Tostron, 1975, Vol. 13, PP. 333-338 . Peraamon Pros. Printed in Great Hrltain .
SENSITIVITY OF CULTURED EMBRYONIC HEART CELLS TO CARDIOTOXIN OBTAINED FROM NAJA NAJA SIAMENSIS VENOM KAREN ARl~ts
and D. McPI~srats
Section of Neurobiology and Behavior, Langmuir Laboratory, Cornell University, Ithaca, New York 14853, U.S.A . (Acceptedfor publication 11 March 1975)
ICxleerl Aars and D. McPllsgralts. Sensitivity of cultured embryonic heart cells to cardiotoxin obtained from Naja raja siomensis venom. Toxicon 13, 333-338, 1975 .-Chunps of embryonic chickheartcells in culture were exposed to cardIotozin prepared by Sephadex CM-50 fractionation from snake (Naja ngla siamensis) venom. Cardiotoxin irreversibly depolarized these cells. More of the cells cultured from 4-day embryos, than from 12-13-day embryos, wntinued to beat spontaneously after 30 min exposure to the toxin at a concentration of 15-20kg per ml. INTRODUCITON
venom contains several pharmacologically active polypeptides such as neurotoxins, phospholipase A (phosphatide-aryl hydrolase, EC 3.1 .1 .4) and cardiotoxins (MELDRUM, 1965 ; LEE, 1972 ; YAxc, 1974). Cardiotoxin (CT3~ stops the beating of the excised toad heart (SARxAR, 1947). More recently it has been found that CTX irreversibly depolarizes the membrane of the striated frog sartorius muscle and the smooth muscle of the guinea-pig ileum (LEE et al., 1968). The work described here shows that cardiotoxin causes membrane depolarization of embryonic heart cells and that, when they are cultured in vitro, cardiac cells from young embryos are less susceptible to the action of the toxin than are those from older embryos. COBRA
MATERIALS AND METHODS Venom from the snake Naja rraja siamensis (Miami Serpentarium) was fractionated on a Sephadex CM-50 column equilibrated with 0~1 M ammonium acetate buffer at pH 6~5. Venom was eluted from the column at approximately 10 ml per hr with a 1000 ml gradient of 0~1-1 M ammonium acetate buffer, pH 6~5. Eluates were pooled into fractions corresponding to the major peaks, dialysed against Tyrode solution (adjusted to pH 6~5) and frozen . Lethality of each fraction was checked by subcutaneous injection into CFl mice (Carworth) . Embryonic hearts were dissected from embryos of White Leghorn chickens . One to ten hearts were finely minced and 5 ml viokase (025 ~; Grand Island Biological) was added to the dispersed tissue and the cells precipitated by centrifugation . The supernatant was discarded and cells resuspended in 1 ml of medium 629A (DsH.~+x, 1967). One-tenth ml of cell suspensions were placed in 35 x 10 mm sterile Falcon Petri dishes which had been coated with 1 ~ gelatin solution and dried. Threeml of medium 629A and 0~3 ml fetal calf serum were added to each dish. Ctiiltllre dishes were kept at 38°C in a humidified incubator gassed with a mixture of S~ CO, in air. Under these conditions, isolated cells and clumps of cells arefound adhering to the culture dish after 24 hr. Cells divide rapidly and many of the cells contract spontaneously. Only cells which had been in culture from 1 to 3 days were used in the present study. No differences were found in the action of CTX on cells cultured for 1 rather than 2 or 3 days. When hearts from embryos which have been inwbated for more than 13 days are treated by our methods, very few spontaneously beating cells are found in culture. For this reason, we have not been able to perform the experiments described here on oldor embryonic cells . (Cells cultured from the hearts of embryos incubated for 7 days are referred to as 7-day cells, etc .) 333 TOXICON 1973 Vot. 13
334
KAREN ARMS and D. McPHEETERS
