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Effects of pirarubicin in comparison with epirubicin and doxorubicin on the contractile function in rat isolated cardiac muscles
Gen. Pharmac.Vol. 26, No. 6, pp.
Pergamon
133%1341, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved
0306-3623(94)003149
0306-3623195 $9.50 + 0.00
EfTects of Pirarubicin in Comparison with Epirubicin and Doxorubicin on the Contractile Function in Rat Isolated Cardiac Muscles SHIN-ICHI
HIRANO,*
NAOKI AGATA, HIROSHI TONE
HIROSHI
IGUCHI
and
Central Research Laboratories, Mercian Corporation, 9-1 Johnan 4 Chome, Fujisawa 251, Japan /Tel: 0466 35 1519; Fax: 0466 35 I5301 (Received 7 November 1994)
Abstract-l. We have examined the effects of pirarubicin (THP), compared with epirubicin (EPI) and doxorubicin (DXR), on the contractile function in papillary muscles isolated from rats. 2. In in vivo experiments, in which the rat was treated once a week for 4 weeks with DXR (total dose 10 mg/kg) and thereafter once a week for 4 weeks with THP, EPI or DXR (total dose 10 mg/kg), a positive instead of negative force-frequency relationship was observed in the muscles treated with EPI and DXR, but not with THP, and an increase in contractile response to extracellular Ca*+ was observed more markedly in the muscles treated with DXR than in those treated with EPI and THP. 3. In in vifro experiments, in which the muscle preparations were incubated with the drugs at 100 or 200 p M for 2 hr, EPI and DXR caused a negative inotropic effect and a prolongation of tension duration, while THP caused a slight positive inotropic effect and a slight prolongation of tension duration. 4. Furthermore, a decrease in the potentiated postrest contraction was observed more markedly in the muscles incubated with EPI and DXR at 200 yM than in those with THP. 5. These results suggest that both EPI and DXR show a cardiotoxicity by impairing the function of cardiac sarcoplasmic reticulum, and that the switching of the treatment from DXR to THP produces less impairing effects. Key Words: Pirarubicin,
epirubicin,
doxorubicin,
cardiac
INTRODUCTION Doxorubicin (DXR) is an anthracycline antibiotic which has been shown to be effective in the treatment of various types of tumor (Blum and Carter, 1974). However, congestive heart failure, cardiomyopathy and alteration in the electrocardiogram (ECG) have been observed following its administration to experimental animals and to patients (Lenaz and Page, 1976). To lower the cardiotoxicity of this antibiotic, great efforts were made to find new derivatives. Pirarubicin [(2”R)-4’-0-tetrahydropyranyladriamycin, THP] a derivative of DXR, was found to have a similarly potent antitumor effect (Matsushita et al., 1985) but lower cardiotoxicity than DXR (Danchev
*To whom GP 26,&N
all correspondence
should
be addressed.
muscle,
contractile
function,
cardiotoxicity
et al., 1979). In the previous paper, we have reported that THP had lower cardiotoxicity in hamsters than DXR from the standpoints of ECG and myocardial structure (Tone et al., 1986), and further that at higher concentrations it only slightly increased the contractile force of the left atria in guinea-pigs, probably mediated through the release of histamine (Agata et al., 1991; Hirano et al., 1991). Epirubicin (EPI), a stereoisomer of DXR, was also found to have a similarly potent antitumor effect (Arcamone et al., 1975; DiMarco et al., 1976) but lower cardiotoxicity than DXR itself (Suzuki et al., 1984). In the comparative study on cardiotoxicity of EPI and DXR, Suzuki et al. (1984) reported that EPI did not so much affect the myocardial structure in rabbits as DXR. In our preliminary experiments, however, we observed that EPI, as well as DXR,
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Shin-ichi Hirano et al.
markedly affected myocardial structure and function; long-term exposure to these two agents produced marked negative inotropic effects in the isolated left atria from guinea-pigs, and i.v. injection of these two agents produced a decrease in heart rate and ECG alterations in hamsters (Agata et al., 1991). Furthermore, the changes in the ECG and myocardial structure of rats induced by the DXR-pretreatment were augmented more greatly by the post-treatment with EPI and DXR than by that with THP (Hirano et al., 1994). The rat has proven to be a useful experimental animal for examining the mechanisms of anthracycline-induced cardiac muscle failure (Lazarus et al., 1980; Jensen et al., 1984; Jensen, 1986; Pelikan et al., 1986; Villani et al., 1991). Jensen (1986) reported that the changes in contractile function of papillary muscles obtained from rats treated with DXR once a week for 8 weeks showed the signs of congestive heart failure after long-term treatment with DXR. In the present study, to elucidate the precise differences in the cardiotoxic actions of anthracyclines, we examined the effects of THP, compared with EPI and DXR, on the contractile response of rat papillary muscles in two different series of experiments. The first one was performed in vivo in the muscle preparations obtained from the rats, which were treated with DXR for 4 weeks and thereafter with THP, EPI or DXR for 4 weeks, because in the clinical cases THP and EPI are often used in patients previously treated with DXR. The second one was performed in the muscles incubated in vitro with the drugs to investigate the direct action of THP in comparison with EPI and DXR. The results of the present study suggest that both EPI and DXR may impair the function of cardiac sarcoplasmic reticulum, and that THP possesses less impairing effects. MATERIALS
AND METHODS
Experimental animals Female Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan) with a starting weight of 170-220 g were used in in vivo experiments. The animals were observed in quarantine for 10 days before drug administration, to document overall good health. In in vitro experiments, female Sprague-Dawley rats (Japan SLC) weighing 250-330g were used. The age of the animals was almost the same as that of rats, used in the in vivo experiments, at the end of the post-treatment period. Contractile tension Rats were killed for isolated tissue study by cervical dislocation and then exsanguinated. The hearts were
rapidly excised, trimmed of both atria, weighed and placed in modified Kreb’s solution for isolation of right ventricular papillary muscles. The right ventricular papillary muscles were mounted in a waterjacketed tissue bath between a fixed rod and the level arm of a force-displacement transducer (TB-651T, Nihon Kohden, Tokyo, Japan) for measurements of isometric contractile tension. Maximum rate of tension development (dT/dt,,,) was obtained by electrically differentiating contractile tension with a differentiator (ED-601G, Nihon Kohden). The tissues were incubated at 31°C with a modified Kreb’s solution of the following composition (in mM concentrations): NaCl, 137; KCl, 3.0; MgCl,, 1.0; CaCl,, 1.0; NaHCO,, 12.0; and glucose, 5.5. The solution was gassed with 95% 0,-Y!! COZ. The resting tension was adjusted to 0.5 g and the muscles were initially stimulated electrically by square wave pulses of 5 msec duration at a frequency of 1 Hz and an intensity of 20% above the contractile threshold via parallel platinum field electrodes. The stimuli were delivered from an electronic stimulator (SEN-7203, Nihon Kohden) through an isolation unit (SS-302J, Nihon Kohden). Contractile tension and dT/dt,,, were recorded on a pen-recorder (WT685G, Nihon Kohden) with a chart speed of 100 mmjsec. The isometric contractile parameters measured were peak isometric tension (PT), resting tension (T,), dT/dt,,, , time-to-peak tension (TPT), time for half-relaxation from PT (TR,,,) and tension duration (T,). In vivo experiments Effects of THP, EPI and DXR on the mechanical function of the papillary muscle preparations were evaluated using the modified method of Jensen (1986), who examined the contractile changes of papillary muscles isolated from the rats treated once a week for 8 weeks with 2.5 mg/kg of DXR. Drug treatment. DXR was previously administered i.v. to 56 rats at a dose level of 2.5 mg/kg once a week for 4 weeks. These rats were separated in 4 groups of 8 or 16 at the end of 4th week. Then, THP, EPI or DXR was administered i.v., respectively, to 16 rats at a dose level of 2.5 mg/kg once a week for 4 weeks, and physiological saline solution was also administered i.v. to 8 rats. The rats were observed through a post-treatment period of 2-3 weeks until they developed the signs of toxicity, such as ascites. Of the 16 rats treated with THP, EPI or DXR, 5-6 surviving rats were selected at random and killed for the isolated muscle experiments described below. Eight age-matched control rats were given i.v. injection of physiological saline solution once a week for 8 weeks, and also 6 rats were killed for the isolated muscle
Effects of anthracyclines on the contractile function in cardiac muscles experiments. The rats were weighed weekly and observed daily for general symptoms. Isolated muscle preparations. The right ventricular papillary muscle was removed from surviving rat at the end of post-treatment period. The papillary muscles were allowed to equilibrate for at least 1 hr before contractile measurements were taken. During this period, the resting length of the muscle was gradually increased until the maximum developed tension was obtained. The length of muscle at this point is defined as L,,,. The cross-sectional area of the papillary muscle was determined from measurement of L,,, and wet weight of the preparation. After the equilibration period, baseline contractile parameters were measured in the presence of 1.0 mM Ca2+, with the muscles being driven at a frequency of 1 Hz. Mean values of the various contractile parameters were obtained from about 20 individual twitch recordings for at least 10 min. The inotropic responsiveness of the preparations was then evaluated as mentioned below. Tissues were driven at a frequency of 0.1 Hz in a 1.OmM Ca2+-Kreb’s solution. Frequency-dependent changes in contractile activity were determined by increasing the stimulus frequency in discrete steps from 0.1 Hz to 0.2, 1.O and 2.0 Hz returning to 0.1 Hz at the end of a series. Three to four frequency-tension curves were obtained with each preparation. Then, the inotropic effect of varying the bath Ca2+ concentration (0.5, 1.0,2.0 and 4.0 mM Ca2+) was determined at a stimulation frequency of 0.1 Hz. The tissues were incubated for 15 min in Ca2+-free Kreb’s solution, and then allowed to equilibrate for at least 15 min in each Ca2+ solution before measurements were taken. For all parameters examined, the response of the muscle to Ca2+ was expressed as a percentage of the mean value determined in 0.5 mM Ca2+-Kreb’s solution (100%). In vitro experiments
Right ventricular papillary muscles were removed from untreated rats and equilibrated at a stimulation frequency of 1 Hz for 1 hr as described above. After the equilibration period, THP, EPI or DXR was applied, respectively, to the incubation medium. The final concentration of each drug was 100 or 200 PM. The vehicle (deionized water) was applied to the medium for control preparations. Relatively high concentration of each drug was used in these studies, so that their effects could be observed within 2 hr. In another series of experiments, after 1 hr exposure to each drug at 200 PM, potentiated postrest contraction was examined. The stimulator was turned off, and postrest contraction was elicited by stimulating the muscles again after various resting periods ranging from 5-60 sec.
