d i n g o f m y o c a r d i a l cells, t h e s e r e s u l t s s u g g e s t that d o x o r u b i c i n - i n d u c e d dep r e s s i o n o f the C a 2+ t r a n s i e n t is u n l i k e l y to r e s u l t f r o m the C a 2÷ o v e r l o a d i n g . I n s t e a d it is e x p e c t e d t h a t d o x o r u b i c i n m a y c a u s e a d e p l e t i o n o f C a 2+ stored in t h e s a r c o p l a s m i c r e t i c u l u m w h i c h t h e n in turn c a u s e s a d e c r e a s e in t h e r e l e a s e o f C a 2÷. U n d e r c o n d i t i o n s that r e s u l t in a d e c r e a s e in C a 2+ r e l e a s e f r o m s a r c o p l a s m i c r e t i c u l u m , C a 2+ c h a n nel a n t a g o n i s t s m a y e v e n facilitate d o x o r u b i c i n - i n d u c e d c a r d i o - t o x i c i t y ( K l u g m a n n et al., 1981; R a b k i n et al., 1983; S r i d h a r et al., 1992). In s u m m a r y , d o x o r u b i c i n initially i n c r e a s e d a n d s u b s e q u e n t l y d e c r e a s e d the size o f the C a 2+ t r a n s i e n t s in m y o c y t e s i s o l a t e d f r o m g u i n e a - p i g hearts. T h e d i a s t o l i c [ C a 2 + ] i a n d t h e t i m e to p e a k C a 2÷ t r a n s i e n t s i n c r e a s e d m o n o t o n o u s l y . T h e s e c h a n g e s r e s u l t e d in a b i p h a s i c c h a n g e s in the size o f t w i t c h c o n t r a c t i o n , a n d m o n o p h a s i c d e c r e a s e in d i a s t o l i c m y o c y t e l e n g t h a n d an i n c r e a s e in the t i m e to p e a k t w i t c h c o n t r a c t i o n . D o x o r u b i c i n failed to a l t e r the C a 2÷ s e n s i t i v i t y o f s k i n n e d atrial m u s c l e f i b e r s o b t a i n e d f r o m g u i n e a - p i g heart. T h e p o s i t i v e a n d n e g a t i v e i n o t r o p i c e f f e c t s o f d o x o r u b i c i n are a c c o u n t e d f o r b y the c h a n g e s in C a 2+ t r a n s i e n t s . T h e s e effects o f d o x o r u b i c i n m a y b e e x p l a i n e d f r o m an i n c r e a s e in C a 2+ r e l e a s e f r o m the s a r c o p l a s m i c r e t i c u l u m a n d a n i n h i b i t i o n o f C a 2÷ uptake by this organelle.
Acknowledgements We thank Professor Yasuo Ogawa of Juntendo University f o r his h e l p a n d e n c o u r a g e m e n t s . T h i s s t u d y w a s partly s u p p o r t e d b y a G r a n t - i n - A i d for G e n e r a l S c i e n t i f i c R e s e a r c h (no, 0 4 6 6 0 3 3 0 ) f r o m the J a p a n e s e M i n i s t r y o f Education, Sciences and Culture.
References Akera, T., K. Temma, H. Kondo and K. Hagane, 1990, Digital imaging system for recording rapid changes in intracellular Ca 2+ concentrations triggered by electrical stimulation of cardiac myocytes, Keio J. Med. 39, 168. Agata, N., S. Hirano, Y. Hara, H. Iguchi, M. Shirai, H. Tone and N. Urakawa, 1991, A comparison of pirarubicin with other anthracyclines on the cardiovascular system, Pharmacometrics 41,467. Azuma, J., N. Sperelakis, H. Hasegawa, Y. Tanimoto, S. Vogel, K. Ogura, N. Awata, A. Sawamura, H. Harada, T. lshiyama, Y. Morita and Y. Yamamura, 1981, Adriamycin cardiotoxicity: possible pathogenic mechanisms, J. Mol. Cell. Cardiol. 13, 381. Bernardini, C. T.M. Del, R. Danesi and T.M. Della, 1986, The influence
of amnnone on cardiac toxicity induced by adriamycin in rats, Arch. Int. Pharmacodyn. Ther. 283, 243. Binah, O., I.S. Cohen and M.R. Rosen, 1983, The effects of adriamycin on normal and ouabain-toxic canine Purkinje and ventricular muscle fibers, Circ. Res. 53, 655. Bosnjak, Z.J., A. Aggarwal, L.A. Turner, J.M. Kampine and J.P. Kampine, 1992, Differential effects of halothane, enflurane, and isoflurane on Ca 2÷ transients and papillary muscle tension in guinea pigs, Anesthesiology 76, 123. Boucek, R.J., S.H. Buck, F. Scott, N.L. Oquist, S. Fleicber, R.D. Olson and E. Okediji, 1993, Anthracycline-induced tension in permeabilized cardiac fibers: evidence for the activation of the calcium release channel of sarcoplasmic reticulum, J. Mol. Cell. Cardiol. 25, 249. Caroni, P., F. Villani and Carafoli, 1981, The cardiotoxic antibiotic doxorubicin inhibits the Na÷/Ca 2÷ exchange of dog heart sarcolemmal vesicles, FEBS Lett. 130, 184. Combs, A.B., D. Acosta and K. Ramos, 1985, Effects of doxornbicin and verapamil on calcium uptake in primary cultures of rat myocardial cells, Biochem. Pharmacol. 34, 1115. Endoh, M. and J.R. Blinks, 1988, Actions of sympathomimetic amines on the Ca 2+ transients and contractions of rabbit myocardium: reciprocal changes in myofibrillar responsiveness to Ca 2+ mediated through orand /3-adrenoceptors. Circ. Res. 62, 247. Fabiato, A., 1983, Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum, Am. J. Physiol. 245, C1. Ferroni, C., O. Hano, C. Ventura, E.G. Lakatta, M. Klockow, H. Spurgeon and M.C. Capogrossi, 1991, A novel positive inotropic substance enhances contractility without increasing the Ca 2+ transient in rat myocardium, J. Mol. Cell. Cardiol. 23, 325. Frampton, J.E., C.H. Orchard and M.R. Boyett, 1991, Diastolic, systolic and sarcoplasmic reticulum [Ca2+ ] during inotropic interventions in isolated rat myocytes, J. Physiol. (London) 437, 351. Gwathmey, J.K. and R.J. Hajjar, 1990, Intracellular calcium related to force development in twitch contraction of mammalian myocardium, Cell Calcium 11, 531. Hagane, K., T. Akera and J.R. Berlin, 1988, Doxorubicin: mechanism of cardiodepressant actions in guinea pigs, J. Pharmacol. Exp. Ther. 246, 655. Harada, H., B.J. Cusack, R.D. Olson, W. Stroo, J. Azuma, T. Hamaguchi and S.W. Schaffer, 1990, Taurine deficiency and doxorubicin: interaction with the cardiac sarcolemmal calcium pump, Biochem. Pharmacol. 