For electrophysiological recording, medium was removed from the cells and replaced by Tyrode solution warmed to 38°C. G~Ilture dishes were placed on the warmed stage (maintained at 38 f 1°C) of an inverted phase microscope . No deterioration in appearance or electrical activity of the cells occurs after S hr under these conditions. Intracellular recordings were obtained by standard electrophysiological techniques using a glass micrcelectrode filled with 3 M KCI. Signals were led via Ag-AgCI electrodes and an electrometer (W. P. Instruments Inc., Model M4-A) for display on an oscilloscope . Each microelectrode could be used for stimulation and intracellular recording. The ground electrode was a Ag-AgCI wire immersed in the bath. All electrodes had resistances between 30-60 Miè and most were between 4x50 MSl. It is exceedingly düficult to record from a cardiac myoblast which is completely isolated from its fellows (see also DEHAAN and GOTTLIEB, 1968) and we have found no differences between the electrical activity of cells isolated from one another, those in confluent monolayers and those in clumps . Recordings from cells in these different situations are not distinguished in this paper. Protein concentrations were measured by the Lowry method (LowRY et al., 1951). Phospholipasc A prepared from Nr~a raja sfamensfs venom was a gift from Dr . M. E. Eldefrawi. RESULTS
The toxin fractions eluted from the Sephadex column are shown in Fig. 1 ; fraction IX is GTX. All of the CTX used in the present series of experiments came from one pre-
LI 1A d
n IX
IV
.4
V
VIII VI
.8 COIQC. NH~Ac (M)
.8
1.0
FI(i . I . PROFII.E OF VHNOM FRACTIONSELUTED FROM SEP}3ADEX COLUMN . (See Methods for details.)
partition which was more toxic than any other batch we have used. Lethality per mg protein varies somewhat from one preparation to another for unknown reasons. When CI'X is added to a dish of cultured heart cells at a concentration of 10 pg per ml, nearly all the cells in the dish cease to beat spontaneously within 60 min. Some clumps cease beating with no prior rate change and a few slow down before beating ceases, but most clumps display a characteristic increase in rate until the clump stops, usually in a state of contraction. Occasionally the clump resumes beating and this pattern repeats itself. Heart cells from a single embryo show considerable variation in their susceptibility to CI'X. Some cease to beat within 30 sec of exposure to CTX (10 lIg per ml), others continue to beat for up to 60 min. When cells are in contact with each other in culture, they often beat synchronously (DEHAAN and HmnxOW, 1972). We find that clumps which are joined by a monolayer of cells on the bottom of the dish may also beat synchronously. Only clumps which were not in visible contact or synchrony with adjacent clumps were used in the present study. We have used tissue culture preparations rather than intact heart in TOXICON 1973 Yol. I3
Effect of cardiotoxin on embryonic heart
33 5
the present work in order to increase exposure of the cells to the toxin. When cardiotoxin is applied to the intact heart, cells on the outside are affected much more rapidly than those inside a mass of tissue . CHANG and LEe (19C~ found a similar effect when they applied whole cobra venom to rat diaphragm and frog sartorius muscles. Tissue culture is thus necessary to reduce the error introduced by the difference in size of 4-day and l3-day embryonic hearts . We made electrophysiological recordings from many heart cells after CTX application. Although it is not always possible to record from a single heart cell for more than 2 min, we have frequently held cells for 15 min or more. Loss of an electrode from the cell can usually be distinguished in the oscilloscope record from depolarization since in the former case, the cell continues to beat and the application of depolarizing stimuli usually restores the position of the electrode and the recording. Records were discarded where there was any ambiguity in this matter. Where we recorded from a cell whose beat was seen to stop under the influence of CTX, the electrophysiological recording precisely paralleled the behavior of the cell, as recorded visually . Figure 2 shows the electrophysiological recording from a cell which stopped beating within 2 min after CTX was added. Since slightly de-
so
mv
1
min
FIG . 2 . E18CIROPHYStOIAGICAL RECORDING FROId A 7-DAY CFIICK HEART CELL IN CULTURE.
