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(Circulation Research. 1995;76:645-653.)
© 1995 American Heart Association, Inc.

Articles

Effects of Doxorubicin on Excitation-Contraction Coupling in Guinea Pig Ventricular Myocardium

Yong-Xiao Wang, Michael Korth

From the Institut für Pharmakologie und Toxikologie der Technischen, Universität München (Germany).

Correspondence to Dr Michael Korth, Institut für Pharmakologie und Toxikologie der Technischen, Universität München, Biedersteiner Str 29, D-80802 München, Germany.

	Abstract

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Abstract Doxorubicin, an anticancer drug, was recently shown to release Ca²⁺ from cardiac sarcoplasmic reticulum (SR) by increasing the open probability of Ca²⁺ release channels. In the present study, we investigated the effects of doxorubicin on excitation-contraction coupling of guinea pig heart preparations. In papillary muscles contracting at 0.5 Hz, 100 µmol/L doxorubicin produced within 3 hours the following effects: it increased the force of contraction by 269.3±19.8% (n=6) and prolonged the time to peak force by 75.1±8.7% (n=6), relaxation time by 54.7±8.7% (n=6), and action potential duration (APD) at 90% repolarization (APD₉₀) by 38.6±2.9% (n=3). Despite its positive inotropic effect, doxorubicin depressed the early contraction component by increasing the latency between stimulus and the onset of force development. In single myocytes, 100 µmol/L doxorubicin prolonged APD₉₀ by 62.1% (n=18) and blocked time-dependent delayed rectifier K⁺ current (I_K) by 44% (n=9). Ca²⁺ inward current and inward rectifier K⁺ current were not affected by doxorubicin. Ca²⁺ transients elicited in myocytes loaded with the fluorescent Ca²⁺ indicator fura 2 were strongly suppressed by doxorubicin in their initial rising phase. Thereafter, doxorubicin produced a delayed rise in intracellular Ca²⁺, which reached a late peak exceeding that of the control peak by 52±8% (n=5). The results suggest that doxorubicin decreases Ca²⁺-induced Ca²⁺ release from cardiac SR, probably by increasing the SR Ca²⁺ leak. On the other hand, prolongation of APD due to inhibition of I_K allows more Ca²⁺ to enter the cell. After being only temporarily buffered by the SR, Ca²⁺ may accumulate in the cytosol as long as depolarization is maintained and lead to a more complete activation of contractile proteins.

Key Words: doxorubicin • ventricular myocytes • positive inotropic effect • sarcoplasmic reticulum Ca²⁺ release • action potential

	Introduction

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The anthracycline derivative doxorubicin is a highly effective antineoplastic agent that is widely used in the treatment of leukemia as well as a variety of solid tumors.^{1 2} Its clinical usefulness, however, is limited by its cardiotoxicity, which has long been recognized as a serious drawback of chemotherapy with anthracyclines.^{3 4} The clinical manifestations of cardiotoxicity from anthracyclines can be divided into two categories: (1) acute effects, manifested mainly by atrial and ventricular dysrhythmias,^{5 6} and (2) a chronically developing cumulative dose-dependent cardiomyopathy, which may lead to congestive heart failure.^{7 8 9} The mechanism of doxorubicin toxicity is still uncertain, but recent research indicates that an impaired myocardial Ca²⁺ homeostasis is likely to play an important role.^{10 11} There is compelling evidence that anthracyclines alter the function of cardiac sarcoplasmic reticulum (SR). Doxorubicin has been shown to bind to the Ca²⁺ release channel in fractions enriched in terminal cisternae,¹² to increase the open probability of Ca²⁺ release channels reconstituted in lipid bilayers,^{13 14 15 16} and to induce Ca²⁺ release from SR vesicles^{17 18} and from SR of permeabilized cardiac fibers by an iron-catalyzed reversible sulfhydryl redox reaction.¹⁹ In a chronic rabbit model, doxorubicin-induced cardiomyopathy was associated with a decrease in Ca²⁺ release channel density of the SR, which was probably due to a downregulation in response to chronic stimulation.²⁰ This change was accompanied by a severe dilatation of sarcotubular structures and a depressed cardiac contractility. Distension of SR was also the earliest morphological change observed in myocardial biopsies from patients receiving anthracyclines.^{21 22} Since Ca²⁺ released from the SR is thought to represent the predominant source of activator Ca²⁺ for contraction,²³ its decreased availability can be held responsible for the contractile dysfunction in anthracycline-induced cardiomyopathy. On the other hand, if Ca²⁺ release channels of cardiac SR are the main cellular targets of anthracyclines, depressed contractility should also be an early manifestation of cardiotoxicity. Contrary to this view, doxorubicin produced persistent positive inotropic effects in isolated cardiac preparations^{19 24 25 26} and has been reported to increase left ventricular systolic and diastolic function 4 and 24 hours after the treatment of tumors in patients with normal ventricular function.^{22 27} The purpose of the present study was to determine the short-term effects of doxorubicin on Ca²⁺ homeostasis of isolated cardiac preparations. We investigated, for the first time, the effects of extracellularly and intracellularly applied anthracyclines on Ca²⁺ transients of membrane-intact and voltage-controlled ventricular myocytes. In addition, membrane currents and action potentials of single cells and electromechanical effects of multicellular preparations were also examined.

