Proc. Natl. Acad. Sci. USA Vol. 89, pp. 1256-1260, February 1992 Medical Sciences Cardiostimulatory and antiarrhythmic activity of tubulin-binding agents (/vlnblastne/taxol/navelbine/podophykotoxin) THEODORE J. LAMPIDIS*t, DESPINA KOLONIAS*, NIRAMOL SAVARAJA, AND ROBERT W. RUBIN§ *Department of Oncology, Sylvester Comprehensive Cancer Center, IDepartment of Anatomy and , University of Miami, School of Medicine, and ?Veterans Administration Hospital, Miami, FL 33136 Communicated by Keith R. Porter, August 26, 1991

ABSTRACT Rhythmic, spontaneously pulsating cardiac tubulin-binding drugs, and/or nocodazole, and cells cultured from newborn rats are imediately stmulated to thus it is not known whether other tubulin-binding agents beat faster by addition of a number of tubulin-binding agents have effects on cardiac contractility. but not by their non-tubulin-binding an . The tubulin- In this study, we have addressed the hypothesis that the binding agents tested incde vinbastine, crie, navel- stimulation of cardiac muscle beating rate is a general prop- bine, two analogs of (S12362 and S12363), nocoda- erty of tubulin-binding agents. In addition, by using taxol, a zode, colchicne, and podophylotoxin. In addi to binding tubulin-binding compound that does not depolymerize mi- tubulin, all ofthe above agents also depolymerize microtubules. crotubules, we have investigated whether cardiostimulation In contrast, taxol, a tubulin-binding agent that stabile is associated with depolymerization. The phys- microtubules, does not stimulate cardiac cells. Moreover, the ical association of tubulin with the contractile elements of immediate and ensuing cardiac stimulation by vinbastine at cardiac muscle cells has also been studied by use of a 0.05 jug/ml Is completely blocked by pre- and cotreatment with monoclonal antibody (mAb) specific for a-tubulin. Further- taxol at 1.0 pg/mi. The time necey to reverse the cardio- more, antiarrhythmic properties oftubulin-binding agents are stimulatory effect ofvinbiatn is sign tly r than that presented, which opens the possibility of uncovering an required for nocodazole, further implicating depomrization additional class of antiarrhythmic drugs. ofmicrotubules in the cardiac activity ofthese agents. All ofthe tubulin-binding agents tested (including tol) also immedi- ately reverse adriamycin-induced arrhythms. By using a MATERIALS AND METHODS monoclonal antibody to a-tubulin, typical liamentou micro- Establishment ofPriary Cardiac Cultures. The procedure tubules are visualized in cardiac muscle and coultued non- used here for isolating heart cells from newborn rats is a muscle cells by i o When cells are treated for modification of that of Mark and Strasser (6) and has been 2 hr with vinblastine at 0.05 pg/mi, fluorescence is d d in described (7). Plastic plates (35-mm diameter) were seeded cross-striated patterns in cardiac muscle cells. Overall, these with 0.1-ml droplets of the primary cardiac cell suspension data open the possibility of uncovering an addition relation- (2.3 x 106 cells per ml) and incubated at 3TC (5% C02/95% ship between cytoskeletal elements (other than actin and my- air) for 18 hr, at which time fresh medium (2 ml) was added osin) and the contractility ofcardiac muscle. They also suggest to each plate. For fluorescence microscopy studies, cells an alternative mechais for affecting cardiac cell function in were seeded on 12-mm glass coverslips. Medium changes vitro (namely, by tubulin-binding agents). If these agents are were made every 48 hr thereafter. Drug treatments began 7 shown to be cardioactive in vivo, they may provide another days after the cells were seeded, at which time cultures approach to the treatment and management of cardiac ar- beated rhythmically and synchronously and had fully recov- rhythmias. ered from trypsinization (8). Since individual cultures had different initial rates, the data presented throughout the Tubulin-binding agents such as colchicine, the vinca alka- manuscript are from individual cultures, which are repre- loids, and benzimidazole derivatives have been extremely sentative of results from at least three different preparations useful