Cr7{ at 40 Eig per ml was added at the beginning of the recording . An effect upon the resting potential and the amplitude of the action potential is visible within 30 sec . This recording shows the increase in frequency of the action potential immediately before the all stopped beating, which is characteristic of the action of CI'X .
polarized pacemaker cells may exhibit an increase in rate (Lai-u~txuxi, and SP>~ .axts, 196, the observed acceleration in beating rate prior to cessation may be due to depolarization of the cell membrane. Subsequent penetrations of other cells within the clump yielded a variety of greatly reduced resting potentials . Electrical stimulation occasionally led to a brief resumption of weak beating in cells whose spontaneous beat had just ceased under the influence of CTX but stimulation of a cell whose beat had stopped for more than 15 min never resulted in contraction. When the resting potential reached a value of -15 to --5 mV, the action potential ceased . The effect of CTX in normal Tyrode solution is practically irreversible . If the toxin is washed off the cells with three changes of Tyrode solution within 2 min after the cells have stopped beating, the spontaneous activity of the cells can be partially restored, al though we have never found the action potential and resting potential restored to their previous magnitudes . If the toxin is left in contact with the cells for more than 10 min, we have never been able to restore contraction by subsequent washing with normal Tyrode. TOXlCON 1973 Vol . I 3
336
KAREN ARMS and D. McPHEETERS
This irreversibility is true for all ages of embryonic heart from 4-14 days of incubation and for cells which have been reincubated for up to 3 days after thorough washing. We tested the possibility that the effect of the CTX which we used might be due to contamination of the fraction with phospholipase A known to be present in the venom. Phospholipase A was added to 8-day heart cells (at a concentration of 15 Fig per ml) during intracellular recording . No alteration in any aspect of the action or resting potentials was seen. After 2 hr in phospholipase, many of the clumps were still beating although nearly all of the single cells had become detached from the culture dish. We also found (Leung, Arms and Gray, unpublished observations), as have EA1tL and EXCELL (1971) and CHnxc et al . (1972), that the effect of CTX is completely antagonized by high concentrations of Caa+. The effect of phospholipase A, on the other hand, is enhanced by increased Cas+ concentrations (LoxG and PENNY, 1957 ; Cxnxc et al., 1972). Although contamination of our CTX fraction with phospholipase A has not been ruled out, this evidence suggests that the effects we observe are largely those of CTX and not of the enzyme. The effects of all the other fractions from the Sephadex column upon the beating of 7-day cells in culture was also tested . At a concentration of 20 Fig protein per ml, none of the fractions I-VIII significantly reduced the number of cell clumps which contract spontaneously in culture over a period of 90 min. Fraction I has little toxic effect on cells in culture or when injected into mice. Fractions IV-VII cause death when injected into mice at 025 Fig per g body weight but autopsy showed that the atria of these mice were still beating after death. These fractions include the a, ß, and y neurotoxins of the venom and death is probably a result ofparalysis of the skeletal muscles involved in respiration. We conclude that none of these fractions has any cardiotoxic effect . Fraction VIII is slightly cardiotoxic in that it causes slight irregularity of the beat of cultured heart cells although none of them stopped beating during a 2 hr exposure to this fraction of 20 N~g per ml . In order to test the susceptibility of cells from embryos of different ages to CTX, beating cell clumps were identified in culture and examined at 10 min intervals in the presence of CTX and under control conditions. Table 1 shows the result of this study. A significantly greater number of 4-day than of 7-8 day cells continues to beat after 10 min exposure to TABLE I . EFFECT OF CARDIOTOXIN (CTX) ON BEATIIVO OF HEART CLUMPS
Min after addition of CTX 10 20 30
-
Age of embryos (days incubation)
4 82 f 6 (re et al., 1968) and cardiac muscle . Possibly as a result of this action, it also irreversibly prevents spontaneous contraction by cultured embryonic heart cells and by these cells in vivo. The only agent we have found which completely antagonizes the action of cardiotoxin is a high concentration of divalent cations (Leung, Arms and Gray, unpublished observations) . The results presented here show that early embryonic heart is less susceptible than older heart to the inhibitory effect of G7X. Other workers have shown that beating and action potentials of cells from early embryonic hearts are more resistant to the inhibitory effects of such factors as tetrodotoxin . low Na+ (ISHIMA, 1968) and high K+ concentration (DsHAntv, 1970) than are cells from older embryos. The resistance of early heart cells to toxic effects may be correlated with the fact that the action potential recorded from young embryonic hearts has certain features which distinguish it from the potential characteristic of cells from older hearts (SPERELAKIS and SHIGENOBU, 1972 ; MCDONALD and DEHAAN, 1973). Resistance to toxic effects by the early heart of submammalian vertebrates may be an adaptation to the fact that the ionic composition of the extra-cellular fluid cannot be regulated to the fine limits which are maintained in later life when the embryonic kidney starts to function (see also DaHnnN, 1967). Acknowledgements-This work was supported by a Grant-in-Aid from the American Heart Association
and with funds contributed in part by the Southern Tier Heart Association . We thank Dr . M. E. E1.or.FtuW1, Mr . S. L:W. LEUNa and Mr. D. B . GRAY for help and discussion throughout the course of this work .