1341
Drugs
THP was synthesized from daunorubicin in our laboratories. EPI and DXR were obtained from the Kyowa Hakko Kogyo Co. Ltd (Tokyo, Japan). These drugs were dissolved in physiological saline solution and in deionized water for in vivo and in vitro experiments, respectively. Statistical analysis
Results of the experiments were expressed as mean + SEM. The data were analyzed by Student’s paired or unpaired t-test and P < 0.05 was defined as significant. RESULTS In vivo experiments Clinical features. No dead animals were observed in the treatment with DXR + saline (control) during the entire observation period. One dead animal was observed in the treatment with DXR + THP, and eight with DXR + EPI or DXR + DXR. At the end of the 4th treatment, a suppression of body weightgain was observed in the rats pre-treated with DXR, when compared with those of saline. During the post-treatment period, the body weight also remained unchanged in the groups post-treated with saline and THP, while it decreased with EPI and DXR. As for general symptoms, all the rats pre-treated with DXR showed a ruffled coat and alopecia after 3rd posttreatment with saline or each drug, and some of these animals showed diarrhea. At necropsy, atrophy of the thymus and development of ascites were observed more markedly in the rats post-treated with EPI and DXR than in those treated with THP (data not shown). Effects on force-frequency relationships. Figure 1 shows force-frequency relationships in papillary muscle preparations, isolated from rats treated in vivo with each drug. The data were represented as an absolute value (g). The PT at a stimulation frequency of 0.1, 0.2 and 1.0 Hz was significantly smaller in the muscle preparations treated with DXR + saline (control), DXR + EPI or DXR + DXR when compared with saline + saline (untreated) group. Also, in the muscles treated with DXR + THP, the PT at 0.1 and 0.2 Hz was smaller than that of the saline + saline group, which was less prominent when compared with the case of DXR + saline, DXR + EPI and DXR + DXR. Figure 2 shows force-frequency relationships in papillary muscle preparations, which are represented as a percentage of the values obtained under stimulation at a frequency of 0.1 Hz. As shown in Figs 1 and 2, in the muscle preparations isolated from
Shin-ichi Hirano et al.
1342 0.8 0.7 0.8 3
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0.4 0.3 0.2 0.1 0
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Frequency (Hz) Fig. I. Effects on force-frequency relationships in papillary muscle preparation isolated from rats, pre-treated for 4 weeks with DXR (total dose 10 mg/kg, i.v.) and then further treated for 4 weeks with THP, EPI or DXR (total dose lOmg/kg of each), respectively. Tissues were driven at a frequency of 0.1 Hz in a 1.0 mM Ca*+-Kreb’s solution. Frequency-dependent changes in contractile activity were determined by increasing the stimulus frequency in discrete steps from 0.1 Hz to 0.2, 1.0 and 2.0 Hz, returning to 0.1 Hz at the end of a series. The data were represented as an absolute value (g), PT is peak tension. Each point represents the mean (f SEM) of 5d experiments: 0, saline + saline; 0, DXR + saline; A, DXR + THP; 0, DXR + EPI; 0, DXR + DXR. *P c 0.05 and **P < 0.01 vs saline + saline, respectively. *P < 0.05 and **P i 0.01 vs DXR + saline, respectively.
As shown in Fig. 4, Td increased with increasing extracellular Ca2+ concentrations in all the groups pre-treated with DXR, while it rather tended to ’ decrease in the saline + saline group. In 1.0-4.0 mM Ca2+, the increase in T, was significantly greater in the DXR + DXR group than in the saline + saline group. At a high concentration of Ca2+ (4.0 mM), the increases in Td were greater in all the group pretreated with DXR than in the saline + saline group. No statistical difference was observed in TR,,, among all the groups, although an increase in TR,,, tended to be more prominent in the DXR + DXR group than in the other groups. In vitro experiments Effects on contractile tension. Effects of THP, EPI and DXR on contractile tension of rat papillary muscles were examined in vitro. Effects of these drugs at 100 and 200 PM are shown in Figs 5-8. THP at 100 and 200 PM produced a slight increase in dT/dt,,, and a prolongation of T, and TPT without affecting TR,,,. In contrast, EPI at 100 and 200 PM produced a marked decrease in dT/dt,,,, although at 100 PM the decrease was preceded by a slight and transient 180
untreated rats (saline + saline), the contractile force (PT and dT/dt,,) decreased with increasing stimulation frequency (negative force-frequency relationships), which is normally observed in this species (Benforado, 1958; Langer et al., 1975; Lazarus et al., 1980). On the contrary, in the muscles isolated from rats treated with DXR + EPI or DXR + DXR, the contractile force increased with increasing stimulation frequency (positive force-frequency relationships). In the case of DXR + saline (control) or DXR + THP, the contractile forces were little influenced by varying the stimulation frequency (Figs 1 and 2). Effects of calcium. Effects of varying extracellular Ca2+ concentrations on PT and dT/dt,, were shown in Fig. 3. The data were represented as a percentage of the values obtained in 0.5 mM Ca2+. In all preparations examined, PT and dT/dt,,, increased with increasing extracellular Ca2+ concentrations (0.5-4.0 mM). Increases in PT in 1.W.O mM Ca2+ and in dT/dt,,, in 4.0 mM Ca2+ were significantly greater in the DXR + DXR group than in the saline + saline (untreated) group. The increases in PT and d T/d t,,,,, in the DXR + THP and DXR + EPI groups were also greater than those in the saline + saline group, although these effects were less marked when compared with DXR + DXR group.
160 140 120 100 80 60 -
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100 80 60 0
0.5
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Frequency (Hz) Fig. 2. Effects on force-frequency relationships in papillary muscle preparations, which are represented as a percentage of the values obtained under stimulation at a frequency of 0.1 Hz. See legend to Fig. 1.
1343
Effects of anthracyclines on the contractile function in cardiac muscles
g
which was preceded by a slight crease in dT/dt,,,, and transient increase. In addition, DXR prolonged Td and TPT at 100 or 200pM, and a TR,,, at 200 /IM. Eficts on postrest contraction. Effects of THP, EPI and DXR on potentiated postrest contraction were examined after 1 hr exposure to each drug (200 PM) (Fig. 9). The potentiated postrest contraction was significantly decreased by the treatment with EPI and DXR, and conversely became the suppressed postrest contraction (smaller than the steady-state contraction, below 100%) at longer rest periods. On the other hand, although the potentiated postrest contraction was also decreased by the treatment with THP, it was always greater than the steady-state contraction (above 100%).