in demonstrating the large number of diverse cellular of primary cultures, done at least in triplicate for each drug functions that depend either directly or indirectly upon the and dose. Standard deviations are presented for the intervals proper functioning of microtubules. These include a number of time between beats. ofprocesses that involve movement-i.e., movement ofcilia Microcomputer Analysis of Cardiac Cell Function: Chrono- and flagella, movement of chromosomes, and intracellular topic Changes and Arrhythmlas. Cardiac muscle cells are movement ofvesicles and organelles (1, 2). Although a sliding unique in that they maintain their ability to beat in culture for filament mechanism has been proposed to explain the role of prolonged periods. To utilize the beating as a measure of microtubules in the movement of the above-mentioned pro- physiological function, a closed microscopic stage that con- cesses, which is similar to that shown for actin in the trols temperature and pH (two variables to which cardiac cell movement involved in muscle contraction, any relationship beating is particularly sensitive) has been constructed. We oftubulin and/or microtubules to the mechanism of contrac- have previously described in detail a computerized image- tion of muscle remains unknown. processing system developed to characterize heart cell beat- There have been, however, three scattered reports (3-5), ing (5). This system allows for the quantitative measurement one of which comes from the laboratory of T.J.L. (5), and computation of the interval of time (to the millisecond suggesting that tubulin-binding agents can affect the rate of level) between beats (which is instantaneously converted to spontaneously beating cardiac cells growing in vitro (3-5). beats per min and standard deviations). Under the conditions These previous reports confined their studies to one or two Abbreviation: mAb, monoclonal antibody. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: University of payment. This article must therefore be hereby marked "advertisement" Miami, School of Medicine, Department of Oncology, 1550 N.W. in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10th Avenue, Miami, FL 33136. 1256 Downloaded by guest on September 27, 2021 Medical Sciences: Lampidis et al. Proc. Nat!. Acad. Sci. USA 89 (1992) 1257 we developed for growing healthy cardiac cells, these cells beat with an average standard deviation of less than 2 (8). Thus, the spontaneous contractions of these cells are

o .i-.~A., rhythmic and remain so for prolonged periods in culture (>10 B AlA V iA " 01,' days) (9). Therefore, studies at the cellular and subcellular level on the genesis ofarrhythmias as well as on the potencies } cs l~i Allbt~tl~lltlul of antiarrhythmic drugs can be addressed using this system. q Immunofluorescence. The anti-a-tubulin mAb (Sigma) used 16 C to label tubulin in our cardiac cell preparations was derived from the hybridoma produced by fusion of mouse myeloma cells and splenocytes from an immunized mouse. Purified chicken embryo brain microtubules were used as the immu-

nogen. This preparation was shown to be immunospecific for L!~_~ ~ ~ ~!j ~ ~ 2 0 | a-tubulin by indirect immunofluorescent staining and immu- noblotting procedures. The second antibody, fluorescein isothiocyanate-labeled mouse polyvalent immunoglobulin, -REQUENCY COEATS/MIN) was prepared in goat (Sigma). The staining procedure is a FIG. 1. Early effects of vinblastine on cardiac cell contractility. modification of that originally used to demonstrate the spec- A histogram of intervals of time between beats and polygraph ificity of this mAb (10). Cells were rinsed three times in cold recordings (5 mm/sec, 15-sec samplings) of synchronously beating phosphate buffer, fixed with cold methanol (-200C) for 20 cardiac cell cultures treated continuously with vinblastine at 0.5 min, rinsed again, air dried, and labeled overnight with the ,tg/ml are shown. Time A, before treatment; the beating rate = 65.64 appropriate dilution of a-tubulin antibody. The following ± 2.2 (mean ± SD). Time B, 1 min after treatment; the beating rate day, unreacted antibody was rinsed off the