REFERENCES Cxwrtc, C. C. and Lr~e, C. Y. (1966) Electrophysiological study of neuromuscular blocking action of cobra neurotoxin . Br . J. Pharmac . Chemotherap . 28, 172. Cxwxc, C. C., Cxuexc, S:T., Le>?, C. Y. and Wst, J. W. (1972) Role of cardiotoxin and phospholipase A in the blockage of nerve conduction and depolarization of skeletal muscle induced by cobra venom. Br . J. Pharmac. 44, 752. DsHenx, R. L. (1%7) Regulation of spontaneous activity and growth of embryonic chick heart cells in tissue culture. Devl Biol. 16, 216. DeH.~.~n, R. L. (1970) The potassium-sensitivity of isolated embryonic heart cells increases with development . Devl Biol. 23, 226. DeHMrr, R. L. and GOSH, S . H . (1968) The electrical activity of embryonic chick heart cells isolated in tissue culture singly or in interconnected cell sheets . J. gen . Physiol . 52, 643. DHHentv, R. L. and Hnt~xow, R. (1972) Synchronization of pulsation rates in isolated cardiac myocytes. F.xpl Cell Res . 70, 214. Eext., J. E. and Excets,, B . J . (1971) The action of a depolarizing fraction from Nrtja nivea venom on frog skeletal muscle. J. Physiol . Lond. 214, 27 . I~uKn, Y. (1968) The effect of tetrodotoxin and sodium substitution on the action potential in the course of development of the embryonic chick heart. Proc . Jap. Acad. 44, 170. LeE, C. Y. (1972) Chemistry and pharmacology of polypeptide toxins in snake venoms. A . Rev . Pharmac. 12, 26s. Lss, C. Y., CHANG, C. C., Cxtu, T. H., Cxw, P. J. S., TsFaic, T. C. and LEE, S.Y . (1968) Pharmarnlogical properties of cardiotoxin isolated from Formosan cobra venom. Arch . Pharmak . exp. Path. 299, 360. LektAncuHr.., D. and SrExRr N. (1%7) Electrical activity of cultured heart cell . In : Factors .erecting Myocardial Contractility (T~xz, R. D., ICnvni.J;x, F. and RosexTS, J., F.ds.). New York : Academic Press. Lorca, C. and PEtvrtv, I. F. (19s7) The structure of the naturally occurring phosphoglycerides . 3. Action of mocassin-venom phospholipase A on ovolecithin and related substances. Biochemistry 66, 382. LowxY, O. H., Ros®xoucR, N. J., Faxx, A. L. and Rarmau,, R. G. (1951) Protein measurement with the Folin phenol reagent. J. biol . Chem . 193, 265.
"~
TOXICON 1975 Vo 1. 13
338
KAREN ARMS and D. McPHEETERS
McDoxni.n, T. F. and DaHnnx, R. L. (1973) Ion levcls and membrane potentials in chick heart tissue and cultured cells. J. gen. Physiol. 61, 89 . MELDRUM, B. S. (1965) The actions of snake venom on nerve and muscle. The pharmacology of phospholipase A and of polypeptide toxins. Pharmac. Rev. 17, 393. SARKAR, N. K. (1947) Isolation of cardiotoxin from cobra venom (Naja tripudimis, monocellate variety) . J. Ind. them. Soc. 24, 227. SkItcetvosu, K. and Srmer nxis, N. (1971) Development of sensitivity to tetrodotoxin of chick embryonic hearts with age. J. molec. Cell Cardiol. 3, 271 . SPEItELAKIS, N. and $HIQENOHU, K. (1972) Changes in membrane properties of chick embryonic hearts during development . J. gen. Physiol. 60, 430. YANG, C. C. (1974) Chemistry and evolution of toxins in snake venoms. Toxicon 12, 1 .