	Materials and Methods

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Materials
Doxorubicin hydrochloride was kindly provided by Farmitalia Carlo Erba and was dissolved in distilled water to give a 10 mmol/L stock solution. Appropriate portions of this stock solution were added to the bath or to the pipette solution just before use to achieve final concentrations. (-)-Isoprenaline hydrochloride and 3-isobutyl-1-methylxanthine (IBMX, Sigma) were dissolved in distilled water to give 1 and 10 mmol/L stock solutions, respectively. To avoid inactivation of isoprenaline, bath solutions contained 40 µmol/L CaNa₂ EDTA (Merck AG). (±)-Propranolol hydrochloride (Sigma) was added to the bath solution 60 minutes before the addition of doxorubicin.

Multicellular Preparations
Guinea pigs of either sex weighing 250 to 350 g were killed by cervical dislocation. Right ventricular papillary muscles (diameter, 0.5 to 0.8 mm) were rapidly excised from the isolated heart and mounted in a two-chambered organ bath with internal circulation of the bath solution (volume, 50 mL). The bath solution was constantly gassed and kept in circulation by 95% O₂/5% CO₂; the temperature was maintained at 35°C, pH 7.4. The bath solution was a modified Krebs-Henseleit solution of the following composition (mmol/L): NaCl 115, KCl 4.7, MgSO₄ 1.2, CaCl₂ 2.0, NaHCO₃ 25, KH₂PO₄ 1.2, and glucose 10.

Measurement of Contractility and Action Potential
The muscles were stimulated at their base through two punctate platinum electrodes with square-wave pulses of 1 ms and an intensity slightly above threshold. The force of contraction was measured isometrically by means of an inductive force transducer (Q-11, 10p, Hottinger Baldwin Meßtechnik) connected to an oscilloscope and a pen recorder. The resting force was kept constant at 4 mN throughout the experiment. An equilibration of at least 1 hour at a stimulation frequency of 1 Hz preceded each experiment. Subsequently, the frequency of stimulation was lowered to 0.5 Hz, and the drug intervention was started as soon as the force of contraction had reached a steady state. The following parameters of the isometric contraction were evaluated: peak force of contraction, time to peak force (measured from the stimulus to peak force), and relaxation time.

Transmembrane electrical activity was recorded with conventional glass microelectrodes, which were filled with 3 mol/L KCl and had tip resistances of 10 to 30 M. Transmembrane potentials were measured by means of an electrometer amplifier (model 773, World Precision Instruments), stored together with the respective contraction signals on a digital audio tape (DAT-recorder DTR-1202, Bio-Logic), and subsequently evaluated by a computer. The maximum rate of rise of the action potential (_max) was obtained by an electronic differentiator with linear differentiation in the range 0 to 1000 V/s.

Single-Cell Isolation
Isolated myocytes were prepared from ventricles of adult guinea pigs by enzymatic dissociation according to a method previously described.²⁸ Briefly, the heart was retrogradely perfused at 37°C and at a constant rate of 10 mL/min with the following solutions: 15 minutes with a modified Krebs-Henseleit solution (see above); then 4 minutes with a nominally Ca²⁺-free Joklik solution (Joklik-MEM, Biochrom); and finally, 10 minutes with the same solution to which had been added 50 µmol/L CaCl₂, collagenase (Worthington type II, 30 mg/50 mL, Biochrom), protease (type XIV, 15 mg/50 mL, Sigma), trypsin (7 mg/50 mL, Serva), and 0.1% bovine serum albumin (fraction V, Sigma). All solutions were gassed with 5% CO₂ in O₂; pH was 7.4. The cells were then disaggregated from the ventricles by gentle mechanical agitation. After filtration through a nylon mesh, the cells were centrifuged at 37g for 3 minutes and then resuspended in modified Krebs-Henseleit solution containing 2% bovine serum albumin and kept for use at room temperature under a continuous stream of 5% CO₂ in O₂.

Whole-Cell Voltage Clamp
A drop of cell suspension was added to the modified Krebs-Henseleit solution in the recording chamber (volume, 0.5 mL) mounted on an inverted microscope. After the cells had attached to the bottom, the bath was perfused at a flow rate of 4 mL/min with prewarmed modified Krebs-Henseleit solution continuously gassed with 5% CO₂ in O₂. The temperature in the bath (35°C to 36°C) was continuously monitored.