39, 745. Holmberg, S.R.M. and A.J. Williams, 1990, Patterns of interaction between anthraquinone drugs and the calcium-release channel from cardiac sarcoplasmic reticulum, Circ. Res. 67, 272. Jensen, R., 1986, Doxorubicin cardiotoxicity: contractile changes after long-term treatment in the rat, J. Pharmacol. Exp. Ther. 236, 197. Jiang, J., K. Temma and T. Akera, 1994a, Dual effects of nicardipine: evidence from intracellular Ca 2+ transients and twitch contractions observed in single myocytes obtained from rat heart, Eur. J. Pharmacol. 266, 155. Jiang, J., K. Temma and T. Akera, 1994b, Doxorubicin-induced changes in intracellular Ca2÷ transients observed in cardiac myocytes isolated from guinea-pig heart, Can..1. Physiol. Pharmacol. 72, 622. Kim, D.-H., A.B. Landry 111, Y.S. Lee and A.M. Katz, 1989, Doxornbicin-induced calcium release from cardiac sarcoplasmic reticulum vesicles, J. Mol. Cell. Cardiol. 21,433. Kitazawa, T., 1984, Effect of extracellular calcium on contractile activation in guinea-pig ventricular muscle, J. Physiol. (London) 355, 635. Klugmann, S., B.F. Klugmann, G. Decorti, D. Gori, F. Silvestri and F. Camerini, 1981, Adriamycin experimental cardiomyopathy in Swiss mice. Different effects of two calcium antagonistic drugs on ADM-induced cardiomyopathy, Pharmacol. Res. Commun. 13, 769. Konishi, M., S. Kurihara and T. Sakai, 1984, The effects of caffeine on tension development and intracellular calcium transients in rat ventricular muscle, J. Physiol. (London) 355, 605.
K. Temma et al. / Environmental Toxicology and Pharmacology 1 (1996) 131-139 Koyama, T., K. T e m m a and T. Akera, 1991, Reperfusion-induced contracture develops with a decreasing [Ca 2+ ]i in single heart cells, Am. J. Physiol. 261, HI 115. Kurebayashi, N. and S. Ogawa, 1991, Discrimination of calcium-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian ATPase activity, J. Musc. Res. Cell Motil. 1214, 355. Kusuoka, H., S. Futaki, Y. Koretsune, A. Kitabatake, H. Suga, T. Kamada and M. lnoue, 1991, Alterations of intracellular calcium homeostasis and myocardial energetics in acute adriamycin-induced heart failure, J. Cardiovasc. Pharmacol. 18, 437. Lattanzio, F.A., R G . Schlatterer, M. Nicar, K.P. Cambell and J.L. Sutko, 1987, The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes, J. Biol. Chem. 262, 2711. Liu, Q. and M. Vassalle, 1991, On the mechanism by which doxorubicin abolishes the oscillatory events induced by Ca overload in single cardiac myocytes, J. Cardiovasc. Pharmacol. 18, 711. Meissner, G., 1986, Ryanodine activation and inhibition of the Ca 2+ release channel of sarcoplasmic reticulum, J. Biol. Chem. 261, 6300. Olson, R.D. and P.S. Mushlin, 1990, Doxorubicin cardiotoxicity: analysis of prevailing hypotheses, FASEB J. 4, 3076. Olson H.M., D.M. Young, D.J. Prieur, A.F. LeRoy and R.L. Reagan, R.L, 1974, Electrolyte and morphologic alterations of myocardium in adriamycin-treated rabbits, Am. J. Pathol. 77, 439. Ondrias, K., L. Borgatta, H.H. Kim and B.E. Ehrlich, 1990, Biphasic effects of doxorubicin on the calcium release channel from sarcoplasmic reticulum of cardiac muscle, Circ. Res. 67, 1167. O'Nell, S.C.O. and D.A. Eisner, 1990, A mechanism for the effects of caffeine on Ca 2 ÷ release during diastole and systole in isolated rat ventricular myocytes, J. Physiol. (London) 430, 519.
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Pessah, I.N., E.L. Durie, M.J. Schiedt and I. Zimanyi, 1990, Anthraquinone-sensitized Ca 2+ release channel from rat cardiac sarcoplasmic reticulum: possible receptor-mediated mechanism of doxorubicin cardiomyopathy, Mol. Pharmacol. 37, 503. Rabkin, S., M. Otten and P.I. Polimeni, 1983, Increased mortality with cardiotoxic doses of adriamycin after verapamil pretreatment despite prevention of myocardial calcium accumulation, Can. J. Physiol. Pharmacol. 61, 1050. Salviati, G. and P. Volpe, 1988, Ca 2+ release from sarcoplasmic reticulum of skinned fast- and slow-twitch muscle fibers, Am. J. Physiol. 254, C459. Sridhar, R., C. Dwivedi, 1. Anderson, P.E. Baker, H.M. Sharma, P. Desai and F. N. Engineer, 1992, Effects of verapamil on the acute toxicity of doxorubicin in vivo, J. Nat. Cancer Inst. 84, 1653. Temma, K., T. Akera, A. Chugun, H. Kondo, K. Hagane and S. Hirano, 1993 Comparison of cardiac actions of doxorubicin, pirarubicin and aclarubicin in isolated guinea-pig heart, Ear. J. Pharmacol. 234, 173. Temma, K., T. Akera, A. Chugun, S. Ozawa and H. Kondo, 1994, Cellular Ca 2+ loading and inotropic effects of doxorubicin in atrial muscle preparations isolated from rat or guinea-pig hearts, Ear. J. Pharmacol. 252, 173. Thompson, R.B., K.D. Warber and J.D. Potter, 1990, Calcium at the myofilaments, In: Calcium and the Heart, ed. G A . Langer (Raven Press, New York) p. 127. Wen&, IR. and D.G. Stephenson, 1983, Effects of caffeine on Caactivated force production in skinned cardiac and skeletal muscle fibres of the rat, Pflugers Arch. 398, 210.