150 -
-0 100
-
0
1
2
3
4
5
DISCUSSION
Ca2’concentration (mM) Fig. 3. Effects of varying extracellular Ca2+ concentration on peak tension (PT) and maximum rate of tension development (dT/df,,,) in various groups. The inotropic effect of varying the bath Ca2+ concentration (0.5, 1.0, 2.0 and 4.0 mM Ca2+) was determined at a stimulation frequency of 0.1 Hz. The tissues were incubated for 15 min in Ca2+-free Kreb’s solution, and then allowed to equilibrate for at least 15 min in each Ca2+ solution before measurements were taken. The response of the muscle to Ca2+ was expressed as a percentage of the mean value determined in 0.5 mM Ca’+-Kreb’s solution (100%). See legend to Fig. 1.
In the present study, the in uivo effects of THP, EPI and DXR on the contractile function of rat papillary
T @ iJ E
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,
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increase. EPI at 100 or 200 PM also significantly prolonged Td, TPT and TR,,,. Similarly to EPI, DXR at 100 and 200pM produced a marked deI
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Ca2+conc8ntration (mM) Fig. 4. Effects of varying extracellular Ca2+ concentration on tension duration (Td) and time for half relaxation from peak tension (TRI12) in various groups. The data represented the values obtained in 0.5 mM Ca2+. See legends to Figs 1 and 3.
Fig. 5. Effects of THP (A), EPI the (O), and DXR (0) at 100 (upper) or 200 PM (lower) on maximum rate of tension development (dT/df,,) of papillary muscle preparations. Papillary muscle preparations obtained from rat hearts were electrically stimulated at 1.0 Hz at 31°C. Vehicle (0) or 100 or 2OOpM THP, EPI or DXR (final concentration) was added to the incubation medium at zero time. Data points represent the mean (k SEM) of five experiments. *P < 0.05 and **P < 0.01 vs control, respectively.
1344
Shin-ichi Hirano et
60
’
I 0
20
40
60
60
100
120
Time (min) 260 240 220
f
200 g
160
P
160 140 120 100 60
1 ’
0
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??
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20
I
40
60
60
100
120
al.
higher stimulation frequencies as a result of the suppression of Ca2+ release from sarcoplasmic reticulum (Borzak et al., 1991). In other words, the contractile tension at lower stimulation frequencies highly depends on Ca2+ release from the sarcoplasmic reticulum. Indeed, it was demonstrated in the rat ventricular muscles that the negative force-frequency relationship was abolished by ryanodine, an agent known to inhibit Ca*+ release from the sarcoplasmic reticulum (Stemmer and Akera, 1986; Tanaka and Shigenobu, 1989). In the present study with rat, the muscle preparations post-treated with EPI and DXR showed positive force-frequency relationships, suggesting that EPI and DXR suppress the function of the sarcoplasmic reticulum. On the other hand, the muscle preparations post-treated with THP showed neither positive nor negative force-frequency relationships, which was similar to the case of the pre-treatment with DXR alone, suggesting that THP has little or less effect on the sarcoplasmic reticulum function. The effects of THP, EPI and DXR on the responsiveness of the muscles to extracellular Ca2+ were also investigated. The post-treatment with DXR more greatly enhanced Ca2+-induced contraction and
Time (min)
Fig. 6. Effects of THP, EPI and DXR at 100 or 200 PM on tension duration (Td) of papillary muscle preparations. See
100 pM
legend to Fig. 5. muscles were examined. We used the muscle preparations from the rats treated once a week for 4 weeks with 2.5 mg/kg of DXR (total dose 10 mg/kg) and thereafter once a week for 4 weeks with 2.5 mg/kg of THP, EPI or DXR (total dose lOmg/kg), because clinically THP and EPI are often applied to the patients previously treated with DXR. A single dose (2.5 mg/kg) of each drug was used to compare the magnitude of the effects on contractile function of papillary muscles at the same dose level. The dose of the drugs was determined from the results of our similarly designed experiment (Hirano et al., 1994), which compared the effects of 1.25 and 2.5 mg/kg of each drug on ECG and myocardial structure. Several investigators have reported that the cumulative dose of DXR-treatment for 8 weeks (20 mg/kg) is sufficient to produce the progressive morphologic destruction using the rat model (Olson and Capen, 1978; Jensen et al., 1984; Jensen, 1986). It is well known that a ventricular muscle isolated from rat, unlike guinea-pig, shows the negative instead of positive force-frequency relationships (Benforado, 1958; Koch-Weser and Blinks, 1963; Langer et al., 1975). This phenomenon has been explained by a decrease in contractile tension at
1
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’ 0
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Time (min) 160
,
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Time (min)
Fig. 7. Effects of THP, EPI and DXR at 100 or 200 PM on time to peak tension (TPT) of papillary muscle preparations. See legend to Fig. 5.
Effects of anthracyclines
on the contractile
280 260
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I.
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120 100 80
i
’ 0
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Time (min) Fig. 8. Effects of THP, EPI and DXR at 100 or 200 PM on time for half relaxation from peak tension (TRm) of papillary muscle preparations. See legend to Fig. 5.
prolonged Td when compared with THP and EPI, suggesting that intracellular Ca2+ concentration in the DXR-treated muscles is higher and its decrease is slower than in the THP- and EPI-treated muscles. Because intracellular Ca2+ concentration depends on the relative capability of Ca2+-releasing system and Ca’+-sequestering system, although the results can not be simply interpreted, it is suggested that the Ca’+-sequestering system such as the sarcoplasmic reticulum is more greatly impaired by DXR than by THP and EPI. The results are consistent with a previous electron and light microscopic study with hamster hearts that DXR produced more marked structural alterations of the sarcoplasmic reticulum than did THP (Danchev et al., 1979). We further applied THP, EPI and DXR to the isolated papillary muscle incubated in the organ bath, to examine their own direct actions on the cardiac muscle. In this experiment, the drugs were applied at relatively high concentrations in order to produce the in vitro effects even by their short-term exposure. The present results obtained in the isolated papillary muscle could be useful in predicting the long-term effects of these drugs, because papillary muscle preparations were incubated for a relatively short period,
function
in cardiac
muscles
1345
whereas human hearts are chronically exposed to these drugs. The higher concentration of these drugs used in the present study are consistent with the drug concentrations used by Hagane et al. (1988), who examined the effects of DXR on atria1 muscle preparations at concentrations of 100 or 200 PM. In the in vitro experiments, EPI and DXR at 100 or 200 PM produced a marked negative inotropic effect, which was preceded by a slight and transient positive inotropic effect. On the other hand, THP at the same concentrations produced only a slight positive inotropic effect. These results indicate that the effect of THP on cardiac muscle is different from those of EPI and DXR, and that EPI and DXR greatly impair myocardial contractile function. Similar effects were reported in guinea-pig left atria (Hagane et al., 1988; Agata et al., 1991). The THPand DXR-induced positive inotropic effects are considered to be mediated by endogenous histamine and/or norepinephrine (Kobayashi et al., 1972; Bristow et al., 1980, 1981; Hirano et al., 1991). Also, the prolongations of Td, TPT and TR,:, were produced by the drugs, which were more prominent with EPI and DXR than with THP. These results are consistent with those in in vivo experiments described above with the exception of EPI. The reason for the difference in the effects of EPI on T,, between the in vitro and the in uivo experiments is not clear at present. The results suggest that EPI and DXR more greatly impair the contractile function and Ca2+sequestering system as compared with THP. Several investigators also observed DXR-induced
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’
** I
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I
I
I
0
10
20
30
40
50
60
Rest
I
period (set)
Fig. 9. Effects of THP, EPI and DXR on potentiated postrest contraction of papillary muscle preparations. After 1 hr application of 200 FM (final concentration) THP (A), EPI (0) or DXR (0) to the incubation medium, potentiated postrest contraction was examined after various resting periods ranging from 540 sec. In control experiments (O), deionized water was added. The data were shown as percentage of the values obtained after 1 hr exposure of each drug. Data points represent the mean (&SEM) of five experiments. *P -c0.05and **Pi 0.01vscontrol, respectively.
Shin-ichi HIirano et al.
