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Maintained differentiation in rat cardiac monolayer cultures: Tetrodotoxin sensitivity and ultrastructure
Journal of Molecular and Cellular Cardiology (1980) 12, 493-498
RAPID
COMMUNICATIONS
Maintained Differentiation in Rat Cardiac Monolayer Cultures : Tetrodotoxin Sensitivity and Ultrastructure While cardiac cell culture would seem to be a promising technique for examining the development of the myocardial cell and its modification by the extracellular environment, a continuing concern is the loss in monolayer culture of certain characteristics of the intact source cardiac tissue, often attributed to cell “dedifferentiation” to an embryologically younger stage [17]. The most frequently cited example is loss of the tetrodotoxin (TTX) sensitive fast upstroke to the action potential [16]. It has been determined that the enzymes employed in tissue disaggregation can destroy or inactivate the TTX sensitive membrane channels; recovery of functional channels occurs in aggregate, but not monolayer, cultures and is .dependent on protein synthesis [12]. However, cardiac cells in monolayer culture are capable of performing unscheduled DNA synthesis after radiation induced damage [7] and monolayers of other cell types have been demonstrated to resynthesize certain membrane receptors that were lost upon trypsinization [18]. It is therefore puzzling that cardiac cells fail to repair or resynthesize the TTX sensitive fast channels in monolayer cultures. Several studies have questioned the concept of dedifferentiation in cardiac cultures. Polinger concluded from studies of DNA synthesis that overgrowth by fibroblasts rather than dedifferentiation was occurring in cardiac cultures [II]. Galper and Catterall demonstrated that the Na+ ionophore veratridine-which may act at the rapid Na+ channel [3]- increases Na+ flux across the membrane by a TTX sensitive mechanism [4], suggesting that inactive TTX sensitive fast channels were present. A recent article in this journal reported the presence of active fast channels in cardiac monolayer cultures [9]. While the upstroke values (pm,,) in this latter study were only 65 V/s, and the TTX concentration was high (10 mg/l), the report is significant in providing the first electrophysiologic support for the possibility that cardiac cells in monolayer culture do not revert to a more primitive form. Although the majority of studies have been conducted on cultures derived from embryonic chick heart, the concept of dedifferentiation has been extended to cultures from mammalian sources such as the neonatal rat heart. The developing * This work is supported by USPHS-NHLBI Heart Association. OOZO-2828/80/050493+06
$02.00/O
grant HL 12738 and a grant from the New York 0 1980 Academic Press Inc. (London)
Limited
494
R. B. ROBINSON
AND
M. J. LEGATO
rat heart is qualitatively similar to the chick in that the earliest recorded action potentials in both intact hearts are of a slow response, TTX insensitive form [Z, 151. In both cases, the source tissue has usually developed TTX sensitive fast response activity prior to culturing. Studies of rat heart monolayer cultures, like earlier studies on the chick, report slow upstroke action potentials [I, 141 which are TTX insensitive [14]. We now report culture conditions for rat cardiac monolayers which permit the recording of fast upstroke action potentials which are highly TTX sensitive. These cells are also well differentiated ultrastructurally, so that by both morphological and functional criteria they have not dedifferentiated in culture. The cultures were produced by a modification of an earlier method [lo]. Ventricles from 2- to 3-dayold rats are minced and dispersed by 12 serial exposures (15 min each) to 0.375 mg/ml trypsin (ICN, 1 : 250) in a HEPES buffered Ca2+-Mg2+ free salt solution at 37°C. The last 10 digests are collected, diluted 10% with cold horse serum, pooled and centrifuged at 200 x g for 5 min. The pellet is resuspended in growth medium (MEM in Hank’s Balanced Salt Solution, 12 mM additional NaHCO,, 2 pg/ml hypoxanthine, 20 pg/ml gentamycin and 10% horse serum) and plated at 5 x 105 cells/ml. After 14 to 