ETAP
Environmental Toxicology and Pharmacology 1 (1996) 131-139
Doxorubicin alters
Ca 2+
transients but fails to change contractile proteins
Ca 2+
sensitivity of
Kyosuke Temma a,,, Akihito Chugun a, Tai Akera b, Hiroshi Kondo a, Nagomi Kurebayashi c Department of Veterinary Pharmacology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Higashi 23-bancho, Towada-shi. Aomori 034, Japan b MSD Research Laboratories-Japan, Minato-ku, Tokyo 107, Japan c Department of Pharmacology, School of Medicine, Juntendo University, Bunkyo-ku, Tokyo 113. Japan Received 15 August 1995; revised 31 August 1995; accepted 1 December 1995
Abstract Doxorubicin produced a transient increase and a subsequent decrease in the amplitude of twitch contraction in myocytes isolated from guinea-pig heart and loaded with fura-2. These changes were associated with an increase and a subsequent decrease, respectively, in the amplitude of Ca 2+ transients (peak minus diastolic C a 2+ concentrations). Doxorubicin increased the diastolic Ca 2+ concentration with a concomitant shortening of the diastolic myocyte length. The time to peak Ca 2+ transients and the time to peak twitch contraction increasedin parallel. Doxorubicin failed to affect the Ca 2+ concentration-contraction curve in skirined fibers obtained from atrial muscle. We conclude that biphasic inotropic effects of doxorubicin result from biphasic changes in Ca 2+ transients, and that doxorubicin fails to alter C a 2+ sensitivity of contractile proteins. These findings are consistent with the hypothesis that doxorubicin enhances Ca 2+ release and impairs Ca z+ uptake by the sarcoplasmic reticulum. Keywords: Doxorubicin; Ca 2+ transient; Ca 2 + sensitivity of contractile proteins; Myocyte; Skinned fiber
I. Introduction Doxorubicin, an efficacious anticancer drug, causes a dose-dependent and often lethal cardiotoxicity. The exact mechanism responsible for cardiotoxic actions of doxorubicin is unknown. Olson et al. (1974) by using an atomic absorption spectrophotometry and Combs et al. (1985) from the 45Ca2+ uptake studies showed that doxorubicin increases intracellular Ca 2+ concentration ([Ca 2+ ]i). These investigators postulated that an increase in [Ca 2+ ]i may be the cause of doxorubicin-induced cardiotoxicity. Doxorubicin restored the slow action potentials in isolated chickheart preparations exposed to tetrodotoxin (Azuma et al., 1981) suggesting that doxorubicin stimulates sarcolemmal C a 2+ channels. Moreover, doxorubicin elevates the resting tension in isolated chick heart preparations (Azuma et al., 1981) indicating that heart muscle cells exposed to doxorubicin are overloaded with Ca 2+. Kusuoka et al. (1991)
Abbreviations: IOC, index of contraction; ([Ca 2 + ]i, intracellular Ca 2+ concentration; MOPS, 3-(N-morphinolino)propanesulfonic acid * Corresponding author. Tel.: Japan-176-23-4371; Fax: Japan-176-238703. 1382-6689/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved 1-6
SSDI 1 3 8 2 - 6 6 8 9 ( 9 5 ) 0 0 0 2
showed that doxorubicin increases [Ca2+]i in ventricular muscle preparations obtained from ferret hearts. Several investigators, however, reported contradicting results. For example, Klugmann et al. (1981), Rabkin et al. (1983) and Sridhar et al. (1992) showed that verapamil failed to protect the animals from doxorubicin-induced cardiotoxicity, and instead increased the mortality. Consistent with these findings, Bernardini et al. (1986) and Jensen (1986) reported that doxorubicin inhibits Ca 2+ influx across the cell membrane. We recently observed that doxorubicin decreases, instead of increases, the amplitude of C a 2+ transients (peak minus diastolic [Ca 2+ ]i) in fura2-loaded myocytes isolated from guinea-pig heart (Jiang et al., 1994b). Pretreatment with verapamil failed to modify the effect of doxorubicin to depress C a 2+ transients indicating that doxocubicin-induced depression of myocardial functions is not secondary to the C a 2+ overload caused by enhanced Ca z+ influx. Many inotropic drugs alter the developed tension primarily by changing the amplitude of C a 2+ transients (Thompson et al., 1990). Several of these drugs, however, alter the affinity of cardiac contractile proteins for Ca 2+, thereby modifying the magnitude of the inotropic effects
K. Temma et al. / Environmental Toxicology and Pharmacology I (1996) 131-139
132
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K. Temma et al. / Environmental Toxicology and Pharmacology I (1996) 131-139
(Konishi et al., 1984; Endoh and Blinks, 1988; Gwathmey and Hajjar, 1990; Ferroni et al., 1991; Bosnjak et al., 1992). In the present study, the effects of doxorubicin on Ca 2+ transients and on Ca 2+ sensitivity of contractile proteins were examined. In isolated heart muscle preparations, relatively high concentrations of doxorubicin ( 1 0 0 - 2 0 0 /xM) cause a transient positive and a subsequent negative inotropic effect (Hagane et al., 1988; T e m m a et al., 1994). The negative inotropic effect of high concentrations of doxorubicin observed in isolated heart muscle preparations in vitro is likely to be mediated by the same mechanism responsible for the cardiotoxicity observed in vivo after a prolonged exposure to a lower concentration of doxorubicin in plasma (Hagane et al., 1988; Olson and Mushlin, 1990; Agata et al., 1991; Temma et al., 1994). The results of the present study show that the positive and negative inotropic effects of doxorubicin are caused by an increase and a decrease in the amplitude of Ca 2+ transients, and that doxorubicin fails to alter Ca 2+ sensitivity of the contractile proteins.