1346
prolongation of TPT and TR,,, and suggested that they are due to the suppression of the sarcoplasmic reticulum function (Jensen, 1986; Hagane et al., 1988; Temma et al., 1993). Furthermore, it was revealed that the postrest contractions and
were more markedly
DXR
postrest
than
by THP.
contraction
the amount reticulum
has been
of Ca*+ release (Nayler
manganese,
pressed postrest these
the steady contraction agents.
proposed
of the to reflect
from the sarcoplasmic 1971; Bayer et al.,
and Iijima (1981) reported
D600 and
inhibit transsarcolemmal
by EPI
magnitude
and Merrillees,
1975; Bers, 1985). Endoh that
suppressed
The
lanthanum,
known
Ca*+ influx, markedly
to sup-
state contraction, whereas the was not or was less affected by
In the present
study,
EPI and DXR
markedly suppressed both the postrest contraction and the steady state contraction. Therefore, it is most likely that these effects by EPI and DXR are due to the suppression of the sarcoplasmic reticulum function
rather
than
colemmal
to the inhibition
Ca2+
suppressed
influx.
the postrest
of the transsar-
Although
contraction
THP
also
to a lesser extent
as compared
with EPI and DXR, THP is unlikely to
impair
sarcoplasmic
the
reticulum
function
so
severely because it did not decrease but rather slightly increased the steady state contraction. In summary, EPI and DXR more greatly the function
of the cardiac
compared
with THP.
and DXR
produce
the sarcoplasmic mented
sarcoplasmic
It is suggested
cardiotoxic
reticulum
less cardiotoxicity
impair
reticulum that both
as EPI
effects by impairing
function.
Also, well-docu-
of THP may be explained,
in part, by its less impairing
effects on the sarcoplas-
mic reticulum. In addition, switching of the treatment
it must be noted that the from DXR to THP pro-
duces less impairing
effects on cardiac
muscles.
Acknowledgements-The authors wish to thank Drs Y. Hara, H. Kuboki and H. Izumi of our laboratories for their technical help.
REFERENCES Agata N., Hirano S., Hara U., Iguchi H., Shirai M., Tone H. and Urakawa N. (1991) A comparison of pirarubicin with other antharacyclines on the cardiovascular system. Pharmacometrics 41, 467414.
Arcamone F., Fransceshi G., Minghetti A., Pence S., Redaelli S. and DiMarco A. (1975) Synthesis and antitumor properties of new glycosides of daunomycinone and adriamycinone. J. Med. Chem. 18, 103-707. Bayer R., Henneckers R., Kaufmann R. and Mannhold R. (1975) Inotropic and electrophysiological actions of verapamil and D-600 in mammalian myocaridium: pattern of inotropic effects of the racemic compounds. NaunynSchmiedeb. Arch. Pharmacol. 290, 49-68.
Benforado J. M. (1958) Frequency-dependent pharmacological and physiological effects on the rat ventricle strip. J. Pharmacol. Exp. Ther. 122, 86-100.
Bers D. (1985) Ca2+ influx and sarcoplasmic reticulum Ca*+ release in cardiac muscle activation during postrest recovery. Am. J. Physiol. 248, H36&H381. - _ Blum R. H. and Carter J. K. (1974) . , Adriamvcin: a new anticancer drug with significant clinical activity. Ann Intern. Med. 80, 249-259.
Borzak S., Murphy S. and Marsh J. D. (1991) Mechanisms of staircase in rat ventricular cells. Am. J. Physiol. 260, H884H892. Bristow M. R., Sagemann W. S., Scott R. H., Billingham M. E., Bowder R. E., Kernoff R. S., Snidow G. H. and Daniels J. R. (1980) Acute and chronic cardiovascular effects of doxorubicin in dogs: the cardiovascular pharmacology of drug-induced histamine release. J. Cardiovast. Pharmacol. 2, 487-515.
Bristow M. R., Minobe W. A., Billingham M. E., Marmor J. B., Johnson G. A., Ishimoto B. M., Sagemann W. S. and Daniels J. R. (1981) Anthracycline-associated cardiac and renal damage in rabbits: evidence for mediation by vasoactive substances. Lab. Invest. 45, 157-168. Danchev D., Paintrand M., Hayat M., Bourut C. and Math+ G. (1979) Low heart and skin toxicity of a tetrahydropyranyl derivative of Adriamycin (THP-ADM) as observed by electron and light microscopy. J. Antibiot. 32, 1085-1086.
DiMarco A., Casazza A. M., Gambetta R., Supino R. and Zunino F. (1976) Relationship between activity and aminosugar stereochemistry of daunomycin and adriamycin derivatives. Cancer Res. 36, 1962-1966. Endoh M. and Iijima T. (1981) Twitch potentiation by rest in canine ventricular muscle: effects of theophylline. Am. J. Physiol. 241, H583-H590. Hagane K., Akera T. and Berlin J. R. (1988) Doxorubicin: mechanism of cardiodepressant actions in guinea pigs. J. Pharmacol. Exp. Ther. 246, 655-661.
Hirano S., Agata N., Hara Y., Iguchi H., Shirai M., Tone H. and Urakawa N. (1991) Effects of pirarubicin, an antitumor antibiotic, on the cardiovascular system. Cancer Chemofher. Pharmacol. 28, 266-272.
Hirano S.. Wakazono K.. Aaata N.. Ieuchi H. and Tone H. (1994) Comparison of cardiotoxi&;of pirarubicin, epirubicin and doxorubicin in the rat. Drugs Exp/ Clin. Res. 20, 153-160.
Jensen R. A., Acton E. M. and Peters J. H. (1984) Doxorubicin cardiotoxicity in the rat: comparison of electrocardiogram, transmembrane potential, and structural effects. Cardiovas. Pharmacol. 6, 186-200.
Jensen R. A. (1986) Doxorubicin cardiotoxicity: contractile changes after long-term treatment in the rat. J. Pharmacol. Exp. Ther. 236, 197-203.
Kobayashi T., Nakayama R., Takatani 0. and Kimura K. (1972) Positive chronotropic and inotropic actions of new antitumor agent Adriamycin and its cardiotoxicity: its special references to myocardial contractile force and the change of the transmembrane action potential. Jap. Circ. J. 36, 259-265.
Koch-Weser J. and Blinks J. R. (1963) The influence of the interval between beats on myocardial contractility. Pharmacol. Rev. 15, 601-652.
Langer G. A., Brady A. J., Tan S. T. and Serena S. E. (1975) Correlation of the glycoside response, the force staircase and the action potential configuration in the neonatal rat heart. Circ. Res. 36, 744752. Lazarus M. L., Rossner K. L. and Anderson K. M. (1980) Adriamycin-induced alterations of the action potential in rat papillary muscle. Cardiovas Res. 14, 44&450. Lenaz L. and Page J. (1976) Cardiotoxicity of adriamycin and related anthracyclines. Cancer Treaf. Rev. 3, 11I-120. Matsushita Y., Kumagai H., Yoshimoto A., Tone H., Ishikura T., Takeuchi T. and Umezawa H. (1985) Antitumor activities of (2”R)-4’-0-tetrahydropyranyladnamycin (THP) and its combination with other antitumor agents on murine tumors. J. Antibiot. 38, 1408-1419.
Effects of anthracyclines on the contractile function in cardiac muscles Nayler W. G. and Merrillees N. C. R. (1971) Cellular exchange of calcium. In Calcium and rhe Heart (Edited by Harris P. and Opie L. H.), pp. 24-32. Academic Press, New York. Olson H. M. and Capen C. C. (1978) Chronic cardiotoxicity of doxorubicin (adriamycin) in the rat: morphologic and biochemical investigations. Toxicol. Appl. Pharmacol. 44, 60416.
Pelikan P. C. D., Weisfeldt M. L., Jacobus W. E., Miceli M. V., Bulkley B. H. and Gerstenblith G. (1986) Acute doxorubicin cardiotoxicity: functional, metabolic, and morphologic alterations in the isolated, perfused rat heart. Cardiovas. Pharmacol. 8, 1058-1066. Stemmer P. and Akera T. (1986) Concealed positive forcefrequency relationships in rat and mouse cardiac muscle revealed by ryanodine. Am. J. Physiol. 251, HI106HlllO. Suzuki T., Yamamoto H., Iwasaki T., Okamoto S., Iizuka T., Kanda H. and Murata K. (1984) A comparative study of 4’-epi-doxorubicin and doxorubicin cardiotoxic-
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ities. Jap. J. Cancer Chemother. 11, 2170-2176 (in Japanese). Tanaka H. and Shigenobu K. (1989) Effect of ryanodine on neonatal and adult rat heart: developmental increase in sarcoplasmic reticulum function. J. Mol. Cell Cardiol. 21, 1305-1313.
Temma K., Akera T., Chugun A., Kondo H., Hagane K. and Hirano S. (1993) Comparison of cardiac actions of doxorubicin, pirarubicin and aclarubicin in isolated guinea-pig heart. Eur. J. Pharmacol. 234, 173-181.