Voltage-clamp experiments were performed in the whole-cell clamp configuration.²⁹ Patch electrodes were fabricated from borosilicate capillary tubes (Hilgenberg GmbH) and filled with prefiltered solution containing (mmol/L) potassium aspartate 80, KCl 50, KH₂PO₄ 10, MgSO₄ 3, NaCl 5, HEPES 5, K₂ATP 5, and EGTA 0.1. The pH was adjusted to 7.4 by adding KOH. The resistance of the electrodes ranged from 1.5 to 3 M. The whole-cell voltage clamp was achieved by the use of a patch-clamp amplifier (EPC7, List Medical Electronics). The cell capacitance and series resistance were compensated. The L-type Ca²⁺ current (I_Ca) was recorded by applying a test pulse of 300-ms duration every 5 s from a holding potential of -80 mV. To inactivate both fast Na⁺ and T-type Ca²⁺ currents, a prepulse to -40 mV of 40-ms duration preceded the test pulses. The amplitude of I_Ca was measured as peak inward current with respect to the zero current level. Steady state membrane currents were obtained by applying hyperpolarizing and depolarizing clamp steps for 5 s from a holding potential of -40 mV at a rate of 0.1 Hz. The steady state membrane current was measured as the net current at the end of the clamp step with respect to the zero current level. The delayed rectifier K⁺ current (I_K) was obtained by measuring the outward tail currents elicited on repolarization to -30 mV at the end of 5-s depolarizing clamp steps. The amplitude of the deactivating I_K tail was measured as the difference between the peak outward tail current and the steady state current level after decay of the tail current. When measuring K⁺ currents, a holding potential of -40 mV was used to inactivate fast Na⁺ and T-type Ca²⁺ currents, and the external bath solution contained 0.3 mmol/L CdCl₂ in order to block interfering I_Ca. Current and voltage signals were digitized on-line at 3 kHz by a 12-bit analog-to-digital converter (CED 1401, Cambridge Electronic Design) and stored in a computer for later analysis.

Ca²⁺ Transients
Ca²⁺ transients were recorded by using the Ca²⁺-sensitive dye fura 2. The whole-cell voltage-clamp technique (see above) was used to record I_Ca and Ca²⁺ transients simultaneously from single cells. Myocytes were loaded with 3 µmol/L fura 2-AM (Molecular Probes) dissolved in dimethyl sulfoxide. Loading proceeded for 15 minutes in modified Krebs-Henseleit solution at room temperature. The cells were then transferred to the recording chamber mounted on an inverted microscope equipped with epifluorescence illumination. To allow for intracellular dye conversion and to wash away extracellular fura 2-AM, cells were superfused with prewarmed (35°C to 36°C) modified Krebs-Henseleit solution at a constant rate of 4 mL/min for >45 minutes. The dye was alternately (200 Hz) excited at 340- and 380-nm wavelengths of light generated by a Deltascan illumination system (AMKO). Emission fluorescence at 510 nm was detected with a photon-counting photomultiplier tube (R928, Hamamatsu). The fluorescence ratio (340/380 nm) was calculated as a measure of [Ca²⁺].

Statistics
Where appropriate, results are presented as mean±SEM. Significance tests were performed using Student's t test for paired or unpaired observations. Differences between means were regarded statistically significant at P<.05.

	Results

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Inotropic Effects
Fig 1A shows that the anthracycline derivative doxorubicin produced a concentration-dependent (30 to 300 µmol/L) positive inotropic effect, which developed slowly over 3 to 4 hours. The increase in force of contraction began 5 minutes after the addition of 300 µmol/L doxorubicin to the bath solution but was delayed by >90 minutes when doxorubicin was applied at a 10 times lower concentration. After 4 hours, the positive inotropic effect amounted to 4.1±2.1, 8.2±2.2, and 13.2±0.3 mN at 30 (n=6), 100 (n=6), and 300 (n=5) µmol/L doxorubicin, respectively. Propranolol (1 µmol/L) did not significantly diminish the positive inotropic effect of 100 µmol/L doxorubicin (7.6±2.6 mN after 4 hours, n=4), indicating that ß-adrenoceptors were not involved. Experiments carried out in the absence of doxorubicin showed a continuous decline in the force of contraction over time. After 4 hours, force had decreased to 78.3±3.1% of the control level (n=7). The doxorubicin-induced increase in the force of contraction was accompanied by a marked prolongation of contraction duration. As shown in Fig 1B, this effect was due to a prolongation of the time to peak force and, to a lesser degree, also of the relaxation time. In a total of 17 preparations, time to peak force was increased after 180 minutes by 52.7±9.8%, 75.1±8.7%, and 82.4±7.5% and relaxation time by 29.1±5.1%, 54.7±7.3%, and 68.4±4.3% at 30 (n=6), 100 (n=6), and 300 (n=5) µmol/L doxorubicin, respectively. The inset of Fig 1B depicts three representative isometric contraction curves that had been obtained before (control), 180 minutes after the addition of 100 µmol/L doxorubicin, and 20 minutes after the additional application of 100 nmol/L ryanodine. In contrast to the control curve, which increased immediately after the stimulus and reached its maximum after 145 ms, contraction in the presence of doxorubicin developed slowly after a latency of 60 ms and reached its peak after 245 ms. Thus, when compared with the control curve, doxorubicin suppressed contractile force during the initial 136 ms of contraction although peak force was increased 2.6-fold. As can be further seen from the inset of Fig 1B, additional application of ryanodine in a concentration that induced depletion of SR Ca²⁺ stores barely affected the doxorubicin-induced isometric contraction curve.

View larger version (22K): [in this window] [in a new window]   Figure 1. A, Graph showing the time-dependent positive inotropic effect of 30 (), 100 (), and 300 () µmol/L doxorubicin. The time-dependent decline of contraction force in the absence of doxorubicin is also shown (). Symbols represent the arithmetic means, with SEM shown as vertical bars of six ( and ), five (), and seven () papillary muscles. B, Graph showing the prolongation of time to peak force () and of relaxation time () produced by 100 µmol/L doxorubicin. Symbols represent the arithmetic means, with SEM shown as vertical bars of six muscles. Inset, Superimposed isometric contraction curves of a papillary muscle recorded before (C), 240 minutes after the addition of 100 µmol/L doxorubicin (D), and 20 minutes after the additional application of 100 nmol/L ryanodine (D+R). Contraction frequency was 0.5 Hz in panels A and B.