TOXICON 1973 Vol. 13
SENSITIVITY OF CULTURED EMBRYONIC HEART CELLS TO CARDIOTOXIN OBTAINED FROM NAJA NAJA SIAMENSIS VENOM KAREN ARl~ts
and D. McPI~srats
Section of Neurobiology and Behavior, Langmuir Laboratory, Cornell University, Ithaca, New York 14853, U.S.A . (Acceptedfor publication 11 March 1975)
ICxleerl Aars and D. McPllsgralts. Sensitivity of cultured embryonic heart cells to cardiotoxin obtained from Naja raja siomensis venom. Toxicon 13, 333-338, 1975 .-Chunps of embryonic chickheartcells in culture were exposed to cardIotozin prepared by Sephadex CM-50 fractionation from snake (Naja ngla siamensis) venom. Cardiotoxin irreversibly depolarized these cells. More of the cells cultured from 4-day embryos, than from 12-13-day embryos, wntinued to beat spontaneously after 30 min exposure to the toxin at a concentration of 15-20kg per ml. INTRODUCITON
venom contains several pharmacologically active polypeptides such as neurotoxins, phospholipase A (phosphatide-aryl hydrolase, EC 3.1 .1 .4) and cardiotoxins (MELDRUM, 1965 ; LEE, 1972 ; YAxc, 1974). Cardiotoxin (CT3~ stops the beating of the excised toad heart (SARxAR, 1947). More recently it has been found that CTX irreversibly depolarizes the membrane of the striated frog sartorius muscle and the smooth muscle of the guinea-pig ileum (LEE et al., 1968). The work described here shows that cardiotoxin causes membrane depolarization of embryonic heart cells and that, when they are cultured in vitro, cardiac cells from young embryos are less susceptible to the action of the toxin than are those from older embryos. COBRA
MATERIALS AND METHODS Venom from the snake Naja rraja siamensis (Miami Serpentarium) was fractionated on a Sephadex CM-50 column equilibrated with 0~1 M ammonium acetate buffer at pH 6~5. Venom was eluted from the column at approximately 10 ml per hr with a 1000 ml gradient of 0~1-1 M ammonium acetate buffer, pH 6~5. Eluates were pooled into fractions corresponding to the major peaks, dialysed against Tyrode solution (adjusted to pH 6~5) and frozen . Lethality of each fraction was checked by subcutaneous injection into CFl mice (Carworth) . Embryonic hearts were dissected from embryos of White Leghorn chickens . One to ten hearts were finely minced and 5 ml viokase (025 ~; Grand Island Biological) was added to the dispersed tissue and the cells precipitated by centrifugation . The supernatant was discarded and cells resuspended in 1 ml of medium 629A (DsH.~+x, 1967). One-tenth ml of cell suspensions were placed in 35 x 10 mm sterile Falcon Petri dishes which had been coated with 1 ~ gelatin solution and dried. Threeml of medium 629A and 0~3 ml fetal calf serum were added to each dish. Ctiiltllre dishes were kept at 38°C in a humidified incubator gassed with a mixture of S~ CO, in air. Under these conditions, isolated cells and clumps of cells arefound adhering to the culture dish after 24 hr. Cells divide rapidly and many of the cells contract spontaneously. Only cells which had been in culture from 1 to 3 days were used in the present study. No differences were found in the action of CTX on cells cultured for 1 rather than 2 or 3 days. When hearts from embryos which have been inwbated for more than 13 days are treated by our methods, very few spontaneously beating cells are found in culture. For this reason, we have not been able to perform the experiments described here on oldor embryonic cells . (Cells cultured from the hearts of embryos incubated for 7 days are referred to as 7-day cells, etc .) 333 TOXICON 1973 Vot. 13
334
KAREN ARMS and D. McPHEETERS