Tone H., Hirano S., Shirai M., Kumagai H., Okajima Y. and Wakabayashi T. (1986) Effect of (2”R)4’-O-tetrahydropyranyladriamycin, a new antitumor antibiotic, on the cardiac function of hamsters. Jap. J. Antibiotic 39, 547-568 (in Japanese). Villani F., Galitnberti M., Monti E., Rozza A., Lanza E., Favalli L. and Posai __ P. (1991) . , Prevention of doxorubicininduced cardiomyopathy by reduced glutathione. Cancer Chemorher. Pharmacol. 28, 365-369.
Pergamon
133%1341, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved
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EfTects of Pirarubicin in Comparison with Epirubicin and Doxorubicin on the Contractile Function in Rat Isolated Cardiac Muscles SHIN-ICHI
HIRANO,*
NAOKI AGATA, HIROSHI TONE
HIROSHI
IGUCHI
and
Central Research Laboratories, Mercian Corporation, 9-1 Johnan 4 Chome, Fujisawa 251, Japan /Tel: 0466 35 1519; Fax: 0466 35 I5301 (Received 7 November 1994)
Abstract-l. We have examined the effects of pirarubicin (THP), compared with epirubicin (EPI) and doxorubicin (DXR), on the contractile function in papillary muscles isolated from rats. 2. In in vivo experiments, in which the rat was treated once a week for 4 weeks with DXR (total dose 10 mg/kg) and thereafter once a week for 4 weeks with THP, EPI or DXR (total dose 10 mg/kg), a positive instead of negative force-frequency relationship was observed in the muscles treated with EPI and DXR, but not with THP, and an increase in contractile response to extracellular Ca*+ was observed more markedly in the muscles treated with DXR than in those treated with EPI and THP. 3. In in vifro experiments, in which the muscle preparations were incubated with the drugs at 100 or 200 p M for 2 hr, EPI and DXR caused a negative inotropic effect and a prolongation of tension duration, while THP caused a slight positive inotropic effect and a slight prolongation of tension duration. 4. Furthermore, a decrease in the potentiated postrest contraction was observed more markedly in the muscles incubated with EPI and DXR at 200 yM than in those with THP. 5. These results suggest that both EPI and DXR show a cardiotoxicity by impairing the function of cardiac sarcoplasmic reticulum, and that the switching of the treatment from DXR to THP produces less impairing effects. Key Words: Pirarubicin,
epirubicin,
doxorubicin,
cardiac
INTRODUCTION Doxorubicin (DXR) is an anthracycline antibiotic which has been shown to be effective in the treatment of various types of tumor (Blum and Carter, 1974). However, congestive heart failure, cardiomyopathy and alteration in the electrocardiogram (ECG) have been observed following its administration to experimental animals and to patients (Lenaz and Page, 1976). To lower the cardiotoxicity of this antibiotic, great efforts were made to find new derivatives. Pirarubicin [(2”R)-4’-0-tetrahydropyranyladriamycin, THP] a derivative of DXR, was found to have a similarly potent antitumor effect (Matsushita et al., 1985) but lower cardiotoxicity than DXR (Danchev
*To whom GP 26,&N
all correspondence
should
be addressed.
muscle,
contractile
function,
cardiotoxicity
et al., 1979). In the previous paper, we have reported that THP had lower cardiotoxicity in hamsters than DXR from the standpoints of ECG and myocardial structure (Tone et al., 1986), and further that at higher concentrations it only slightly increased the contractile force of the left atria in guinea-pigs, probably mediated through the release of histamine (Agata et al., 1991; Hirano et al., 1991). Epirubicin (EPI), a stereoisomer of DXR, was also found to have a similarly potent antitumor effect (Arcamone et al., 1975; DiMarco et al., 1976) but lower cardiotoxicity than DXR itself (Suzuki et al., 1984). In the comparative study on cardiotoxicity of EPI and DXR, Suzuki et al. (1984) reported that EPI did not so much affect the myocardial structure in rabbits as DXR. In our preliminary experiments, however, we observed that EPI, as well as DXR,
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Shin-ichi Hirano et al.
markedly affected myocardial structure and function; long-term exposure to these two agents produced marked negative inotropic effects in the isolated left atria from guinea-pigs, and i.v. injection of these two agents produced a decrease in heart rate and ECG alterations in hamsters (Agata et al., 1991). Furthermore, the changes in the ECG and myocardial structure of rats induced by the DXR-pretreatment were augmented more greatly by the post-treatment with EPI and DXR than by that with THP (Hirano et al., 1994). The rat has proven to be a useful experimental animal for examining the mechanisms of anthracycline-induced cardiac muscle failure (Lazarus et al., 1980; Jensen et al., 1984; Jensen, 1986; Pelikan et al., 1986; Villani et al., 1991). Jensen (1986) reported that the changes in contractile function of papillary muscles obtained from rats treated with DXR once a week for 8 weeks showed the signs of congestive heart failure after long-term treatment with DXR. In the present study, to elucidate the precise differences in the cardiotoxic actions of anthracyclines, we examined the effects of THP, compared with EPI and DXR, on the contractile response of rat papillary muscles in two different series of experiments. The first one was performed in vivo in the muscle preparations obtained from the rats, which were treated with DXR for 4 weeks and thereafter with THP, EPI or DXR for 4 weeks, because in the clinical cases THP and EPI are often used in patients previously treated with DXR. The second one was performed in the muscles incubated in vitro with the drugs to investigate the direct action of THP in comparison with EPI and DXR. The results of the present study suggest that both EPI and DXR may impair the function of cardiac sarcoplasmic reticulum, and that THP possesses less impairing effects. MATERIALS
AND METHODS
Experimental animals Female Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan) with a starting weight of 170-220 g were used in in vivo experiments. The animals were observed in quarantine for 10 days before drug administration, to document overall good health. In in vitro experiments, female Sprague-Dawley rats (Japan SLC) weighing 250-330g were used. The age of the animals was almost the same as that of rats, used in the in vivo experiments, at the end of the post-treatment period. Contractile tension Rats were killed for isolated tissue study by cervical dislocation and then exsanguinated. The hearts were
rapidly excised, trimmed of both atria, weighed and placed in modified Kreb’s solution for isolation of right ventricular papillary muscles. The right ventricular papillary muscles were mounted in a waterjacketed tissue bath between a fixed rod and the level arm of a force-displacement transducer (TB-651T, Nihon Kohden, Tokyo, Japan) for measurements of isometric contractile tension. Maximum rate of tension development (dT/dt,,,) was obtained by electrically differentiating contractile tension with a differentiator (ED-601G, Nihon Kohden). The tissues were incubated at 31°C with a modified Kreb’s solution of the following composition (in mM concentrations): NaCl, 137; KCl, 3.0; MgCl,, 1.0; CaCl,, 1.0; NaHCO,, 12.0; and glucose, 5.5. The solution was gassed with 95% 0,-Y!! COZ. The resting tension was adjusted to 0.5 g and the muscles were initially stimulated electrically by square wave pulses of 5 msec duration at a frequency of 1 Hz and an intensity of 20% above the contractile threshold via parallel platinum field electrodes. The stimuli were delivered from an electronic stimulator (SEN-7203, Nihon Kohden) through an isolation unit (SS-302J, Nihon Kohden). Contractile tension and dT/dt,,, were recorded on a pen-recorder (WT685G, Nihon Kohden) with a chart speed of 100 mmjsec. The isometric contractile parameters measured were peak isometric tension (PT), resting tension (T,), dT/dt,,, , time-to-peak tension (TPT), time for half-relaxation from PT (TR,,,) and tension duration (T,). In vivo experiments Effects of THP, EPI and DXR on the mechanical function of the papillary muscle preparations were evaluated using the modified method of Jensen (1986), who examined the contractile changes of papillary muscles isolated from the rats treated once a week for 8 weeks with 2.5 mg/kg of DXR. Drug treatment. DXR was previously administered i.v. to 56 rats at a dose level of 2.5 mg/kg once a week for 4 weeks. These rats were separated in 4 groups of 8 or 16 at the end of 4th week. Then, THP, EPI or DXR was administered i.v., respectively, to 16 rats at a dose level of 2.5 mg/kg once a week for 4 weeks, and physiological saline solution was also administered i.v. to 8 rats. The rats were observed through a post-treatment period of 2-3 weeks until they developed the signs of toxicity, such as ascites. Of the 16 rats treated with THP, EPI or DXR, 5-6 surviving rats were selected at random and killed for the isolated muscle experiments described below. Eight age-matched control rats were given i.v. injection of physiological saline solution once a week for 8 weeks, and also 6 rats were killed for the isolated muscle
Effects of anthracyclines on the contractile function in cardiac muscles experiments. The rats were weighed weekly and observed daily for general symptoms. Isolated muscle preparations. The right ventricular papillary muscle was removed from surviving rat at the end of post-treatment period. The papillary muscles were allowed to equilibrate for at least 1 hr before contractile measurements were taken. During this period, the resting length of the muscle was gradually increased until the maximum developed tension was obtained. The length of muscle at this point is defined as L,,,. The cross-sectional area of the papillary muscle was determined from measurement of L,,, and wet weight of the preparation. After the equilibration period, baseline contractile parameters were measured in the presence of 1.0 mM Ca2+, with the muscles being driven at a frequency of 1 Hz. Mean values of the various contractile parameters were obtained from about 20 individual twitch recordings for at least 10 min. The inotropic responsiveness of the preparations was then evaluated as mentioned below. Tissues were driven at a frequency of 0.1 Hz in a 1.OmM Ca2+-Kreb’s solution. Frequency-dependent changes in contractile activity were determined by increasing the stimulus frequency in discrete steps from 0.1 Hz to 0.2, 1.O and 2.0 Hz returning to 0.1 Hz at the end of a series. Three to four frequency-tension curves were obtained with each preparation. Then, the inotropic effect of varying the bath Ca2+ concentration (0.5, 1.0,2.0 and 4.0 mM Ca2+) was determined at a stimulation frequency of 0.1 Hz. The tissues were incubated for 15 min in Ca2+-free Kreb’s solution, and then allowed to equilibrate for at least 15 min in each Ca2+ solution before measurements were taken. For all parameters examined, the response of the muscle to Ca2+ was expressed as a percentage of the mean value determined in 0.5 mM Ca2+-Kreb’s solution (100%). In vitro experiments
Right ventricular papillary muscles were removed from untreated rats and equilibrated at a stimulation frequency of 1 Hz for 1 hr as described above. After the equilibration period, THP, EPI or DXR was applied, respectively, to the incubation medium. The final concentration of each drug was 100 or 200 PM. The vehicle (deionized water) was applied to the medium for control preparations. Relatively high concentration of each drug was used in these studies, so that their effects could be observed within 2 hr. In another series of experiments, after 1 hr exposure to each drug at 200 PM, potentiated postrest contraction was examined. The stimulator was turned off, and postrest contraction was elicited by stimulating the muscles again after various resting periods ranging from 5-60 sec.