cells, and the = 102.91 ± 11.32. Time C, 20 min after treatment; the beating rate second fluorescein isothiocyanate-labeled mouse polyclonal = 145.13 ± 2.62. immunoglobulin antibody was added for 1 hr. The cells were of effects on cardiac cell contractility of each of these drugs then rinsed with phosphate buffer, air dried, and fixed onto will be presented elsewhere. a slide with warmed glycerol gelatin (Sigma) that hardens and Effects of Taxol on Cardiac Ceil Contractility. Although makes a permanent seal when left at room temperature. taxol binds tubulin, it increases rather than decreases the Fluorescence Microscopy. A phase-contrast Olympus BHZ polymerization of tubulin in vitro (11), and when added to epifluorescent microscope with fluorescence excitation from cells (12), it causes much of the free tubulin to assemble into a mercury HBO 100-watt bulb and with an excitation filter microtubules. Since it also has been shown to bind tubulin at (IF-490), dichroic mirror, and a 0.515 barrier filter combina- tion was used in our immunofluorescent studies. A photo- a different site than the vinca alkaloids (13), we investigated micrographic system allowed for documentation and com- the effects ofthis compound on cardiac cell beating. At doses parison of our results. equivalent to and higher than those tested for the vinca Drugs. Vinblastine derivatives S12362 and S12363 (Institut alkaloids, taxol had little or no stimulatory effect on cardiac des Recherches Internationales Servier, Neuilly-sur-Seine, contractility (Table 2). In fact at very high doses for pro- France) and navelbine (Pierre Fabre Inc., Paris) were gifts longed exposure (24 hr), taxol produced a slight negative from H. Tapiero (Institut de Cancdrologie et d'Immuno- chronotropic effect. Gdndtique, Villejuif, France). The vinblastine and Blockage ofthe Cardiac Stimulatory Effect ofVinbastine by used were commercial preparations from Eli Lilly. Colchi- Pre- and Cotreatment with Taxol. Since it is known that taxol cine, nocodazole, and podophylotoxin were obtained from can block the depolymerization effects oflow temperature or Sigma. Taxol was a gift from the National Cancer Institute, Ca2l on microtubules (11, 12, 14), we investigated whether and was purchased from Bristol-Myers (Walling- taxol could attenuate vinblastine's stimulatory effect on ford, CT). cardiac cell beating. It is clear that both early and late

RESULTS 9 A Early and Late Effects of Vinca Alkaloids on Cardiac 8 Contractility. When vinblastine (0.5 ,g/ml) was added to spontaneously beating cells, immediate stimulatory effects .p 2 6 3 on pulsation frequency were detected (Fig. 1). At higher 0 L,' Y3. .\' doses (5 ,g/ml), the early chronotropic effects were accom- 6 5 B panied by negative inotropic effects (Fig. 2). At lower doses C3 z 4 (<0.5 ug/ml), inotropic effects were greatly reduced or w A absent and the cells continued to be stimulated when treated continuously for >24 hr. Similar experiments were per- formed for each of the following other tubulin-binding vinca alkaloids: vincristine, navelbine, and vinblastine analogs S12363 and S12362 (both known to bind tubulin at the same site as vinblastine and to be equipotent in depolymerizing :ODG0ZZ microtubules). At 0.5 ug/ml, all of these vinca alkaloids REOUENCY (BE'. TS/M I N stimulated the frequency of cardiac beating in <5 min after FIG. 2. Early chronotropic and inotropic effects ofa high dose of addition of drug, and with increasing time of drug exposure vinblastine. A histogram of intervals of time between beats and the beating rate increased and the stimulation continued for polygraph recordings (5 mm/sec, 15-sec samplings) of synchro- 24 hr (Table 1). Differences in degree of stimulation as well nously beating cardiac cell cultures treated continuously with vin- as the time to induce statistically significant stimulatory blastine at 5.0 ,ug/ml are shown. Time A, before treatment; the effects differed according to the vinca alkaloids tested as well beating rate = 66.61 + 1.23 (mean + SD). Time B, 5 min after as to the individual cultures used. Detailed data and analysis treatment; the beating rate = 186.5 + 4.04. Downloaded by guest on September 27, 2021 1258 Medical Sciences: Lampidis et al. Proc. Natl. Acad. Sci. USA 89 (1992)