2. M a t e r i a l s a n d m e t h o d s
2.1. Single cell preparations
Male Hartly guinea pigs, weighing approximately 400 g, were used in this study. Cardiac myocytes were obtained from ventricular muscle by using collagenase as previously described (Jiang et al., 1994b). The myocytes were loaded with fura-2 by incubating at 30°C for 30 min in a Hepes buffer solution containing 1-1.5 /zM fura2 / A M (Molecular Probes, Eugene, OR, USA) and bovine serum albumin (20 m g / m l ) . The myocytes were washed 3 times to remove extracellular fura 2 / A M , and stored at room temperature (18-23°C) up to 4 h before use. 2.2. Measurements o f [ C a 2 + ] i concentrations and cell motions
The methods used to record and analyze Ca 2+ transients in fura-2 loaded myocytes were similar to those previously described (Akera et al., 1990; Koyama et al., 1991; Jiang et al., 1994a, 1994b). In the present study, however, we used a CCD video camera with the picture elements vertically reduced to one fourth (slit-scan video camera; C-2400-77, Hamamatsu Photonics, K.K., Hamamatsu, Japan). This camera is capable of recording fluores-
133
cence images at 4 ms intervals. Myocytes were placed in an open chamber with a thin quartz-glass bottom (0.2 ml in volume) on the stage of an inverted epifluorescence microscope (Nikon TMD, Nihon Kogaku, Tokyo, Japan). Myocytes were superfused at a flow rate of 0.1 m l / m i n with a Krebs-Henseleit bicarbonate buffer solution containing (mM); 118 NaCI, 27.2 NaHCO 3, 4.8 KCI, 1.2 MgSO 4, 1.2 CaCI2, 1.0 KH2PO4, 11.1 glucose and 2.5 sodium pyruvate. Myocytes were electrically stimulated at 2 Hz at 25°C by means of a pair of platinum electrodes for electrical field stimulation. In this system, a microcomputer controls the following: an electrical stimulator (SEN-3301, Nihon Kohden Kogyo, Tokyo, Japan), the timer for the CCD slit-scan video camera and a filter controller. The filter controller was used to excite myocytes with the ultraviolet light of either 340 or 380 nm wavelength using a 100 W xenon arc lamp. Fluorescence images resulting from 340 nm excitation were recorded at 500 nm at 4 ms intervals for 533 ms (33 ms before to 500 ms after the electrical stimulation). A total of 133 images were taken during one cycle of the Ca 2+ transient. Immediately after a set of fluorescence images were taken with 340 nm excitation, the process was repeated with 380 nm excitation. Fura-2 loading was minimized to reduce the chelation of intracellular Ca 2+. The intensity of the ultraviolet light was reduced using 30% and 50% neutral density filters to avoid damages to myocytes. The filters were placed before and after the 340 or 380 nm excitation filter. To obtain clear pictures, four fluorescence signals were averaged. The images were digitalized and stored in an Argus-50 system (Hamamatsu Photonics, Hamamatsu, Japan) for later analyses. The ratio of fluorescence intensities observed at 340 and 380 nm excitation ( 3 4 0 / 3 8 0 nm) was calculated at each pixel on the screen after subtracting the back ground fluorescence. The [Ca2+]i in myocyte was calculated as described in our previous reports (Akera et al., 1990; Jiang et al., 1994a, Jiang et al., 1994b). The calibration curves for Ca z+ concentrations were obtained using the 'activation solution' for skinned fibers (see below). Fura-2 pentapotassium salt (final concentration, 10 /xM) was added to the activation solution. The activation solution is a Ca2+-EGTA buffer solution containing 22 nM to 2.36 M Ca 2÷ (pCa from 7.66 to 5.63). Twitch contraction of myocyte was estimated from changes in myocyte length using the fluorescence ratio images on the monitor. The diastolic length of myocytes (immediately before the electrical stimulation, i.e., time
Fig. 1. Initiation, propagation and restoration of Ca2+ transients and the motion of myocytes before (A) and after doxorubicin treatment (B). Fura-2 loaded myocytes were stimulated at 2 Hz at 25°C. After a 30 rain equilibration period, fluorescence images were recorded at 500 nm with excitation wave lengths of 340 or 380 nm (A). Subsequently, 100 ~M doxorubicin (final concentration) was added to the medium. The fluorescenceimages were recorded 60 rain later (B). The number at the upper left comer of each panel represents the time in msec after electrical stimulation. Typical pictures from seven experiments.
K Temma et al. / Environmental Toxicology and Pharmacology I (1996) 131-139
134 [Ca2*l i (nM) 900
I' I
Length (ixm) '
'
'
'
i 1°2 I
~
~
0
100
~
~
200
300
loo 7oo 99 600
98
500
97 96
400 95 300
94
200
93 0
100
200
300
400
500
Time (msec)
400
500
Time (msec)
Fig. 2. The time course of Ca 2+ transients and twitch contractions. Fura-2 loaded myocytes were stimulated at 2 Hz at 25°C. After the myocytes were incubated with ([7, II) or without (©, O ) 100 /xM doxorubicin for 60 min, fluorescence images were recorded at 500 nm with excitation wave lengths of 340 or 380 nm. The values for [Ca2+ ]i and myocyte length were plotted against time after electrical stimulation. The left panel represents [Ca 2+ ]i, the right panel represents the length of the myocytes. Each point represents the mean of seven experiments.
recorder (model WI 840G; Nihon Kohden Kogyo, Tokyo, Japan). The Ca 2+ concentration-contraction curves for the skinned fiber were obtained using a series of Ca2+-EGTA buffer solutions (activation solutions) having the Ca 2÷ concentration of between 47 nM and 64.57/xM (pCa from 7.33 to 4.19). The solutions were prepared by mixing basic solutions A and B in varying portions. Solution A contained (mM); 10 EGTA, and solution B; 10 EGTA and 10 calcium methanesulfonate, respectively. In addition, both solutions contained (mM); 20 3-(N-morphinolino)propanesulfonic acid (MOPS), 4.3 Na2ATP and 5.0 magnesium methanesulfonate. The pH value was adjusted to 7.0 using KOH. Ionic strength was adjusted to 0.17 with potassium methanesulfonate. The skinned fiber was exposed to one of the activation solutions for 2 - 4 min, and then relaxed using the relaxation solution containing (mM); 20 MOPS, 2.0 EGTA, 4.3 Na2ATP, 112.7 potassium methanesulfonate and 5.0 magnesium methanesulfonate. The next activation of the skinned fiber was performed 4 - 5 min later.