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Drugs
THP was synthesized from daunorubicin in our laboratories. EPI and DXR were obtained from the Kyowa Hakko Kogyo Co. Ltd (Tokyo, Japan). These drugs were dissolved in physiological saline solution and in deionized water for in vivo and in vitro experiments, respectively. Statistical analysis
Results of the experiments were expressed as mean + SEM. The data were analyzed by Student’s paired or unpaired t-test and P < 0.05 was defined as significant. RESULTS In vivo experiments Clinical features. No dead animals were observed in the treatment with DXR + saline (control) during the entire observation period. One dead animal was observed in the treatment with DXR + THP, and eight with DXR + EPI or DXR + DXR. At the end of the 4th treatment, a suppression of body weightgain was observed in the rats pre-treated with DXR, when compared with those of saline. During the post-treatment period, the body weight also remained unchanged in the groups post-treated with saline and THP, while it decreased with EPI and DXR. As for general symptoms, all the rats pre-treated with DXR showed a ruffled coat and alopecia after 3rd posttreatment with saline or each drug, and some of these animals showed diarrhea. At necropsy, atrophy of the thymus and development of ascites were observed more markedly in the rats post-treated with EPI and DXR than in those treated with THP (data not shown). Effects on force-frequency relationships. Figure 1 shows force-frequency relationships in papillary muscle preparations, isolated from rats treated in vivo with each drug. The data were represented as an absolute value (g). The PT at a stimulation frequency of 0.1, 0.2 and 1.0 Hz was significantly smaller in the muscle preparations treated with DXR + saline (control), DXR + EPI or DXR + DXR when compared with saline + saline (untreated) group. Also, in the muscles treated with DXR + THP, the PT at 0.1 and 0.2 Hz was smaller than that of the saline + saline group, which was less prominent when compared with the case of DXR + saline, DXR + EPI and DXR + DXR. Figure 2 shows force-frequency relationships in papillary muscle preparations, which are represented as a percentage of the values obtained under stimulation at a frequency of 0.1 Hz. As shown in Figs 1 and 2, in the muscle preparations isolated from
Shin-ichi Hirano et al.
1342 0.8 0.7 0.8 3
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Frequency (Hz) Fig. I. Effects on force-frequency relationships in papillary muscle preparation isolated from rats, pre-treated for 4 weeks with DXR (total dose 10 mg/kg, i.v.) and then further treated for 4 weeks with THP, EPI or DXR (total dose lOmg/kg of each), respectively. Tissues were driven at a frequency of 0.1 Hz in a 1.0 mM Ca*+-Kreb’s solution. Frequency-dependent changes in contractile activity were determined by increasing the stimulus frequency in discrete steps from 0.1 Hz to 0.2, 1.0 and 2.0 Hz, returning to 0.1 Hz at the end of a series. The data were represented as an absolute value (g), PT is peak tension. Each point represents the mean (f SEM) of 5d experiments: 0, saline + saline; 0, DXR + saline; A, DXR + THP; 0, DXR + EPI; 0, DXR + DXR. *P c 0.05 and **P < 0.01 vs saline + saline, respectively. *P < 0.05 and **P i 0.01 vs DXR + saline, respectively.
As shown in Fig. 4, Td increased with increasing extracellular Ca2+ concentrations in all the groups pre-treated with DXR, while it rather tended to ’ decrease in the saline + saline group. In 1.0-4.0 mM Ca2+, the increase in T, was significantly greater in the DXR + DXR group than in the saline + saline group. At a high concentration of Ca2+ (4.0 mM), the increases in Td were greater in all the group pretreated with DXR than in the saline + saline group. No statistical difference was observed in TR,,, among all the groups, although an increase in TR,,, tended to be more prominent in the DXR + DXR group than in the other groups. In vitro experiments Effects on contractile tension. Effects of THP, EPI and DXR on contractile tension of rat papillary muscles were examined in vitro. Effects of these drugs at 100 and 200 PM are shown in Figs 5-8. THP at 100 and 200 PM produced a slight increase in dT/dt,,, and a prolongation of T, and TPT without affecting TR,,,. In contrast, EPI at 100 and 200 PM produced a marked decrease in dT/dt,,,, although at 100 PM the decrease was preceded by a slight and transient 180
untreated rats (saline + saline), the contractile force (PT and dT/dt,,) decreased with increasing stimulation frequency (negative force-frequency relationships), which is normally observed in this species (Benforado, 1958; Langer et al., 1975; Lazarus et al., 1980). On the contrary, in the muscles isolated from rats treated with DXR + EPI or DXR + DXR, the contractile force increased with increasing stimulation frequency (positive force-frequency relationships). In the case of DXR + saline (control) or DXR + THP, the contractile forces were little influenced by varying the stimulation frequency (Figs 1 and 2). Effects of calcium. Effects of varying extracellular Ca2+ concentrations on PT and dT/dt,, were shown in Fig. 3. The data were represented as a percentage of the values obtained in 0.5 mM Ca2+. In all preparations examined, PT and dT/dt,,, increased with increasing extracellular Ca2+ concentrations (0.5-4.0 mM). Increases in PT in 1.W.O mM Ca2+ and in dT/dt,,, in 4.0 mM Ca2+ were significantly greater in the DXR + DXR group than in the saline + saline (untreated) group. The increases in PT and d T/d t,,,,, in the DXR + THP and DXR + EPI groups were also greater than those in the saline + saline group, although these effects were less marked when compared with DXR + DXR group.
160 140 120 100 80 60 -
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Frequency (Hz) Fig. 2. Effects on force-frequency relationships in papillary muscle preparations, which are represented as a percentage of the values obtained under stimulation at a frequency of 0.1 Hz. See legend to Fig. 1.
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Effects of anthracyclines on the contractile function in cardiac muscles
g
which was preceded by a slight crease in dT/dt,,,, and transient increase. In addition, DXR prolonged Td and TPT at 100 or 200pM, and a TR,,, at 200 /IM. Eficts on postrest contraction. Effects of THP, EPI and DXR on potentiated postrest contraction were examined after 1 hr exposure to each drug (200 PM) (Fig. 9). The potentiated postrest contraction was significantly decreased by the treatment with EPI and DXR, and conversely became the suppressed postrest contraction (smaller than the steady-state contraction, below 100%) at longer rest periods. On the other hand, although the potentiated postrest contraction was also decreased by the treatment with THP, it was always greater than the steady-state contraction (above 100%).