Table 1. Cardiac stimulation by tubulin-binding vinca alkaloids 130

120

Dose, Treatment time, min 110 Drug 0 1 5 15 120 I&g/ml 1440 100 ~// None (control) 0 57 59 64 58 65 53 90o S12363 0.5 63 75 97 120 142 122

S12362 0.5 58 74 88 91 110 90 Z 70 Vincristine 0.5 69 71 71 82* 146 103 Vinblastine 0.5 69 94 140 133 161 154 I- < 50 Vinblastine 0.5 37 41 69 74t NT 94 Go 40. Navelbine 0.5 34 45 67 71t NT 101 301- The values given are the frequencies in beats per min after 20 treatment with drugs for the times indicated. NT, not tested. 10 *Measured at 20 min. U0A lII I tMeasured at 10 min. 1 5 10 15 20 " 120 TIME OF TREATMENT (minutes) cardiostimulatory effects of vinblastine at 0.05 Fg/ml on cardiac cells were completely blocked by 20 min of pretreat- FIG. 3. Taxol blocks cardiac stimulation by vinblastine. Syn- ment and subsequent cotreatment with taxol at 1 ,ug/ml (Fig. chronously beating cardiac cell cultures were treated with vinblastine 3). This phenomenon followed a dose-response relationship at 0.05 ,ug/ml (e) or taxol at 1 jug/ml (C>) or pretreated with taxol at 1 pSg/ml for 20 min and then cotreated with vinblastine at 0.05 g/ml (at higher vinblastine and lower taxol doses, the blocking (o). Note the complete blockage of vinblastine's cardiostimulatory activity oftaxol became less effective). These results support effect by pre- and cotreatment with taxol. Each point represents the the hypothesis that depolymerization of microtubules is a average of >100 measurements of intervals of time between beats t necessary step in the stimulation ofcardiac cell contractility. SD. Comparative Effects of Podophylotoxin and Etoposide (Vpl6) on Cardiac Cell Beating. Since podophylotoxin binds Adriamycin-Induced Arrhythmias Are Reversed by Vinblas- tubulin but its analogue etoposide (Vpl6) does not, these two tine. Previously, it has been shown that the arrhythmias agents were tested to determine whether the ability to bind induced by Adriamycin in patients could be stimulated in an tubulin could be correlated to effects on cardiac cell beating. in vitro cardiac cell system (9). Here we found that vinblas- We found podophylotoxin at 0.5 ,ug/ml increased cardiac cell tine at 0.5 pug/ml immediately reversed the arrhythmias beating shortly after being added to the cells (before addition, induced by pretreating the cells with Adriamycin at 0.05 32 beats per min; 10 min after addition, 102 beats per min), pug/ml for 2 days (Fig. 5). At lower doses, in which cardiac and the effects increased with time ofexposure similar to the cells are stimulated by vinblastine, arrhythmias could not be other tubulin-binding agents that depolymerize microtubules reversed. Table 3 summarizes the cardiac stimulatory and that were tested. In sharp contrast, there was no effect on antiarrhythmic effects ofthe tubulin-binding and non-tubulin- beating when the non-tubulin-binding analogue Vpl6 was binding analogues tested. It is clear from these results that added to the cells at the same dose (0.5 ,ug/ml) (before tubulin binding is a necessary requirement of the agents addition, 42 beats per min; 10 min after addition, 40 beats per studied to reverse Adriamycin-induced arrhythmias. min). Thus, tubulin binding as had been reported for col- chicine and its non-tubulin-binding analogue lumicolchicine 260- (4, 5) appears to be essential for the cardiostimulatory 240- properties of this group of compounds. Effect ofNocodazole on Cardiac Cell Contractility. Nocoda- 220- zole, another tubulin-binding agent that effectively depoly- merizes microtubules (15), also stimulates cardiac cell beat- 180 INE ing shortly after being applied (Fig. 4). It has previously been 016 demonstrated that nocodazole's binding to tubulin and de- polymerization of microtubules is more quickly reversible ~140- than that of colchicine and vinblastine (16). To determine -J120- whether this was true for cardiostimulation these by agents, Z 100 20 0 20 3 we compared the reversibility of effects of nocodazole at 0.5 0.T c ,ug/ml to that of vinblastine at 0.5 ug/ml in the cardiac cells.