For electrophysiological recording, medium was removed from the cells and replaced by Tyrode solution warmed to 38°C. G~Ilture dishes were placed on the warmed stage (maintained at 38 f 1°C) of an inverted phase microscope . No deterioration in appearance or electrical activity of the cells occurs after S hr under these conditions. Intracellular recordings were obtained by standard electrophysiological techniques using a glass micrcelectrode filled with 3 M KCI. Signals were led via Ag-AgCI electrodes and an electrometer (W. P. Instruments Inc., Model M4-A) for display on an oscilloscope . Each microelectrode could be used for stimulation and intracellular recording. The ground electrode was a Ag-AgCI wire immersed in the bath. All electrodes had resistances between 30-60 Miè and most were between 4x50 MSl. It is exceedingly düficult to record from a cardiac myoblast which is completely isolated from its fellows (see also DEHAAN and GOTTLIEB, 1968) and we have found no differences between the electrical activity of cells isolated from one another, those in confluent monolayers and those in clumps . Recordings from cells in these different situations are not distinguished in this paper. Protein concentrations were measured by the Lowry method (LowRY et al., 1951). Phospholipasc A prepared from Nr~a raja sfamensfs venom was a gift from Dr . M. E. Eldefrawi. RESULTS
The toxin fractions eluted from the Sephadex column are shown in Fig. 1 ; fraction IX is GTX. All of the CTX used in the present series of experiments came from one pre-
LI 1A d
n IX
IV
.4
V
VIII VI
.8 COIQC. NH~Ac (M)
.8
1.0
FI(i . I . PROFII.E OF VHNOM FRACTIONSELUTED FROM SEP}3ADEX COLUMN . (See Methods for details.)
partition which was more toxic than any other batch we have used. Lethality per mg protein varies somewhat from one preparation to another for unknown reasons. When CI'X is added to a dish of cultured heart cells at a concentration of 10 pg per ml, nearly all the cells in the dish cease to beat spontaneously within 60 min. Some clumps cease beating with no prior rate change and a few slow down before beating ceases, but most clumps display a characteristic increase in rate until the clump stops, usually in a state of contraction. Occasionally the clump resumes beating and this pattern repeats itself. Heart cells from a single embryo show considerable variation in their susceptibility to CI'X. Some cease to beat within 30 sec of exposure to CTX (10 lIg per ml), others continue to beat for up to 60 min. When cells are in contact with each other in culture, they often beat synchronously (DEHAAN and HmnxOW, 1972). We find that clumps which are joined by a monolayer of cells on the bottom of the dish may also beat synchronously. Only clumps which were not in visible contact or synchrony with adjacent clumps were used in the present study. We have used tissue culture preparations rather than intact heart in TOXICON 1973 Yol. I3
Effect of cardiotoxin on embryonic heart
33 5
the present work in order to increase exposure of the cells to the toxin. When cardiotoxin is applied to the intact heart, cells on the outside are affected much more rapidly than those inside a mass of tissue . CHANG and LEe (19C~ found a similar effect when they applied whole cobra venom to rat diaphragm and frog sartorius muscles. Tissue culture is thus necessary to reduce the error introduced by the difference in size of 4-day and l3-day embryonic hearts . We made electrophysiological recordings from many heart cells after CTX application. Although it is not always possible to record from a single heart cell for more than 2 min, we have frequently held cells for 15 min or more. Loss of an electrode from the cell can usually be distinguished in the oscilloscope record from depolarization since in the former case, the cell continues to beat and the application of depolarizing stimuli usually restores the position of the electrode and the recording. Records were discarded where there was any ambiguity in this matter. Where we recorded from a cell whose beat was seen to stop under the influence of CTX, the electrophysiological recording precisely paralleled the behavior of the cell, as recorded visually . Figure 2 shows the electrophysiological recording from a cell which stopped beating within 2 min after CTX was added. Since slightly de-
so
mv
1
min
FIG . 2 . E18CIROPHYStOIAGICAL RECORDING FROId A 7-DAY CFIICK HEART CELL IN CULTURE.