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DISCUSSION
Ca2’concentration (mM) Fig. 3. Effects of varying extracellular Ca2+ concentration on peak tension (PT) and maximum rate of tension development (dT/df,,,) in various groups. The inotropic effect of varying the bath Ca2+ concentration (0.5, 1.0, 2.0 and 4.0 mM Ca2+) was determined at a stimulation frequency of 0.1 Hz. The tissues were incubated for 15 min in Ca2+-free Kreb’s solution, and then allowed to equilibrate for at least 15 min in each Ca2+ solution before measurements were taken. The response of the muscle to Ca2+ was expressed as a percentage of the mean value determined in 0.5 mM Ca’+-Kreb’s solution (100%). See legend to Fig. 1.
In the present study, the in uivo effects of THP, EPI and DXR on the contractile function of rat papillary
T @ iJ E
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increase. EPI at 100 or 200 PM also significantly prolonged Td, TPT and TR,,,. Similarly to EPI, DXR at 100 and 200pM produced a marked deI
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Ca2+conc8ntration (mM) Fig. 4. Effects of varying extracellular Ca2+ concentration on tension duration (Td) and time for half relaxation from peak tension (TRI12) in various groups. The data represented the values obtained in 0.5 mM Ca2+. See legends to Figs 1 and 3.
Fig. 5. Effects of THP (A), EPI the (O), and DXR (0) at 100 (upper) or 200 PM (lower) on maximum rate of tension development (dT/df,,) of papillary muscle preparations. Papillary muscle preparations obtained from rat hearts were electrically stimulated at 1.0 Hz at 31°C. Vehicle (0) or 100 or 2OOpM THP, EPI or DXR (final concentration) was added to the incubation medium at zero time. Data points represent the mean (k SEM) of five experiments. *P < 0.05 and **P < 0.01 vs control, respectively.
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Shin-ichi Hirano et
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’
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higher stimulation frequencies as a result of the suppression of Ca2+ release from sarcoplasmic reticulum (Borzak et al., 1991). In other words, the contractile tension at lower stimulation frequencies highly depends on Ca2+ release from the sarcoplasmic reticulum. Indeed, it was demonstrated in the rat ventricular muscles that the negative force-frequency relationship was abolished by ryanodine, an agent known to inhibit Ca*+ release from the sarcoplasmic reticulum (Stemmer and Akera, 1986; Tanaka and Shigenobu, 1989). In the present study with rat, the muscle preparations post-treated with EPI and DXR showed positive force-frequency relationships, suggesting that EPI and DXR suppress the function of the sarcoplasmic reticulum. On the other hand, the muscle preparations post-treated with THP showed neither positive nor negative force-frequency relationships, which was similar to the case of the pre-treatment with DXR alone, suggesting that THP has little or less effect on the sarcoplasmic reticulum function. The effects of THP, EPI and DXR on the responsiveness of the muscles to extracellular Ca2+ were also investigated. The post-treatment with DXR more greatly enhanced Ca2+-induced contraction and
Time (min)
Fig. 6. Effects of THP, EPI and DXR at 100 or 200 PM on tension duration (Td) of papillary muscle preparations. See
100 pM
legend to Fig. 5. muscles were examined. We used the muscle preparations from the rats treated once a week for 4 weeks with 2.5 mg/kg of DXR (total dose 10 mg/kg) and thereafter once a week for 4 weeks with 2.5 mg/kg of THP, EPI or DXR (total dose lOmg/kg), because clinically THP and EPI are often applied to the patients previously treated with DXR. A single dose (2.5 mg/kg) of each drug was used to compare the magnitude of the effects on contractile function of papillary muscles at the same dose level. The dose of the drugs was determined from the results of our similarly designed experiment (Hirano et al., 1994), which compared the effects of 1.25 and 2.5 mg/kg of each drug on ECG and myocardial structure. Several investigators have reported that the cumulative dose of DXR-treatment for 8 weeks (20 mg/kg) is sufficient to produce the progressive morphologic destruction using the rat model (Olson and Capen, 1978; Jensen et al., 1984; Jensen, 1986). It is well known that a ventricular muscle isolated from rat, unlike guinea-pig, shows the negative instead of positive force-frequency relationships (Benforado, 1958; Koch-Weser and Blinks, 1963; Langer et al., 1975). This phenomenon has been explained by a decrease in contractile tension at
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Fig. 7. Effects of THP, EPI and DXR at 100 or 200 PM on time to peak tension (TPT) of papillary muscle preparations. See legend to Fig. 5.
Effects of anthracyclines
on the contractile
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120
Time (min) Fig. 8. Effects of THP, EPI and DXR at 100 or 200 PM on time for half relaxation from peak tension (TRm) of papillary muscle preparations. See legend to Fig. 5.
prolonged Td when compared with THP and EPI, suggesting that intracellular Ca2+ concentration in the DXR-treated muscles is higher and its decrease is slower than in the THP- and EPI-treated muscles. Because intracellular Ca2+ concentration depends on the relative capability of Ca2+-releasing system and Ca’+-sequestering system, although the results can not be simply interpreted, it is suggested that the Ca’+-sequestering system such as the sarcoplasmic reticulum is more greatly impaired by DXR than by THP and EPI. The results are consistent with a previous electron and light microscopic study with hamster hearts that DXR produced more marked structural alterations of the sarcoplasmic reticulum than did THP (Danchev et al., 1979). We further applied THP, EPI and DXR to the isolated papillary muscle incubated in the organ bath, to examine their own direct actions on the cardiac muscle. In this experiment, the drugs were applied at relatively high concentrations in order to produce the in vitro effects even by their short-term exposure. The present results obtained in the isolated papillary muscle could be useful in predicting the long-term effects of these drugs, because papillary muscle preparations were incubated for a relatively short period,
function
in cardiac
muscles
1345
whereas human hearts are chronically exposed to these drugs. The higher concentration of these drugs used in the present study are consistent with the drug concentrations used by Hagane et al. (1988), who examined the effects of DXR on atria1 muscle preparations at concentrations of 100 or 200 PM. In the in vitro experiments, EPI and DXR at 100 or 200 PM produced a marked negative inotropic effect, which was preceded by a slight and transient positive inotropic effect. On the other hand, THP at the same concentrations produced only a slight positive inotropic effect. These results indicate that the effect of THP on cardiac muscle is different from those of EPI and DXR, and that EPI and DXR greatly impair myocardial contractile function. Similar effects were reported in guinea-pig left atria (Hagane et al., 1988; Agata et al., 1991). The THPand DXR-induced positive inotropic effects are considered to be mediated by endogenous histamine and/or norepinephrine (Kobayashi et al., 1972; Bristow et al., 1980, 1981; Hirano et al., 1991). Also, the prolongations of Td, TPT and TR,:, were produced by the drugs, which were more prominent with EPI and DXR than with THP. These results are consistent with those in in vivo experiments described above with the exception of EPI. The reason for the difference in the effects of EPI on T,, between the in vitro and the in uivo experiments is not clear at present. The results suggest that EPI and DXR more greatly impair the contractile function and Ca2+sequestering system as compared with THP. Several investigators also observed DXR-induced
250 7 @ :, 200 .c” 3 150 Q $ m $ 100 50 0
’
** I
I
I
I
I
I
I
0
10
20
30
40
50
60
Rest
I
period (set)
Fig. 9. Effects of THP, EPI and DXR on potentiated postrest contraction of papillary muscle preparations. After 1 hr application of 200 FM (final concentration) THP (A), EPI (0) or DXR (0) to the incubation medium, potentiated postrest contraction was examined after various resting periods ranging from 540 sec. In control experiments (O), deionized water was added. The data were shown as percentage of the values obtained after 1 hr exposure of each drug. Data points represent the mean (&SEM) of five experiments. *P -c0.05and **Pi 0.01vscontrol, respectively.
Shin-ichi HIirano et al.
1346
prolongation of TPT and TR,,, and suggested that they are due to the suppression of the sarcoplasmic reticulum function (Jensen, 1986; Hagane et al., 1988; Temma et al., 1993). Furthermore, it was revealed that the postrest contractions and
were more markedly
DXR
postrest
than
by THP.
contraction
the amount reticulum
has been
of Ca*+ release (Nayler
manganese,
pressed postrest these
the steady contraction agents.
proposed
of the to reflect
from the sarcoplasmic 1971; Bayer et al.,
and Iijima (1981) reported
D600 and
inhibit transsarcolemmal
by EPI
magnitude
and Merrillees,
1975; Bers, 1985). Endoh that
suppressed
The
lanthanum,
known
Ca*+ influx, markedly
to sup-
state contraction, whereas the was not or was less affected by
In the present
study,
EPI and DXR
markedly suppressed both the postrest contraction and the steady state contraction. Therefore, it is most likely that these effects by EPI and DXR are due to the suppression of the sarcoplasmic reticulum function
rather
than
colemmal
to the inhibition
Ca2+
suppressed
influx.
the postrest
of the transsar-
Although
contraction
THP
also
to a lesser extent
as compared
with EPI and DXR, THP is unlikely to
impair
sarcoplasmic
the
reticulum
function
so
severely because it did not decrease but rather slightly increased the steady state contraction. In summary, EPI and DXR more greatly the function
of the cardiac
compared
with THP.
and DXR
produce
the sarcoplasmic mented
sarcoplasmic
It is suggested
cardiotoxic
reticulum
less cardiotoxicity
impair
reticulum that both
as EPI
effects by impairing
function.