As shown in Fig. 4, cardiac cells returned to their normal 60 beating rate significantly faster after washing off nocodazole than they did after washing off vinblastine. 40 20 2. on Table Lack of significant effect of taxol cardiac 0 cell contractility 0 5 10 20O 30 TIME (minutes) Dose, Treatment time, min ,ug/ml 0 1 5 15 20 120 1440 FIG. 4. The cardiac effect of nocodazole is more quickly revers- ible than that of vinblastine. Synchronously beating cardiac cell 0 67 72 75 69 63 61 54 cultures were treated with nocodazole at 0.05 pg/ml for 20 min and 10 52 70 77 65 61 76 35 rinsed in the presence (o) or absence (e) of drug, or treated with 5 65 69 78 76 71 72 51 vinblastine at 0.05 ,ug/ml and rinsed with fresh medium in the 1 58 57 66 55 54 60 32 presence (A) or absence (C) of drug. The control culture was rinsed 0.5 45 52 58 52 52 62 NT with fresh medium after 20 min ofincubation (o). Note the immediate 0.1 65 63 66 63 63 69 NT loss of nocodazole's cardiostimulatory effect when rinsed in drug- free medium. In contrast, cardiostimulatory effects ofvinblastine are The values given are the frequencies in beats per min after much more slowly reversible. Each point represents the average of treatment with drug for the times indicated. NT, not tested. >100 measurements of intervals of time between beats + SD. Downloaded by guest on September 27, 2021 Medical Sciences: Lampidis et al. Proc. Nati. Acad. Sci. USA 89 (1992) 1259

En I- z 6 0 rAblk La. A a 5 1%, zd 4 FREQ (BEATS/MIN) - 86.2Z1 STO. DEV - 38.685 J .r I- 3 A w 2

I

0 - L h1 , I ci dz 4 - uvvVVVVV~JVV~JVVYVVYvv V B LaJ 0--- 3 - FFREG(BEATS/MIN) - 98.94 -j STO. DEV - 6.82 al 2 FIG. 6. Cardiac muscle (Upper Left) and nonmuscle (Upper I Right) cells stained with mAb to a-tubulin. Note the typical micro- tubular filamentous staining in both cell types with a very slight 0 , , , , , . a ~IlL1 . I_ 1 -L1 indication of a cross-striated staining pattern in the cardiac muscle En cell. Note the loss of microtubular staining in both cardiac muscle 6 - (Lower Left) and nonmuscle cells (Lower Right) and the appearance of a distinct cross-striated staining pattern in muscle cells when zLc 5 0 - FREO(BEATS/MIN) - 149. 38 cultures are treated for 2 hr with vinblastine at 0.05 ,tg/ml and then STO. DEV - 2.78 stained. (x2275.)