In three papillary muscles, the influence of doxorubicin on rest decay was investigated by interrupting stimulation (1 Hz) for various intervals (data not shown). The force of the postrest contractions induced in the absence of doxorubicin was potentiated after 5 to 20 s of rest. At longer intervals, force declined and reached an equilibrium after 6 minutes of rest, amounting to 15.3±8.1% of the steady state contractions (n=3). After an incubation period of 4 hours, during which 100 µmol/L doxorubicin had produced its positive inotropic effect, postrest potentiation was abolished. After 20 s of rest, the force of contraction amounted to 81.3±5.7% (n=3) of steady state contractions and was not further decreased by longer rest periods. These findings demonstrate that only a small part of the positive inotropic effect of doxorubicin was dependent on preceding contractions, which in addition showed an accelerated decay during rest.

The positive inotropic effect and the prolongation of contraction produced by 100 µmol/L doxorubicin after 4 hours were only slightly (20%) reversible on washing the muscles with drug-free solution for 1 hour.

Action Potential Duration
As shown in Fig 2 and substantiated in two other papillary muscles, 100 µmol/L doxorubicin produced a time-dependent prolongation of action potential duration (APD) at all levels of repolarization. Three hours after the application of doxorubicin, APD measured at 90% repolarization (APD₉₀) had increased from 201.0±5.4 ms (control) to 277.1±8.3 ms, ie, by 32.3% (n=3, P<.01). As further shown in Fig 2, prolongation of APD was accompanied by an increase in the force of contraction and in contraction duration. When the time at which relaxation began was related to the respective level of action potential repolarization, doxorubicin shifted this point to more negative membrane potentials. The graph in Fig 2 summarizes this effect at different times of drug incubation. Before drug application, relaxation began when membrane repolarization was still at positive potentials (6.6±2.4 mV, n=3). When the muscles were incubated for 3 hours with 100 µmol/L doxorubicin, however, relaxation did not begin until action potential repolarization had returned to -41.4±3.8 mV.

View larger version (20K): [in this window] [in a new window]   Figure 2. Top, Recordings showing the time-dependent prolongation of action potential and contraction duration as induced by 100 µmol/L doxorubicin in a papillary muscle contracting at 0.5 Hz. All action potentials were from one continuous microelectrode impalement. Bottom, Graph showing the relation between repolarization potential of action potential and onset of relaxation, measured as the time elapsing between the upstroke of the action potential and the maximum of the contraction curve. Shown are values obtained from three papillary muscles exposed to 100 µmol/L doxorubicin. The symbols correspond with the symbols in the upper recordings and represent different times after the addition of doxorubicin.

Doxorubicin had no significant effect on resting membrane potential, _max, and overshoot of the action potential. The values (n=3) before and 3 hours after the addition of 100 µmol/L doxorubicin were -85.3±1.1 and -86.1±1.2 mV for the resting membrane potential, 219.3±17.1 and 203.0±19.1 V/s for _max, and 35.4±1.2 and 34.2±1.3 mV for the overshoot.

In the next series of experiments, the effect of doxorubicin on APD was investigated in isolated ventricular myocytes that had been current-clamped in the whole-cell clamp configuration. In the absence of anthracyclines, cells had resting potentials in the range of -72 to -82 mV (mean of nine cells, 78.2±1.2 mV). When stimulated, they displayed action potentials with a mean duration of 409.0±9.1 ms when measured at 90% repolarization (n=9, control solution in Fig 3B). In three other cell groups, APD₉₀ was determined after 1 hour of incubation with either 10 µmol/L tetrodotoxin, 100 µmol/L doxorubicin, or 100 µmol/L doxorubicin plus 10 µmol/L tetrodotoxin (see Fig 3B). Contrary to multicellular preparations, doxorubicin (100 µmol/L) exerted its maximal prolonging effect on APD after 1 hour (Fig 3A). As shown in Fig 3B, APD₉₀ of nine cells treated with 100 µmol/L doxorubicin was 663.3±37.4 ms and thus significantly longer than APD in untreated cells (P<.01). The ability of doxorubicin to prolong APD was preserved in the presence of tetrodotoxin. Although 10 µmol/L tetrodotoxin significantly shortened APD₉₀ as has previously been observed,³⁰ doxorubicin prolonged APD₉₀ in the presence of tetrodotoxin to 584.1±39.5 ms (Fig 3B). There was no significant difference in the prolongation of APD by doxorubicin in the presence of tetrodotoxin; doxorubicin increased APD₉₀ by 62.1% in the absence and by 66.4% in the presence of 10 µmol/L tetrodotoxin. Other parameters of the action potential, such as resting potential and overshoot, were not significantly affected by doxorubicin.

View larger version (23K): [in this window] [in a new window]   Figure 3. Prolongation of action potential duration (APD) induced by superfusion of ventricular myocytes with 100 µmol/L doxorubicin (Dox) for 60 minutes. A, Recordings from two current-clamped myocytes (stimulation frequency, 0.5 Hz). B, Bar graph showing APD measured at 90% repolarization (APD90) in control solution (C) and in solutions containing 10 µmol/L tetrodotoxin (TTX), 100 µmol/L Dox, and 100 µmol/L Dox plus 10 µmol/L TTX (TTX+Dox). Bar heights and error limits represent the arithmetic mean±SEM of four different cell groups. Numbers of cells are given in parentheses. *P<.05 and ***P<.001 vs C values; +++P<.001 vs TTX alone.