Also, well-docu-
of THP may be explained,
in part, by its less impairing
effects on the sarcoplas-
mic reticulum. In addition, switching of the treatment
it must be noted that the from DXR to THP pro-
duces less impairing
effects on cardiac
muscles.
Acknowledgements-The authors wish to thank Drs Y. Hara, H. Kuboki and H. Izumi of our laboratories for their technical help.
REFERENCES Agata N., Hirano S., Hara U., Iguchi H., Shirai M., Tone H. and Urakawa N. (1991) A comparison of pirarubicin with other antharacyclines on the cardiovascular system. Pharmacometrics 41, 467414.
Arcamone F., Fransceshi G., Minghetti A., Pence S., Redaelli S. and DiMarco A. (1975) Synthesis and antitumor properties of new glycosides of daunomycinone and adriamycinone. J. Med. Chem. 18, 103-707. Bayer R., Henneckers R., Kaufmann R. and Mannhold R. (1975) Inotropic and electrophysiological actions of verapamil and D-600 in mammalian myocaridium: pattern of inotropic effects of the racemic compounds. NaunynSchmiedeb. Arch. Pharmacol. 290, 49-68.
Benforado J. M. (1958) Frequency-dependent pharmacological and physiological effects on the rat ventricle strip. J. Pharmacol. Exp. Ther. 122, 86-100.
Bers D. (1985) Ca2+ influx and sarcoplasmic reticulum Ca*+ release in cardiac muscle activation during postrest recovery. Am. J. Physiol. 248, H36&H381. - _ Blum R. H. and Carter J. K. (1974) . , Adriamvcin: a new anticancer drug with significant clinical activity. Ann Intern. Med. 80, 249-259.
Borzak S., Murphy S. and Marsh J. D. (1991) Mechanisms of staircase in rat ventricular cells. Am. J. Physiol. 260, H884H892. Bristow M. R., Sagemann W. S., Scott R. H., Billingham M. E., Bowder R. E., Kernoff R. S., Snidow G. H. and Daniels J. R. (1980) Acute and chronic cardiovascular effects of doxorubicin in dogs: the cardiovascular pharmacology of drug-induced histamine release. J. Cardiovast. Pharmacol. 2, 487-515.
Bristow M. R., Minobe W. A., Billingham M. E., Marmor J. B., Johnson G. A., Ishimoto B. M., Sagemann W. S. and Daniels J. R. (1981) Anthracycline-associated cardiac and renal damage in rabbits: evidence for mediation by vasoactive substances. Lab. Invest. 45, 157-168. Danchev D., Paintrand M., Hayat M., Bourut C. and Math+ G. (1979) Low heart and skin toxicity of a tetrahydropyranyl derivative of Adriamycin (THP-ADM) as observed by electron and light microscopy. J. Antibiot. 32, 1085-1086.
DiMarco A., Casazza A. M., Gambetta R., Supino R. and Zunino F. (1976) Relationship between activity and aminosugar stereochemistry of daunomycin and adriamycin derivatives. Cancer Res. 36, 1962-1966. Endoh M. and Iijima T. (1981) Twitch potentiation by rest in canine ventricular muscle: effects of theophylline. Am. J. Physiol. 241, H583-H590. Hagane K., Akera T. and Berlin J. R. (1988) Doxorubicin: mechanism of cardiodepressant actions in guinea pigs. J. Pharmacol. Exp. Ther. 246, 655-661.
Hirano S., Agata N., Hara Y., Iguchi H., Shirai M., Tone H. and Urakawa N. (1991) Effects of pirarubicin, an antitumor antibiotic, on the cardiovascular system. Cancer Chemofher. Pharmacol. 28, 266-272.
Hirano S.. Wakazono K.. Aaata N.. Ieuchi H. and Tone H. (1994) Comparison of cardiotoxi&;of pirarubicin, epirubicin and doxorubicin in the rat. Drugs Exp/ Clin. Res. 20, 153-160.
Jensen R. A., Acton E. M. and Peters J. H. (1984) Doxorubicin cardiotoxicity in the rat: comparison of electrocardiogram, transmembrane potential, and structural effects. Cardiovas. Pharmacol. 6, 186-200.
Jensen R. A. (1986) Doxorubicin cardiotoxicity: contractile changes after long-term treatment in the rat. J. Pharmacol. Exp. Ther. 236, 197-203.
Kobayashi T., Nakayama R., Takatani 0. and Kimura K. (1972) Positive chronotropic and inotropic actions of new antitumor agent Adriamycin and its cardiotoxicity: its special references to myocardial contractile force and the change of the transmembrane action potential. Jap. Circ. J. 36, 259-265.
Koch-Weser J. and Blinks J. R. (1963) The influence of the interval between beats on myocardial contractility. Pharmacol. Rev. 15, 601-652.
Langer G. A., Brady A. J., Tan S. T. and Serena S. E. (1975) Correlation of the glycoside response, the force staircase and the action potential configuration in the neonatal rat heart. Circ. Res. 36, 744752. Lazarus M. L., Rossner K. L. and Anderson K. M. (1980) Adriamycin-induced alterations of the action potential in rat papillary muscle. Cardiovas Res. 14, 44&450. Lenaz L. and Page J. (1976) Cardiotoxicity of adriamycin and related anthracyclines. Cancer Treaf. Rev. 3, 11I-120. Matsushita Y., Kumagai H., Yoshimoto A., Tone H., Ishikura T., Takeuchi T. and Umezawa H. (1985) Antitumor activities of (2”R)-4’-0-tetrahydropyranyladnamycin (THP) and its combination with other antitumor agents on murine tumors. J. Antibiot. 38, 1408-1419.
Effects of anthracyclines on the contractile function in cardiac muscles Nayler W. G. and Merrillees N. C. R. (1971) Cellular exchange of calcium. In Calcium and rhe Heart (Edited by Harris P. and Opie L. H.), pp. 24-32. Academic Press, New York. Olson H. M. and Capen C. C. (1978) Chronic cardiotoxicity of doxorubicin (adriamycin) in the rat: morphologic and biochemical investigations. Toxicol. Appl. Pharmacol. 44, 60416.
Pelikan P. C. D., Weisfeldt M. L., Jacobus W. E., Miceli M. V., Bulkley B. H. and Gerstenblith G. (1986) Acute doxorubicin cardiotoxicity: functional, metabolic, and morphologic alterations in the isolated, perfused rat heart. Cardiovas. Pharmacol. 8, 1058-1066. Stemmer P. and Akera T. (1986) Concealed positive forcefrequency relationships in rat and mouse cardiac muscle revealed by ryanodine. Am. J. Physiol. 251, HI106HlllO. Suzuki T., Yamamoto H., Iwasaki T., Okamoto S., Iizuka T., Kanda H. and Murata K. (1984) A comparative study of 4’-epi-doxorubicin and doxorubicin cardiotoxic-
1347
ities. Jap. J. Cancer Chemother. 11, 2170-2176 (in Japanese). Tanaka H. and Shigenobu K. (1989) Effect of ryanodine on neonatal and adult rat heart: developmental increase in sarcoplasmic reticulum function. J. Mol. Cell Cardiol. 21, 1305-1313.
Temma K., Akera T., Chugun A., Kondo H., Hagane K. and Hirano S. (1993) Comparison of cardiac actions of doxorubicin, pirarubicin and aclarubicin in isolated guinea-pig heart. Eur. J. Pharmacol. 234, 173-181.
Tone H., Hirano S., Shirai M., Kumagai H., Okajima Y. and Wakabayashi T. (1986) Effect of (2”R)4’-O-tetrahydropyranyladriamycin, a new antitumor antibiotic, on the cardiac function of hamsters. Jap. J. Antibiotic 39, 547-568 (in Japanese). Villani F., Galitnberti M., Monti E., Rozza A., Lanza E., Favalli L. and Posai __ P. (1991) . , Prevention of doxorubicininduced cardiomyopathy by reduced glutathione. Cancer Chemorher. Pharmacol. 28, 365-369.