2.4. Chemicals and statistical analyses zero in Fig. 1B and Fig. 2, right panel) decreased with time in the presence of 100 /xM doxorubicin. To compensate the changes in diastolic myocyte length, changes in the myocyte length resulting from twitch contractions were expressed as percentages of the diastolic length observed before each contraction. The twitch contraction was expressed as the index of contraction (IOC) calculated as follow (Jiang et al., 1994a, Jiang et al., 1994b): IOC =
myocyte length at peak contraction ) 1diastolic myocyte length × 100
Collagenase (type A, isolated from Clostridium histolyticum) and doxorubicin (Adriacin) were purchased from Boehringer Mannheim (Mannheim, Germany) and Kyowa Hakko Kogyo (Tokyo, Japan), respectively. All other chemicals used were of reagent grade. Statistical analyses were performed with Student's t test. The criterion for statistical significance is a P value of less than 0.05.
3. Results
(1)
3.1. Ca 2 + transients and twitch contractions observed in myocytes
2.3. Skinned fiber preparations The skinned fibers (150-200 /xm in diameter; 2 - 3 mm in length) were prepared from guinea-pig hearts (male Hartly guinea pigs, weighing approximately 400 g) as previously described (Kitazawa, 1984) and treated with 1% ( v / v ) Triton X-100 for 30 min at 24°C to completely disrupt cellular membranes (Wendt and Stephenson, 1983; Kurebayashi and Ogawa, 1991). One end of the skinned fiber was fixed to the bottom of the chamber with a pin and the other end was mounted on a stainless steel hook that was connected to an isometric force displacement transducer (model TB-625T; Nihon Kohden Kogyo, Tokyo, Japan). The skinned fiber was stretched to a length corresponding to an average sarcomere length of approximately 2.4 /zm. The sarcomere length was monitored using a microscope. Changes in contraction were continuously recorded using a force-displacement transducer and a pen
Fura-2 loaded ventricular myocytes of guinea-pig hearts were superfused with a Krebs-Henseleit bicarbonate buffer solution at 25°C and stimulated at 2 Hz. After a 30 min equilibration, control fluorescence images were recorded. Under these experimental conditions, twitch contractions and Ca 2÷ transients were stable for next 120 min (Koyama et al., 1991; Jiang et al., 1994a, Jiang et al., 1994b). As shown in Fig. 1A, the Ca 2+ transients started 8-12 ms after the electrical stimulation. Initial increases in [Ca2+ ]i were observed at several sites of the myocyte, and rapidly spread to the entire cell volume 30-40 ms after the electrical stimulation (Fig. 1A). The left panel of Fig. 2 shows the time course of a Ca 2+ transient. The Ca 2+ transients reached the peak at 93 ms (the mean of seven myocytes) after the electrical stimulation, and then declined to the diastolic level (Fig. 2, left panel). The start and the peak of twitch contraction was delayed from those
135
K. Temma et al. / Envi ronmental Toxicology and Pharmacology 1 (1996) 131-139
Table I Effects of doxorubicin on Ca 2+ transients in myocytes isolated from guinea-pig hearts
Table 2 Effects of doxorubicin in Ca2+ transients and twitch contraction in myocytes isolated from guinea-pig hearts
Time of doxorubicin exposure (min)
Size of Ca2+ transients
Size of twitch contraction
Time to peak contraction
(%)
(%)
(ms)
100 115.1 ±7.0 97.7±9.2 74.8 ± 12.1 38.1 ± 10.2 a
100
0 8 30 60 90
Diastolic [Ca2+ ]i
Peak [Ca2+ ]i
Time to peak [Ca2÷ ]i
(nM)
(nM)
(ms)
Time of doxorubicin exposure (min)
259± 14 292± 11 346± 18 a
840±61 957±62 898 ±47 841 ±28 778±52
93±7 112±7 a 149_+ 12 a 174± 11 a 190±9 a
0 8 30 60 90
461 ±53
a
533 _+49 a
a
After myocytes were equilibrated at 25°C for 30 min at 2 Hz stimulation, fluorescence images were taken at time zero. Subsequently, doxorubicin (final concentration 100 p~M) was added to the incubation medium. Fluorescence images were recorded at 8, 30, 60 and 90 min later. Values are the mean ± SE. of seven experiments. " Significantly different from values observed at time zero (P < 0.05, paired t test).