I_Ca
In the experiments shown in Fig 4, peak I_Ca was measured in myocytes either in the absence (control) or in the presence of 100 µmol/L doxorubicin. The Na⁺ current was inactivated by using a preceding depolarization step from -80 to -40 mV for 40 ms. The second depolarization to 0 mV activated I_Ca. As shown in Fig 4A, superfusion of myocytes for 1 hour with doxorubicin produced no significant effect on either the peak or the kinetics of I_Ca. Peak I_Ca was 1.4±0.1 nA in the control group (n=14) and 1.7±0.2 nA in the presence of doxorubicin (n=13, Fig 4B). Current-voltage relations obtained from the two cell groups were also not significantly different from each other (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 4. Failure of 100 µmol/L doxorubicin (Dox) to influence the Ca2+ inward current (ICa) of ventricular myocytes. The effect of Dox on ICa was evaluated 60 minutes after addition of the drug. A, Current tracings from two cells. To elicit ICa, 300-ms depolarizations to 0 mV from a holding potential of -80 mV and a prepulse to -40 mV were delivered at 0.2 Hz. B, Bar graph showing peak ICa measured in control solution (C) and in the presence of 100 µmol/L Dox. Bar heights and error limits represent the arithmetic mean±SEM of two different cell groups. Numbers of cells are given in parentheses. ns indicates no significant difference from C value.

In the experiment shown in Fig 5, I_Ca was enhanced by superfusing the myocyte with 100 µmol/L IBMX (a nonspecific phosphodiesterase inhibitor); the current increased from 1.1 to 2.4 nA. The IBMX-stimulated I_Ca was then inhibited by 3 µmol/L carbachol until it was indistinguishable from the predrug control current. Adding 100 µmol/L doxorubicin to the IBMX- and carbachol-containing bath solution resulted in a significant increase of I_Ca to 1.9 nA. After washing out all three drugs, I_Ca returned to the predrug control level. In Fig 5B the original current recordings of the experiment depicted in Fig 5A are superimposed at the times indicated. Results similar to those shown in Fig 5 were obtained in five other cells in which either 100 µmol/L IBMX or 1 µmol/L isoprenaline was used to enhance I_Ca before carbachol and doxorubicin were added (data not shown). As shown in Fig 6, the stimulating effect of doxorubicin on I_Ca was not observed when the current was enhanced in the absence of carbachol by 100 µmol/L IBMX (panel A) or by submaximally effective concentrations of IBMX (50 µmol/L, panel B) or isoprenaline (60 nmol/L, panel C). These findings indicate that doxorubicin enhanced I_Ca most likely by interfering either directly with muscarinic receptors or with components of the signal-transducing pathway.

View larger version (19K): [in this window] [in a new window]   Figure 5. A, Graph showing the increase in Ca2+ inward current (ICa) produced by 100 µmol/L doxorubicin in a cardiomyocyte superfused with 100 µmol/L 3-isobutyl-1-methylxanthine (IBMX) plus 3 µmol/L carbachol. Note that the IBMX-induced increase in ICa is completely inhibited by carbachol. Horizontal bars indicate the time during which each drug was applied to the bath solution. B, Superimposed current tracings corresponding to the time points a, b, c, and d in panel A. To elicit ICa, 300-ms depolarizations to 0 mV from a holding potential of -80 mV and a prepulse to -40 mV were delivered at 0.2 Hz.

View larger version (24K): [in this window] [in a new window]   Figure 6. Graphs showing the failure of 100 µmol/L doxorubicin to influence the Ca2+ inward current (ICa) previously increased by superfusion of a myocyte with either 100 µmol/L 3-isobutyl-1-methylxanthine (IBMX, A), 50 µmol/L IBMX (B), or 60 nmol/L isoprenaline (Iso, C). Note that ICa could be enhanced by increasing the concentration of IBMX (B) or Iso (C). Horizontal bars indicate the time during which each agent was applied to the bath solution. To elicit ICa, 300-ms depolarizations to 0 mV from a holding potential of -80 mV and a prepulse to -40 mV were delivered at 0.2 Hz.

K⁺ Currents
To test for a possible interaction of doxorubicin with the inward rectifier K⁺ current (I_K1), myocytes were clamped from a holding potential of -40 mV to voltages between -120 and +70 mV in 10-mV steps for 5 s. It was found that current-voltage relations obtained from cells in the absence and from cells superfused for 1 hour with 100 µmol/L doxorubicin did not significantly differ from each other at voltages between -120 and -10 mV (data not shown). At more depolarized membrane potentials (when I_K1 showed marked inward rectification), the current in the presence of doxorubicin was seen to be less outward (or more inward). When the possibility of an effect on I_K was tested more directly by measuring I_K as the outward tail on repolarization to -30 mV after depolarizing step potentials from a holding potential of -40 to +70 mV in 10-mV increments for 5 s, doxorubicin (100 µmol/L) was consistently found to suppress I_K when compared with I_K measured in the absence of the anthracycline. Typical recordings obtained from two different cells clamped from -40 to +50 mV are shown in the inset of Fig 7A; the peak tail current was 180 pA in the absence (open circle) and 100 pA in the presence of doxorubicin (closed circle); ie, doxorubicin decreased the current by 44%. In Fig 7A, tail currents were plotted as a function of membrane potential, and it can be seen that currents in the presence of doxorubicin (closed circles) were suppressed at all voltages. In Fig 7B, currents from the control group (open circles) and from the doxorubicin group (closed circles) were normalized relative to the respective maximal tail current amplitude. It can be observed that both curves are superimposable, which excludes a voltage dependence of the action of doxorubicin on I_K.