z 4 tubulin stains in a cross-striated pattern in the cardiac muscle 3 I- cells, suggesting an association of tubulin with the muscle -LJ contractile proteins actin and myosin. 2 DISCUSSION Although we (5) and others (3, 4) have reported that certain

0 1I .jII L_sI I I 1 I i tubulin-binding agents stimulate the spontaneous frequency 100 200 of individual cardiac cells, it was not clear whether this was FREQUENCY (BEATS/M IN) a general property of all tubulin-binding agents. Our studies here demonstrate across a wide range of compounds that FIG. 5. Vinblastine reverses Adriamycin-induced arrhythmias. A those tubulin-binding agents that depolymerize microtubules histogram of intervals of time between beats and polygraph record- and ings (5 mm/sec, 15-sec samplings) of synchronously beating cardiac (i.e., vinca alkaloids, colchicine, nocodazol, podophy- cell cultures treated continuously with Adriamycin at 0.05 Ag/ml for lotoxin) have very early or immediate effects on heart cell 24 hr (time A) and the same culture 5 min (time B) and 10 min (time beating. On the other hand, taxol, which also binds tubulin C) after adding vinblastine at 0.5 ,ug/ml are shown. but increases rather than decreases the polymerization of tubulin and its assembly into microtubules (11-13), has a-Tubulin mAb Staining in Cardiac Cells. In experiments markedly reduced or no effects on cardiac cell contractility. using an anti-a-tubulin mAb, we found tubulin to be arranged In fact at high doses, for prolonged treatment, taxol has in typical microtubular form in our neonatal cardiac cells as negative chronotropic effects (Table 2). Additionally, the well as in cocultured rat fibroblasts (Fig. 6). When treated for non-tubulin-binding podophylotoxin analogue Vpl6 had no 2 hr with vinblastine at 0.05 /&g/ml, a dose that significantly effect on cardiac cell beating, which is in agreement with increases cardiac cell beating, filamentous microtubular previous findings of no cardiac activity with the non-tubulin- staining was lost in both cardiac muscle and nonmuscle cells. binding colchicine analogue lumicolchicine (4, 5). Our data Curiously, however, we observed that under this treatment then implicate the assembly and disassembly ofmicrotubules as a possible mechanism for the cardiostimulatory activity of Table 3. Relationship between tubulin binding and cardiac tubulin-binding agents. cell contractility To investigate this latter point further, we pretreated cells with taxol and then challenged them with vinblastine. As Stimulates shown in Fig. 3, taxol completely blocked the response of Binds Depolymerizes cardiac cell Reverses cardiac cells to vinblastine. This result is in agreement with Agent tubulin microtubules beating arrhythmias in vitro data previously reported, which demonstrated the Vinblastine + + + + ability oftaxol to block the depolymerization ofmicrotubules Vincristine + + + + by the addition of excess calcium or exposure to 4TC (11, 12, S12362 + + + + 14). These data further support the hypothesis that the S12363 + + + + depolymerization of microtubules is involved in the stimu- Navelbine + + + + lation of cardiac cells by tubulin-binding agents. Taxol + - - + Our results with the washing-out effect of nocodazole also Nocodazole + + + + support this hypothesis. That is, since the microtubule- Colchicine + + + + depolymerizing effect of nocodazole is more quickly revers- Lumicolchicine ible than that of vinblastine (16), our data (Fig. 4) showing Podophylotoxin + + + + faster recovery of a normal beating rate after removal of Vpl6 - - - - nocodazole than after removal of vinblastine further impli- Downloaded by guest on September 27, 2021 1260 Medical Sciences: Lampidis et al. Proc. Natl. Acad. Sci. USA 89 (1992) cates depolymerization ofmicrotubules as a necessary step in fifth class ofantiarrhythmic compounds with a different