Cr7{ at 40 Eig per ml was added at the beginning of the recording . An effect upon the resting potential and the amplitude of the action potential is visible within 30 sec . This recording shows the increase in frequency of the action potential immediately before the all stopped beating, which is characteristic of the action of CI'X .
polarized pacemaker cells may exhibit an increase in rate (Lai-u~txuxi, and SP>~ .axts, 196, the observed acceleration in beating rate prior to cessation may be due to depolarization of the cell membrane. Subsequent penetrations of other cells within the clump yielded a variety of greatly reduced resting potentials . Electrical stimulation occasionally led to a brief resumption of weak beating in cells whose spontaneous beat had just ceased under the influence of CTX but stimulation of a cell whose beat had stopped for more than 15 min never resulted in contraction. When the resting potential reached a value of -15 to --5 mV, the action potential ceased . The effect of CTX in normal Tyrode solution is practically irreversible . If the toxin is washed off the cells with three changes of Tyrode solution within 2 min after the cells have stopped beating, the spontaneous activity of the cells can be partially restored, al though we have never found the action potential and resting potential restored to their previous magnitudes . If the toxin is left in contact with the cells for more than 10 min, we have never been able to restore contraction by subsequent washing with normal Tyrode. TOXlCON 1973 Vol . I 3
336
KAREN ARMS and D. McPHEETERS
This irreversibility is true for all ages of embryonic heart from 4-14 days of incubation and for cells which have been reincubated for up to 3 days after thorough washing. We tested the possibility that the effect of the CTX which we used might be due to contamination of the fraction with phospholipase A known to be present in the venom. Phospholipase A was added to 8-day heart cells (at a concentration of 15 Fig per ml) during intracellular recording . No alteration in any aspect of the action or resting potentials was seen. After 2 hr in phospholipase, many of the clumps were still beating although nearly all of the single cells had become detached from the culture dish. We also found (Leung, Arms and Gray, unpublished observations), as have EA1tL and EXCELL (1971) and CHnxc et al . (1972), that the effect of CTX is completely antagonized by high concentrations of Caa+. The effect of phospholipase A, on the other hand, is enhanced by increased Cas+ concentrations (LoxG and PENNY, 1957 ; Cxnxc et al., 1972). Although contamination of our CTX fraction with phospholipase A has not been ruled out, this evidence suggests that the effects we observe are largely those of CTX and not of the enzyme. The effects of all the other fractions from the Sephadex column upon the beating of 7-day cells in culture was also tested . At a concentration of 20 Fig protein per ml, none of the fractions I-VIII significantly reduced the number of cell clumps which contract spontaneously in culture over a period of 90 min. Fraction I has little toxic effect on cells in culture or when injected into mice. Fractions IV-VII cause death when injected into mice at 025 Fig per g body weight but autopsy showed that the atria of these mice were still beating after death. These fractions include the a, ß, and y neurotoxins of the venom and death is probably a result ofparalysis of the skeletal muscles involved in respiration. We conclude that none of these fractions has any cardiotoxic effect . Fraction VIII is slightly cardiotoxic in that it causes slight irregularity of the beat of cultured heart cells although none of them stopped beating during a 2 hr exposure to this fraction of 20 N~g per ml . In order to test the susceptibility of cells from embryos of different ages to CTX, beating cell clumps were identified in culture and examined at 10 min intervals in the presence of CTX and under control conditions. Table 1 shows the result of this study. A significantly greater number of 4-day than of 7-8 day cells continues to beat after 10 min exposure to TABLE I . EFFECT OF CARDIOTOXIN (CTX) ON BEATIIVO OF HEART CLUMPS
Min after addition of CTX 10 20 30
-
Age of embryos (days incubation)
4 82 f 6 (re et al., 1968) and cardiac muscle . Possibly as a result of this action, it also irreversibly prevents spontaneous contraction by cultured embryonic heart cells and by these cells in vivo. The only agent we have found which completely antagonizes the action of cardiotoxin is a high concentration of divalent cations (Leung, Arms and Gray, unpublished observations) . The results presented here show that early embryonic heart is less susceptible than older heart to the inhibitory effect of G7X. Other workers have shown that beating and action potentials of cells from early embryonic hearts are more resistant to the inhibitory effects of such factors as tetrodotoxin . low Na+ (ISHIMA, 1968) and high K+ concentration (DsHAntv, 1970) than are cells from older embryos. The resistance of early heart cells to toxic effects may be correlated with the fact that the action potential recorded from young embryonic hearts has certain features which distinguish it from the potential characteristic of cells from older hearts (SPERELAKIS and SHIGENOBU, 1972 ; MCDONALD and DEHAAN, 1973). Resistance to toxic effects by the early heart of submammalian vertebrates may be an adaptation to the fact that the ionic composition of the extra-cellular fluid cannot be regulated to the fine limits which are maintained in later life when the embryonic kidney starts to function (see also DaHnnN, 1967). Acknowledgements-This work was supported by a Grant-in-Aid from the American Heart Association
and with funds contributed in part by the Southern Tier Heart Association . We thank Dr . M. E. E1.or.FtuW1, Mr . S. L:W. LEUNa and Mr. D. B . GRAY for help and discussion throughout the course of this work .