View larger version (18K): [in this window] [in a new window]   Figure 7. A, Graph demonstrating the current-voltage relations of the delayed rectifier K+ current (IK) in the absence () and in the presence of 100 µmol/L doxorubicin (). The drug effect was evaluated 60 minutes after its addition to the myocytes. Currents were elicited as indicated in the inset of panel B, with cadmium chloride (300 µmol/L) present. Inset, Original recordings showing suppression of the decaying outward tail current by doxorubicin. B, Graph showing the results in panel A normalized to maximum IK before () and after () doxorubicin. Data from two different cell groups are compared. Symbols represent the arithmetic means, with SEM (A) shown as vertical bars of 10 () and 9 () myocytes. *P<.05, **P<.01, and ***P<.001 vs control values.

Intracellular Ca²⁺ Transients
Fig 8 (upper panel) shows the effects of doxorubicin (100 µmol/L) on field-stimulated systolic Ca²⁺ transients from cardiomyocytes loaded with fura 2 (stimulation frequency, 0.2 Hz). Exposure of a cell to doxorubicin for 1 hour resulted in an increase in amplitude of the transient (on the average by 52.0±8.5% in five cells), a marked prolongation of time to peak, and a pronounced slowing of the rate of decay when compared with the control Ca²⁺ transient. Similar to the contraction experiment depicted in the inset of Fig 1B, doxorubicin suppressed the rising phase of the Ca²⁺ transient during the first 200 ms after the stimulus. The lower panel of Fig 8 shows that ryanodine (100 nmol/L) resembled doxorubicin by suppressing the rising phase and by slowing the rate of decay of the Ca²⁺ transient but differed from the anthracycline by its depressant action on peak amplitude.

View larger version (20K): [in this window] [in a new window]   Figure 8. Effects of doxorubicin (top) and of ryanodine (bottom) on Ca2+ transients elicited in cardiomyocytes loaded with the fluorescent Ca2+ indicator fura 2-AM. Extracellular stimulation was at a rate of 0.2 Hz. Recordings were made before (control) and 60 or 20 minutes after the addition of 100 µmol/L doxorubicin or 100 nmol/L ryanodine.

In another series of experiments, Ca²⁺ transients were elicited every 5 s in voltage-clamped myocytes internally dialyzed with doxorubicin. The experiment shown in Fig 9 was carried out at a holding potential of -80 mV, a preceding depolarization step to -40 mV for 40 ms, and a second depolarization to 0 mV. Two minutes after membrane disruption, the Ca²⁺ transient showed its typical configuration: it increased rapidly to its peak amplitude and began to decline while membrane potential was still depolarized. After 20 minutes of dialysis of the cell with 30 µmol/L doxorubicin (higher concentrations were not tolerated by the cells) and a depolarization duration of 800 ms, the initial peak amplitude of the transient was markedly decreased, but cytosolic Ca²⁺ continued to rise slowly until the membrane potential was repolarized to -80 mV. As illustrated in Fig 9 and verified in three additional cells, prolongation of depolarization from 300 to 800 ms in the presence of 30 µmol/L doxorubicin increased within 15 to 20 minutes the amplitude of the Ca²⁺ transient by 21.2±8.5%. Although doxorubicin increased the diastolic Ca²⁺ level slightly in the experiment shown in Fig 9, this was not seen in the three other cells. As illustrated in Fig 10, Ca²⁺ transients elicited in cells dialyzed for 20 minutes with normal electrode filling solution showed still a nearly unchanged initial peak followed by a spontaneous decline. However, when depolarization duration was maintained for 800 ms, cytosolic Ca²⁺ did not return to the level observed at the holding potential but remained elevated. Similar to the experiment with doxorubicin, the time-dependent decline of I_Ca was small.

View larger version (12K): [in this window] [in a new window]   Figure 9. Recordings showing the effects of depolarization prolongation on Ca2+ transients and Ca2+ inward current of a cardiomyocyte loaded with the fluorescent Ca2+ indicator fura 2-AM and dialyzed with 30 µmol/L doxorubicin. Recordings were made 2 (A) and 20 (B) minutes after membrane disruption. Fluorescence ratio and Ca2+ inward current were elicited by depolarizations to 0 mV from a holding potential of -80 mV and a prepulse to -40 mV delivered at 0.2 Hz. Depolarization duration to 0 mV was 300 and 800 ms for panels A and B, respectively. Im denotes membrane currents.