mech- cardiac stimulation by tubulin-binding agents. anism of action-namely, that associated with the binding of The mechanism by which microtubule depolymerization tubulin. How this may interface with the existing identified actually translates into cardiac stimulation, however, remains mechanisms remains to be determined. unknown. Our immunofluorescent results, which indicate a Obviously, the understanding of how tubulin and/or mi- cross-striated pattern of staining with a mAb to a-tubulin, crotubules interact in the control of cardiac contractility and suggest that tubulin is specifically localized with the contrac- rhythmicity could have significant biologic and pharmaco- tile elements of cardiac muscle cells. This is in disagreement logic implications. Moreover, our in vitro findings of both with earlier studies by Rappaport and Samuel (17) in which cardiac cell stimulation and reversal of Adriamycin-induced they found no cross-striated pattern of tubulin antibody stain- arrhythmias by these agents suggest possible cardiac involve- ing in newborn and adult cardiac cells. A possible explanation ment when tubulin-binding drugs are used alone or in com- for the differences in their results and ours is the use of the bination with Adriamycin in the treatment ofcancer patients. mAb specific for a-tubulin, which might be recognizing a In this regard, profound cardiac disturbances have already particular portion of the molecule not recognized by the been reported in 5% of patients treated with taxol (24). antibody used in their studies. Additionally, we could not Asymptomatic bradycardia has also been observed in 29% of detect the cross-striated pattern very easily in untreated cell ovarian cancer patients treated with this drug (24). Although cultures, which suggests that tubulin localized in these sites the mechanism(s) by which taxol induces bradycardia in vivo may be masked by the filamentous microtubular form of may be multifactorial, it is noteworthy that direct application tubulin. By electron microscopy, however, it has been re- of taxol to cardiac cells in vitro slows their beating ported that microtubules can be visualized passing through (Table 2). desmin filaments located between Z disks in cardiac muscle This work was supported in part by National Cancer Institute (18). It is therefore tempting to speculate that the tubulin we Grant 2RO1-37109 and a grant from the Sylvester Comprehensive have found to be located in cross-striated patterns is somehow Cancer Center. involved with the mechanism of cardiac cell contractility. It 1. Porter, K. R. (1966) in Ciba Foundation Symposium on Prin- appears that cardiac stimulation by tubulin-binding agents may ciples of Biomolecular Organization, eds. Wolstenholme, depend on increased availability of free tubulin via the de- G. E. & O'Conner, M. (Churchill, London), pp. 308-345. polymerization of microtubules. Since it is known that Ca2+ 2. Olmsted, J. B. & Borisy, G. G. (1973) Annu. Rev. Biochem. 42, participates in muscle contraction by reversal ofthe tropomy- 507-534. osin-troponin-induced inhibition of 3. Nath, K., Shay, J. W. & Bollon, A. P. (1978) Proc. Natl. Acad. myosin binding to actin Sci. USA 75, 319-323. (19) and that Ca2l regulates the assembly and disassembly of 4. Klein, I. (1983) Cardiovasc. Res. 17, 459-465. microtubules in vitro (20) and in cells (21), it is possible that 5. Lampidis, T. J., Trevorrow, K. W. & Rubin, R. W. (1986) Exp. Ca2+ is delivered to contraction sites by tubulin. Cell Res. 164, 463-470. Previously, we reported that early stimulatory effects of 6. Mark, G. E. & Strasser, F. F. (1966) Exp. Cell Res. 44, colchicine on cardiac cell beating correlated with data sug- 217-233. gesting that cardiac muscle cells contain tubulin on their cell 7. Lampidis, T. J. (1983) in 1981: Current Status surface (5). Therefore, another possibility for the early effects and Future Development, eds. Mathe, G., Maral, R. & de of the tubulin-binding agents we have studied here could be Jager, R. (Masson, New York), pp. 37-48. 