REFERENCES Cxwrtc, C. C. and Lr~e, C. Y. (1966) Electrophysiological study of neuromuscular blocking action of cobra neurotoxin . Br . J. Pharmac . Chemotherap . 28, 172. Cxwxc, C. C., Cxuexc, S:T., Le>?, C. Y. and Wst, J. W. (1972) Role of cardiotoxin and phospholipase A in the blockage of nerve conduction and depolarization of skeletal muscle induced by cobra venom. Br . J. Pharmac. 44, 752. DsHenx, R. L. (1%7) Regulation of spontaneous activity and growth of embryonic chick heart cells in tissue culture. Devl Biol. 16, 216. DeH.~.~n, R. L. (1970) The potassium-sensitivity of isolated embryonic heart cells increases with development . Devl Biol. 23, 226. DeHMrr, R. L. and GOSH, S . H . (1968) The electrical activity of embryonic chick heart cells isolated in tissue culture singly or in interconnected cell sheets . J. gen . Physiol . 52, 643. DHHentv, R. L. and Hnt~xow, R. (1972) Synchronization of pulsation rates in isolated cardiac myocytes. F.xpl Cell Res . 70, 214. Eext., J. E. and Excets,, B . J . (1971) The action of a depolarizing fraction from Nrtja nivea venom on frog skeletal muscle. J. Physiol . Lond. 214, 27 . I~uKn, Y. (1968) The effect of tetrodotoxin and sodium substitution on the action potential in the course of development of the embryonic chick heart. Proc . Jap. Acad. 44, 170. LeE, C. Y. (1972) Chemistry and pharmacology of polypeptide toxins in snake venoms. A . Rev . Pharmac. 12, 26s. Lss, C. Y., CHANG, C. C., Cxtu, T. H., Cxw, P. J. S., TsFaic, T. C. and LEE, S.Y . (1968) Pharmarnlogical properties of cardiotoxin isolated from Formosan cobra venom. Arch . Pharmak . exp. Path. 299, 360. LektAncuHr.., D. and SrExRr N. (1%7) Electrical activity of cultured heart cell . In : Factors .erecting Myocardial Contractility (T~xz, R. D., ICnvni.J;x, F. and RosexTS, J., F.ds.). New York : Academic Press. Lorca, C. and PEtvrtv, I. F. (19s7) The structure of the naturally occurring phosphoglycerides . 3. Action of mocassin-venom phospholipase A on ovolecithin and related substances. Biochemistry 66, 382. LowxY, O. H., Ros®xoucR, N. J., Faxx, A. L. and Rarmau,, R. G. (1951) Protein measurement with the Folin phenol reagent. J. biol . Chem . 193, 265.
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McDoxni.n, T. F. and DaHnnx, R. L. (1973) Ion levcls and membrane potentials in chick heart tissue and cultured cells. J. gen. Physiol. 61, 89 . MELDRUM, B. S. (1965) The actions of snake venom on nerve and muscle. The pharmacology of phospholipase A and of polypeptide toxins. Pharmac. Rev. 17, 393. SARKAR, N. K. (1947) Isolation of cardiotoxin from cobra venom (Naja tripudimis, monocellate variety) . J. Ind. them. Soc. 24, 227. SkItcetvosu, K. and Srmer nxis, N. (1971) Development of sensitivity to tetrodotoxin of chick embryonic hearts with age. J. molec. Cell Cardiol. 3, 271 . SPEItELAKIS, N. and $HIQENOHU, K. (1972) Changes in membrane properties of chick embryonic hearts during development . J. gen. Physiol. 60, 430. YANG, C. C. (1974) Chemistry and evolution of toxins in snake venoms. Toxicon 12, 1 .
TOXICON 1973 Vol. 13