View larger version (13K): [in this window] [in a new window]   Figure 10. Recordings showing the effects of depolarization prolongation on Ca2+ transients and Ca2+ inward current of a cardiomyocyte loaded with the fluorescent Ca2+ indicator fura 2-AM. Recordings were made 2 (A) and 20 (B) minutes after membrane disruption. Fluorescence ratio and Ca2+ inward current were elicited by depolarizations to 0 mV from a holding potential of -80 mV and a prepulse to -40 mV delivered at 0.2 Hz. Depolarization duration to 0 mV was 300 and 800 ms for panels A and B, respectively. Im denotes membrane currents.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The main conclusion of the present study is that both prolongation of APD due to suppression of I_K and activation of SR Ca²⁺ release channels are at least in part responsible for the positive inotropic effect of doxorubicin in isolated ventricular preparations from the guinea pig heart.

The positive inotropic effect produced by doxorubicin in the isometrically contracting papillary muscle was concentration dependent and developed slowly during several hours. Parallel to the inotropic action, contraction duration was markedly prolonged by an increase in time to peak force and relaxation time. Similar effects of doxorubicin have been described in rabbit papillary muscle and atrium^{19 24} and in guinea pig atrium.^{25 26} Contrary to findings in papillary muscle, the positive inotropic effect of doxorubicin in atrial strips has been reported to depend on contraction frequency. Stimulation rates of 1 or 2 Hz favored a positive inotropic effect of doxorubicin,^{19 26} whereas contractions induced at lower rates or postrest contractions were markedly inhibited by doxorubicin.^{19 31} Besides its positive inotropic effect in the papillary muscle, doxorubicin induced a delay in the onset of contraction, resulting in a depressed initial contraction component (see inset of Fig 1B). A similar phenomenon has been observed with the plant alkaloid ryanodine,^{32 33 34} and this points to a common site of action, although a positive inotropic effect was not usually observed with ryanodine. Recent studies, however, have demonstrated that exposure of guinea pig ventricular myocytes or rabbit papillary muscles to ryanodine for >40 minutes results in positive inotropic effects combined with a prolongation of time to peak force.^{34 35} Doxorubicin and ryanodine have been reported to interact with the cardiac and the skeletal SR Ca²⁺ release channel. Both compounds induce Ca²⁺ release from subcellular fractions containing SR from rat and canine hearts.^{17 18 36} Doxorubicin, in micromolar concentrations, increased the open probability of SR Ca²⁺ release channels isolated from skeletal muscle¹⁴ and cardiac muscle^{13 15 16} reconstituted into artificial lipid bilayers. Recently, activation of the Ca²⁺ release channel of SR by doxorubicin has been demonstrated by the induction of a phasic contracture in permeabilized rabbit papillary muscles.¹⁹ In the present study, the influence of doxorubicin on Ca²⁺ transients elicited in myocytes by either field stimulation or by voltage-clamp depolarization has been investigated. Ca²⁺ transients without doxorubicin exhibited a rapid rise and reached a peak within 60 to 80 ms after depolarization. Evidence has been presented that this rapid rise of cytosolic Ca²⁺ reflects Ca²⁺-induced Ca²⁺ release from SR.³⁷ The finding that doxorubicin, whether applied extracellularly or intracellularly via the patch electrode, markedly reduced the early Ca²⁺ peak without affecting I_Ca conclusively shows that the anthracycline decreased the amount of Ca²⁺ that is released on depolarization from SR. This effect is most likely due to Ca²⁺ depletion via SR Ca²⁺ release channels exhibiting a high probability for the open state. Several lines of evidence support this conclusion: (1) Ryanodine failed to influence the force or shape of the isometric contraction curve in the presence of doxorubicin (inset of Fig 1B). It might be argued that doxorubicin prevented ryanodine from binding to its specific receptor. However, it was found that doxorubicin and ryanodine bind to different sites of the SR Ca²⁺ release channel and that doxorubicin actually increased ryanodine binding.³⁸ (2) Ryanodine resembled doxorubicin by suppressing the early peak of the Ca²⁺ transient (Fig 8). (3) Doxorubicin abolished postrest potentiation and accelerated rest decay of steady state contractions (present study), effects that are also characteristic for ryanodine.³⁵

Several mechanisms for the positive inotropic effect of doxorubicin must be considered. Doxorubicin in submicromolar concentrations has been reported to stimulate Ca²⁺ influx through voltage-dependent Ca²⁺ channels and to increase cellular cAMP content.³⁹ In micromolar concentrations, however, doxorubicin inhibited Ca²⁺-dependent action potentials in chick hearts and decreased cAMP levels.³⁹ The activation of ß-adrenoceptors (present study) or of histamine receptors has also been excluded as a mechanism for the positive inotropic effect of doxorubicin.²⁶ In the present study, doxorubicin up to 100 µmol/L had no influence on the maximum or on the kinetics of I_Ca activated in single ventricular myocytes by depolarizing steps to various potentials. Recently, doxorubicin was shown to exhibit an atropine-like inhibitory effect on cardiac muscarinic receptors.⁴⁰ In ventricular myocytes stimulated by catecholamines or other cAMP-elevating drugs, a decrease in I_Ca and hence the force of contraction can be elicited by muscarinic receptor agonists such as carbachol.^{41 42} Conversely, inhibitory effects of doxorubicin either at the receptor level or on the signal transducing pathway restored I_Ca previously suppressed by carbachol as shown in Fig 5. Enhancement of I_Ca in the absence of carbachol was not a prerequisite for doxorubicin to affect the current (Fig 6). Thus, under the combined action of sympathetic and parasympathetic neurotransmitters, doxorubicin may be able to increase I_Ca and hence the force of contraction. At concentrations relevant to the positive inotropic effect, doxorubicin has failed to significantly inhibit Na⁺,K⁺-ATPase in guinea pig hearts.²⁵ Thus, the mechanism of the positive inotropic effect of doxorubicin seems to be different from that of cardioactive steroids and may not involve Na⁺ pump inhibition. Doxorubicin has been reported to inhibit Na⁺-Ca²⁺ exchange in the dog heart.⁴³ Theoretically, a compound that inhibits Ca²⁺ extrusion via the exchanger should be able to load cellular Ca²⁺ stores, from which more Ca²⁺ may be released during subsequent excitations. Contrary to such a mechanism, doxorubicin diminished the capability of the SR to store Ca²⁺ and failed to increase the diastolic Ca²⁺ of isolated cardiomyocytes or the resting force of papillary muscles treated with doxorubicin. Finally, the positive inotropic effect of doxorubicin was not due to a direct action on contractile proteins. In permeabilized papillary muscles, doxorubicin did not shift the pCa-force relation, indicating that the anthracycline had no effect on the Ca²⁺ sensitivity of the myofilaments.¹⁹