o f the C a 2+ transients (Fig. 2). The cell started to shorten at 50 ms and reached the peak at 186 ms (Fig. 2, right panel). A t the peak o f twitch contraction, m y o c y t e s shortened to 92.6 + 1.3% ( n = 7) o f the diastolic length. A s s u g g e s t e d by m a n y investigators, d o x o r u b i c i n increases the diastolic [Ca2+]i ( O l s o n et al., 1974; C o m b s et al., 1985; A z u m a et al., 1981; K u s u o k a et al., 1991). A t 60 min after the addition o f 100 /.tM d o x o r u b i c i n (final concentration), the m y o c y t e s h o w n in Fig. 1B at t i m e zero was brighter than the s a m e m y o c y t e s h o w n in Fig. 1A. The diastolic [Ca2+]i i n c r e a s e d with t i m e in the presence of d o x o r u b i c i n (Table 1). T h e patterns o f the initiation, spreading and restoration o f the Ca 2+ transient were unc h a n g e d by d o x o r u b i c i n (Fig. 1B). D o x o r u b i c i n , h o w e v e r , p r o l o n g e d the time to p e a k Ca 2+ transient and d e l a y e d the decrease in [Ca 2+ ]i f o l l o w i n g the peak (Fig. 2, left panel). A s s o c i a t e d with these c h a n g e s in [Ca2+]i, the diastolic length o f the m y o c y t e s d e c r e a s e d and the time course of the twitch contraction was d e l a y e d in the presence o f 100 p,M d o x o r u b i c i n (Fig. 2, right panel). T h e s e effects o f d o x o r u b i c i n on [Ca2+]i and twitch contraction increased gradually o v e r a 90 m i n e x p e r i m e n t a l period with similar time courses (Tables 1 and 2). D o x o r u b i c i n m o n o t o n o u s l y increased the diastolic [Ca2+ ]i and the t i m e to p e a k C a 2+ "transients; h o w e v e r , the effect o f d o x o r u b i c i n on the p e a k Ca 2+ transient was biphasic (Table 1). T h e p e a k C a 2+ transient increased at 8 min and then decreased. T h e size o f the C a 2+ transients and the index o f contraction ( I O C ) were calculated to allow a direct c o m p a r i s o n b e t w e e n d o x o r u b i c i n - i n d u c e d changes in the a m p l i t u d e s o f C a 2+ transients and twitch contractions (Table 2). T h e size o f C a 2+ transients and I O C initially increased, and then gradually decreased. The t i m e course o f c h a n g e s in the size o f C a 2+ transients was a l m o s t identical to that o f c h a n g e s in the size o f twitch contractions. T h e plots o f the size o f C a 2+ transients against the I O C g a v e a straight line (Fig. 3). T h e r e was a
186_+2 194± 12 238± 16 a 245 ± 14 a 271±5 a
12.1 96.3±11.6 70.9 ± 6.5 a 48.9:t:10.3 a
110.1
4-
The size of the Ca 2+ transients was estimated as [Ca2+ ]i at the peak minus [Ca2+ ]i at diastolic state. The size of twitch contractions was estimated from the IOC values (see Eq. 1). These values are expressed as percentages of the control value observed at time zero. Values are the mean±S.E, of seven experiments, a Significantly different from the control values observed at time zero (P < 0.05, paired t test).
h i g h d e g r e e of correlation b e t w e e n the size o f C a 2+ transients and the size o f twitch contractions ( y = 0.809, 35 points, P < 0.001) 3.2. Ca 2 + s e n s i t i u i t y o f s k i n n e d f i b e r s
Sensitivity o f contractile proteins to C a 2+ was e x a m ined using skinned fibers isolated from left atrial m u s c l e o f g u i n e a - p i g heart and treated with Triton X-100. Atrial muscle, instead o f ventricular muscle, was used to obtain better skinned fiber preparations. T h e t o x i c o l o g i c a l effects
lOG (% of control) 160
~
i
I
I
I
Q
140
I I 00 0/,~
120 IO0 8O 6O 4O 2O 0 0
t
i
1
[
I
I
20
40
60
80
100
120
140
160
Size of Ca2" transient (% of control)
Fig. 3. The relationship between the size of Ca 2+ transients and twitch contractions observed in the presence of doxorubicin. Fura-2 loaded myocytes were stimulated at 2 Hz at 25°C. After a 30 min equilibration, myocytes were exposed to 100 p,M doxorubicin. The size of Ca2+ transients (peak [Ca2+ ]i minus diastolic [Ca2+ ]i) and IOC were calculated at 0, 8, 15, 30, 60 and 90 min later. Each value for the size of Ca2+ transient and IOC was expressed as a percentage of the values observed at time zero (immediately before the doxorubicin addition). Each point represents an individual time point observed in a total of seven preparations.
K. Temma et al. / Environmental Toxicology and Pharmacology 1 (1996) 131-139
136 Tension (%) 120 j
I
I
1
T 10
Ca 2+ activation of the contraction. The value observed in the presence of 100 /xM doxorubicin was 1.25 _ 0.11 ~ M (n = 5) which was not significantly different from the value for the second curve observed in the absence of doxorubicin. These results indicate that Ca 2+ sensitivity of the skinned fibers is not altered by a 60 min exposure to 100 /xM doxorubicin.
80 60 40 20 0 0.1
4. Discussion 100
Ca 2+ concentration fitJM)
Fig. 4. Effects of doxorubicin on the Ca 2+ concentration-response curves in skinned fibers. Skinned fibers obtained from atrial muscle guinea-pig hearts were incubated with 1 ( v / v ) % Triton X-100 for 30 min to disrupt intracellular membranes. Subsequently, a Ca 2+ concentration-contraction curve was generated for control preparations (O). After a 60 min incubation in the absence ([]), or presence of 100 mM doxorubicin (zx), a Ca 2+ concentration-contraction curve was generated again. Values observed at 64.59/~M Ca 2+ in the first curve were set at 100%. Values at each point were calculated for each preparation and then averaged.