8. Masson-Pevet, M., Jongsma, H. J. & Debruine, J. (1976) J. attributed to their association with cell surface tubulin. In a Mol. Cell Cardiol. 8, 747-757. number ofdifferent types of nonmuscle cultured cells (22), it 9. Lampidis, T. J., Henderson, I. C., Israel, M. & Canellos, G. P. is well established that effects of tubulin-binding agents on (1980) Cancer Res. 40, 3901-3909. microtubules cannot be detected within 5 min after addition 10. Blose, S. H., Meltzer, D. J. & Feramisco, J. R. (1984) J. Cell of drug. Thus, the immediate effects we detect with our Biol. 98, 847-859. cardiac cell monitoring system indicate that either (i) the 11. Schiff, P. B., Fant, J. & Horwitz, S. B. (1979) Nature (London) amount of depolymerization that occurs immediately upon 22, 665-667. application of these agents (which is undetectable by tubulin 12. Schiff, P. B. & Horwitz, S. B. (1980) Proc. Natd. Acad. Sci. antibody staining; data not shown) is enough to yield car- USA 77, 1561-1565. 13. Schiff, P. B. & Horwitz, S. B. (1981) Biochemistry 20, 3247- diostimulatory effects or (ii) these agents act at the cell 3252. surface, which in turn may affect ion channels such as Na+, 14. Thompson, W. C., Wilson, L. & Purich, D. L. (1981) Cell K+, or Ca2+ participating in cardiac cell contractility. Motil. 1, 445-454. Many ofthe cardiopathogenic effects described in patients 15. De Brabander, M., Van de Veire, R. M. L., Aerts, F., Borgers, and laboratory animals treated with the anticancer agent M., Desplenter, L. & De Cree, J. (1976) Cancer Res. 36, Adriamycin can be stimulated in vitro. Among these effects 1011-1018. is a dose-related arrhythmia, which is demonstrated by 16. De Brabander, M., Geuens, G., Nuydens, R., Wilebords, R. & continuous exposure to Adriamycin at 0.05 and 0.1 ug/ml (9). DeMey, J. (1980) in Microtubules and Microtubules Inhibitors, Using this procedure to induce arrhythmias in vitro, we have eds. De Brabander, M. & De May, J. (Elsevier, Amsterdam), not their non- pp. 255-281. shown here that tubulin-binding drugs (and 17. Rappaport, L. & Samuel, J. L. (1988) Int. Rev. Cytol. 113, tubulin-binding analogues) have antiarrhythmic activity (see 101-143. Table 3) at doses that stimulate cardiac cell contractility in 18. Watkins, S. C., Samuel, J. L., Marotte, F., Bertier-Savalle, B. control cultures (0.5 Ag/ml). In addition, taxol, which does & Rappaport, L. (1987) Circ. Res. 60, 327-336. not stimulate cardiac cell beating, also displays antiarrhyth- 19. Darnell, J., Lodish, H. & Baltimore, D. (1986) in Molecular Cell mic activity. Thus, as compared to the stimulation of cardiac Biology (Scientific American, New York), pp. 825-828. contractility, the antiarrhythmic activity of tubulin-binding 20. Weisenberg, R. C. (1972) Science 177, 1104-1105. agents does not necessarily depend on their ability to de- 21. Fuller, G. N. & Brinkley, B. R. (1976) J. Supramol. Struct. 5, polymerize microtubules. 497-514. 22. Dustin, P. (1984) in Microtubules (Springer, Berlin), pp. 94- To date four general classes ofantiarrhythmic drugs that are 126, 171-233. used clinically to treat a variety of cardiac conditions associ- 23. Williams, E. M. (1980) inAntiarrhythmicAction and the Puzzle ated with arrhythmias (23) have been identified. Although each of Perhexiline (Academic, London), pp. 3-36. ofthese classes appears to have distinct mechanisms ofaction, 24. Rowinsky, E. K., McGuire, W. P., Guarnieri, T., Fisherman, certain drugs contain overlapping antiarrhythmic mechanisms J. S., Christian, M. C. & Donehower, R. C. (1991) J. Clin. (23). The work presented here has the potential to uncover a Oncol. 9, 1704-1712. Downloaded by guest on September 27, 2021