Doxorubicin prolonged APD in multicellular preparations as well as in single myocytes. Prolongation of APD might have arisen from the elevation of depolarizing plateau currents, but it appears that enhancement by doxorubicin either of fast Na⁺ or of Ca²⁺ currents is not involved, since prolongation of APD persisted in the presence of tetrodotoxin and I_Ca remained unaffected in voltage-clamp experiments. Doxorubicin failed to significantly alter I_K1 but caused a substantial voltage-independent suppression of I_K (measured as maximum amplitude of decaying outward tails following an activating depolarization) in all cells studied. Therefore, suppression of I_K by doxorubicin seems to be responsible for APD prolongation and probably, via a sustained Ca²⁺ influx, for the development of a positive inotropic effect. Interestingly, ryanodine was shown to resemble doxorubicin by inducing in the rabbit papillary muscle a prolongation of APD together with a positive inotropic effect.³⁴ In the presence of doxorubicin, the force of contraction and the Ca²⁺ transient were initially depressed, but both signals continued to increase slowly during depolarization until terminated by repolarization (Figs 2 and 9). Thus, depolarization duration determines to what level both signals will rise. Contrary to this finding, cells with intact SR function respond to long depolarizations with an early Ca²⁺ peak, which declines initially but remains elevated during continued depolarization (Fig 10). A sustained elevation of cytosolic Ca²⁺ during prolonged depolarizations has also been described in rat cardiomyocytes,⁴⁴ but the mechanism responsible for this effect is not clear. Since the ability of the SR to retain Ca²⁺ is impaired by doxorubicin, as has been discussed before, Ca²⁺ transient and contraction must depend on transsarcolemmal Ca²⁺ influx. It has been speculated that in ventricular myocytes of some species including the guinea pig, excitation-dependent influx of Ca²⁺ may be quite substantial.^{35 45} This is supported by the rather moderate decrease in force³⁵ and in the peak Ca²⁺ amplitude observed 20 minutes after the addition of 100 nmol/L ryanodine to cardiomyocytes (Fig 8). In addition, the pronounced prolongation of the Ca²⁺ transient by doxorubicin (Fig 8) could result in a more complete activation of myofilaments and thus contribute to the force development. Contrary to the results found in control cells, prolongation of the Ca²⁺ transient was mainly due to a progressive rise in intracellular Ca²⁺ and a delayed decay on repolarization. These effects may be due to a noninactivating Ca²⁺ influx and an impaired ability of SR to retain accumulated Ca²⁺. Because of the sustained opening of SR Ca²⁺ release channels, Ca²⁺ may only temporarily be buffered by the SR and, after diffusing out into the cytosol, may interact with the myofilaments as long as the depolarization is maintained. Isolated myocytes differ from multicellular preparations in their electrical properties. Therefore, some uncertainty remains in extrapolating from Ca²⁺ transients measured in single myocytes to the contractile behavior of the papillary muscle.

It may be argued that the doxorubicin-induced lengthening of APD may not be sufficient to explain the quite substantial positive inotropic effect in papillary muscles. This discrepancy may arise from an additional inotropic effect of doxorubicin or its metabolites. Doxorubicin is slowly converted in cardiac cells to its C-13 hydroxy derivative doxorubicinol, which still releases Ca²⁺ from the SR but, unlike doxorubicin, effectively inhibits membrane ion pumps such as Na⁺,K⁺-ATPase.^{14 46 47} It remains to be determined whether prolonged treatment with doxorubicin leads to an increase in intracellular Na⁺ activity, which stimulates reversed-mode Na⁺-Ca²⁺ exchange and may thus contribute to an enhanced Ca²⁺ influx. Finally, the possibility has to be considered that doxorubicin or its metabolite may affect intracellular Ca²⁺ binding sites different from SR and thus influence the free cytosolic [Ca²⁺] during depolarization.

	Acknowledgments

 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Ko 659/4-2) to Dr Korth. We appreciate the excellent technical assistance of Roswitha Hell.

Received May 9, 1994; accepted December 17, 1994.

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