of doxorubicin and the effects of doxorubicin on isolated heart muscle preparations are similar in atrial and ventricular muscle (Olson and Mushlin, 1990; Boucek et al., 1993; Temma et al., 1994) justifying the use of atrial muscle in this study. A high concentration of caffeine (50 mM) failed to cause a contraction in these fibers (data not shown) indicating that all cellular membranes, such as sarcoplasmic reticulum, were completely disrupted. Contraction of skinned fibers in the relaxing solution was stabilized within 10 min. At this point, the solution was switched to the activation solution containing an indicated concentration of Ca 2÷ ranging from 47 nM to 64.57/.tM to generate the Ca 2+ concentration-contraction curves. After the first Ca 2+ concentration-response curve was obtained, the skinned fiber was incubated for 60 rain in the presence or absence of 100/xM doxorubicin. Subsequently, the skinned fiber was exposed to the activation solution to produce the second Ca 2+ concentration-response curve. An exposure to the activation solutions produced a typical sigmoidal concentration-response curve in skinned fibers (Fig. 4). In the presence of a high concentration of Ca 2+, the force of contraction was 7.66 _ 0.71 mg (n = 9) in the first concentration-response curve. The maximal value of the twitch contraction for the second curve observed in the absence of doxorubicin was 78.1 ___5.6% (n = 4) of the corresponding value for the first curve. The EDs0 values for C a 2+ w e r e 1.34 _+ 0.03 /,tM (n = 9) and 1.33 + 0 . 0 9 / z M (n = 4) for the first and the second curve, respectively. The addition of 100 /xM doxorubicin failed to alter the maximal response of the second curve (81.8 _ 4.3%, n = 5) compared to the corresponding value of the second curve observed in a doxorubicin-free solution. Moreover, doxorubicin failed to alter the EDso values for
In an earlier study (Temma et al., 1994), we observed that in isolated atrial muscle preparations obtained from guinea-pig hearts and incubated at 36°C, 200 /zM doxorubicin caused a biphasic positive inotropic effect: specifically, an early and a late phase positive inotropic effect. However, the pattern of the inotropic effects was markedly influenced by the experimental conditions. At low temperature or at a high stimulation frequency, doxorubicin caused a gradual negative inotropic effect, instead of a positive inotropic effect, after the initial transient positive inotropic effect. It is postulated that the decrease in developed tension of heart muscle may be causally related to the doxorubicin-induced cardiotoxicity. In the present study, therefore, relatively low temperature and high stimulation frequency were chosen. Using myocytes at 24°C under 2 Hz stimulation, doxorubicin caused a near identical pattern of inotropic effects with that observed at a low temperature and 2 Hz stimulation in isolated left atrial muscle preparations. In these myocytes, size of peak Ca 2+ transients also increased transiently and then decreased with the same time course as that of twitch contraction. The increase and subsequent decrease in the size of Ca R+ transients and twitch contraction showed a good linear correlation. In addition, at a higher temperature (34°C), the significant decrease in twitch contraction and size of Ca 2÷ transients observed at 24°C after the transient increase, was almost abolished (our recent observations, data not shown). At high Ca 2÷ (2.4 mM), the decreases in twitch contraction and the size of Ca 2+ transients were measurable. Further, doxorubicin caused a monotonous increase in the diastolic [Ca 2÷ ]i and the time to peak Ca z+ transients. These changes resulted in monophasic changes in the diastolic myocytes length and the time to peak twitch contraction. Results of the present study are consistent with that of earlier experiments using isolated left atrial muscle preparations of guinea-pig hearts (Temma et al., 1993, 1994), and clearly show that changes in the size of Ca 2÷ transients and in the diastolic [Ca 2÷ ]i are responsible for doxorubicin-induced changes in developed and resting tension, respectively. In the present study, myocytes obtained from guinea-pig heart were stimulated at 2 Hz at 25°C. E s t i m a t e d [Ca2+]i at diastolic state and the peak of Ca 2+ transients was 259 and 840 nM, respectively. The time to peak Ca 2÷ transient
K. Temma et al. / Environmental Toxicology and Pharmacology I (1996) 131-139
was 93.4 ms after the electrical stimulation and the peak twitch contraction was observed at 186 ms. These values appear reasonable for guinea-pig heart muscle preparations stimulated at 2 Hz at 25°C. Moreover, the distribution of [Ca2+]i at diastolic state and also at the peak of the Ca z+ transient was relatively uniform. These results would indicate that potential problems associated with the use of fura-2 for the estimation of [Ca2+]~ in isolated myocytes (Frampton et al., 1991) are minor in the present study. The Ca 2÷ concentration-contraction curves observed in the skinned fibers show a typical sigmoidal curve. Doxorubicin failed to alter the Ca 2÷ concentration-contraction relationships. Under the conditions studied, these results indicate that the inotropic effects of doxorubicin are not modified by changes in Ca 2+ sensitivity of the contractile proteins. Instead, changes in Ca 2+ transients are the cause of the positive and negative inotropic effects of doxorubicin. Recently, Boucek et al. (1993) reported the lack of doxorubicin effects on the concentration-response curves for Ca 2÷ in skinned fibers isolated from rabbit hearts with a lower concentration of doxorubicin and a shorter exposure time. Doxorubicin caused a significant prolongation of the time to peak Ca 2÷ transients confirming the results of our previous study (Jiang et al., 1994b). These results suggest that doxorubicin impairs the mechanism(s) that regulates [Ca2+]i in the cardiac muscle. These mechanisms include an influx or an effiux mechanism across the cell membrane, or an uptake or release mechanism across the sarcoplasmic reticulum membranes. Doxorubicin has been reported to decrease the Ca 2÷ influx across the sarcolemma (Bernardini et al., 1986; Jensen, 1986). Similar to doxorubicin, the Ca 2÷ channel blocker, verapamil, delayed the time course of Ca 2÷ transients in myocytes isolated from guinea-pig hearts (Jiang et al., 1994b). Verapamil, however, decreased the diastolic [CaZ+]i (Jiang et al., 1994b) whereas doxorubicin increased the d i a s t o l i c [Ca2+]i in the present study. These results, therefore, indicate that a decrease in the Ca 2+ influx is unlikely to be the cause of doxorubicin-induced prolongation of the time to peak Ca 2÷ transients. Doxorubicin has been reported to stimulate Ca 2÷ release from the sarcoplasmic reticulum resulting from its specific binding to the Ca 2+ release channels and ensuing increases in open probability of the channels (Kim et al., 1989; Holmberg and Williams, 1990; Ondrias et al., 1990; Pessah et ai., 1990). Boucek et al. (1993) reported a doxorubicin-induced increase in Ca 2+ release in sarcoplasmic reticulum and mitochondria membrane intactskinned fibers. These actions of doxorubicin may resemble actions of low concentrations of ryanodine (Meissner, 1986; Lattanzio et al., 1987). If doxorubicin causes a continuous leak release of Ca 2÷ from the sarcoplasmic reticulum, and when the Ca 2+ release exceeds the reserve capacity of the Ca 2÷ pump of the sarcoplasmic reticulum, the increase in the diastolic [Ca2+]i and the prolongation
137
of the time to peak C a 2+ transients would be anticipated. These results were observed in the present study. Liu and Vassalle (1991) recently suggested that 10-50 /zM doxorubicin impairs Ca 2+ uptake by the sarcoplasmic reticulum in myocytes isolated from ventricular muscle of guinea-pig hearts. Enhanced Ca 2+ release and inhibited Ca z+ uptake by the sarcoplasmic reticulum explain the observed increase in the diastolic [Ca2÷]i and the prolongation of the time to peak of Ca 2+ transients. In addition, the decrease in Ca 2+ uptake may also account for the slower decay of the C a 2+ transients and of the twitch contraction by doxorubicin. In this regard, the actions of doxorubicin resemble some actions of caffeine. Caffeine binds to the ryanodine receptors, increases the open probability of C a 2+ channels, and stimulates Ca 2+ release in membrane preparations obtained from cardiac or skeletal muscle sarcoplasmic reticulum (Salviati and Volpe, 1988; Kim et al., 1989; Ondrias et al., 1990; Pessah et al., 1990). Caffeine increased the diastolic [Ca2+]i (O'Nell and Eisner, 1990; Jiang et al., 1994b) and increased the time to p e a k C a 2+ transients (Konishi et al., 1984). These reports further support the concept that the cause of the prolongation of the time to peak Ca 2+ transients and the increase in the diastolic [Ca2+]i result from the impairment of C a 2+ uptake and release mechanisms in the sarcoplasmic reticulure. It should be noted that the prolongation of the time to peak Ca z+ transients and the increase in diastolic [Ca2+]i developed with almost the same time course in the present study. As described above, myocyte length shortened concomitant with an increase in the diastolic [Ca2+] r Although in isolated heart muscle preparations obtained from chicks, the increase in resting tension was noticed by Azuma et al. (1981), the increase in resting tension by doxorubicin was not observed in isolated left atrial muscle preparations of guinea-pig hearts (Temma et al., 1994). The cause of this discrepancy between myocytes and atrial muscle preparations is presently unknown. An initial increase and a subsequent decrease in the size of C a 2+ transients caused by doxorubicin may also result from the augmentation of Ca 2+ release from the sarcoplasmic reticulum. An enhancement of the Ca 2+ release is likely to initially increase the size of Ca 2+ transients. Subsequently when the Ca 2+ release begins to exceed the capacity of the C a 2+ pump, the amount of releasable Ca 2+ stored in the sarcoplasmic reticulum would decrease. This would result in a decreased size of Ca 2+ transients that are triggered by membrane excitation. In addition, it has been reported that release of C a 2+ from sarcoplasmic reticulum is decreased by an increase in diastolic Ca 2+ due to an inactivation of Ca 2+ release channels (Fabiato, 1983). It has been reported that Ca 2+ overloading of the cardiac cells causes a decrease in C a 2+ transients. Doxorubicin has been shown to cause an enhancement of slow channels, or a decrease in Na +, Ca 2÷ exchange reactions or Ca 2+ pump activities (Azuma et al., 1981; Caroni et al.,
138
K. Temma et al. / Environmental Toxicology and Pharmacology 1 (1996) 131-139
1981: H a r a d a et al., 1990). T h e r e f o r e , it is s u g g e s t e d that d o x o r u b i c i n m a y c a u s e C a 2+ o v e r l o a d i n g as a r e s u l t o f the a b o v e effects, a n d t h e r e b y c a u s e c a r d i o t o x i c i t y . H o w e v e r , d o x o r u b i c i n d i m i n i s h e d , i n s t e a d o f a u g m e n t e d , the oscillatory a f t e r c o n t r a c t i o n p r o d u c e d b y a toxic c o n c e n t r a t i o n o f o u a b a i n ( B i n a h et al., 1983). B e c a u s e , the o s c i l l a t o r y a f t e r c o n t r a c t i o n r e s u l t s f r o m C a 2÷ o v e r l o a d i n g o f m y o c a r d i a l cells, t h e s e r e s u l t s s u g g e s t that d o x o r u b i c i n - i n d u c e d dep r e s s i o n o f the C a 2+ t r a n s i e n t is u n l i k e l y to r e s u l t f r o m the C a 2÷ o v e r l o a d i n g . I n s t e a d it is e x p e c t e d t h a t d o x o r u b i c i n m a y c a u s e a d e p l e t i o n o f C a 2+ stored in t h e s a r c o p l a s m i c r e t i c u l u m w h i c h t h e n in turn c a u s e s a d e c r e a s e in t h e r e l e a s e o f C a 2÷. U n d e r c o n d i t i o n s that r e s u l t in a d e c r e a s e in C a 2+ r e l e a s e f r o m s a r c o p l a s m i c r e t i c u l u m , C a 2+ c h a n nel a n t a g o n i s t s m a y e v e n facilitate d o x o r u b i c i n - i n d u c e d c a r d i o - t o x i c i t y ( K l u g m a n n et al., 1981; R a b k i n et al., 1983; S r i d h a r et al., 1992). In s u m m a r y , d o x o r u b i c i n initially i n c r e a s e d a n d s u b s e q u e n t l y d e c r e a s e d the size o f the C a 2+ t r a n s i e n t s in m y o c y t e s i s o l a t e d f r o m g u i n e a - p i g hearts. T h e d i a s t o l i c [ C a 2 + ] i a n d t h e t i m e to p e a k C a 2÷ t r a n s i e n t s i n c r e a s e d m o n o t o n o u s l y . T h e s e c h a n g e s r e s u l t e d in a b i p h a s i c c h a n g e s in the size o f t w i t c h c o n t r a c t i o n , a n d m o n o p h a s i c d e c r e a s e in d i a s t o l i c m y o c y t e l e n g t h a n d an i n c r e a s e in the t i m e to p e a k t w i t c h c o n t r a c t i o n . D o x o r u b i c i n failed to a l t e r the C a 2÷ s e n s i t i v i t y o f s k i n n e d atrial m u s c l e f i b e r s o b t a i n e d f r o m g u i n e a - p i g heart. T h e p o s i t i v e a n d n e g a t i v e i n o t r o p i c e f f e c t s o f d o x o r u b i c i n are a c c o u n t e d f o r b y the c h a n g e s in C a 2+ t r a n s i e n t s . T h e s e effects o f d o x o r u b i c i n m a y b e e x p l a i n e d f r o m an i n c r e a s e in C a 2+ r e l e a s e f r o m the s a r c o p l a s m i c r e t i c u l u m a n d a n i n h i b i t i o n o f C a 2÷ uptake by this organelle.
Acknowledgements We thank Professor Yasuo Ogawa of Juntendo University f o r his h e l p a n d e n c o u r a g e m e n t s . T h i s s t u d y w a s partly s u p p o r t e d b y a G r a n t - i n - A i d for G e n e r a l S c i e n t i f i c R e s e a r c h (no, 0 4 6 6 0 3 3 0 ) f r o m the J a p a n e s e M i n i s t r y o f Education, Sciences and Culture.
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