24 h, when most non-muscle cells have attached, the muscle cell enriched supernatant is transferred to new dishes containing poly-1-lysine coated glass coverslips. The media is changed the next day to remove any unattached cells, and every three days thereafter. For electrophysiologic experiments coverslips from 3 to 7 day cultures are transferred to an open chamber on the stage of an inverted microscope equipped with interferencecontrast optics [S] and continuously suffused at 37°C with a serum-free solution based on the salt and glucose components of the growth medium. Cells are impaled with 30 to 50 MR glass microelectrodes filled with 3 M KC1 and attached to stage-mounted micromanipulators (E. Licht Company). The action potential is electronically differentiated by a circuit which is linear to beyond 200 V/s. For electron microscopic observation coverslips with attached cells were fixed by standard techniques [8] in gluteraldehyde and osmium, alcohol dehydrated and embedded in Epon by inverting the coverslip over an open ended Beam capsule. The coverslips were removed from the hardened blocks by submersion in warm water. The blocks were stained with lead citrate and uranyl acetate and cut at a shallow angle to the plane of cell growth. The monolayer preparation of rat myoblasts is not completely homogeneous ultrastructurally, with cells exhibiting varying degrees of differentiation in the electron microscope. Figure 1 is an electron micrograph of one of the more advanced myocytes. Such cells are filled with orderly rows of myofibrils and are ultrastructurally very similar to those of newborn rat myocardium. They are dominated by large nuclei (not shown) which have prominent nucleoli and an outer membrane covered with ribosomes. Mitochondria are abundant, with well developed internal cristae. Z-bands are wider and somewhat more irregularly
TETRODOTOXIN
SENSITIVITY
IN RAT
CARDIAC
CULTURES
495
FIGURE 1. An electron micrograph ofa portion of a well-differentiated myoblast from a 7-dayold monolayer preparation. Myofibrils fill the body of the cell and are arranged in orderly parallel array. Z-bands, which resemble those of the in uivo fetal heart, are wider and have a more irregular shape than in the adult myocardial cell. x 16 100.
contoured than in adult myocardium and centers of new myofilament production, with clusters of polyribosomes and accumulated Z-substance, are prominent. Rudimentary intercalated discs and other simpler specialized intercellular connections are abundant. A sarcoplasmic reticular network can be identified and rough endoplasmic reticulum, also a feature of in viuo fetal myocardium, is often seen. The T system is absent; it is a late post-natal development. Electrophysiologic recordings from these cultures demonstrate a high degree of functional differentiation in most impaled cells, with action potential upstroke velocities as great as 150 V/s. The high control pmax values were confirmed by a total of 28 impalements in cells from three separate culture preparations: 90.1 + 4.7 (S.E.) V/s; maximum diastolic potential (MDP) = -69.1 f Pm* = 1.5 (s.E.) mV. The effect of TTX (1 mg/l) on such a cell is illustrated in Figure 2. decreased more than 50% within 1 min. At the same time there was a ulna* marked decrease in overshoot. The TTX sensitivity was tested in a total of 7 impalements that were maintained during drug exposure (Table 1). The average decrease
496
R. B. ROBINSON
(a ) Control
AND
M. J. LEGATO
(b) TTX
Img/L
(c) Return
control
FIGURE 2. Effect of Tetrodotoxin (I mg/l) on rat cardiac cell culture action potential. (a) Control; (b) same impalement, 105 s after beginning suffusion with tetrodptoxin; (c) same impalement, 100 s after returning to control solution. Upper trace: dk’/dt, with I’,,,., indicated by peak of downward deflection (arrows) ; Lower trace: spontaneous transmembrane action potential.
in vm,, was 75.7%, and was highly significant by the t-test (P < 0.001). There was a non-significant (P > 0.1) decrease in MDP, possibly due to partial loss of the impalement during some experiments. This small decrease in MDP would not be expected to directly result in such a marked decrease in vmax, based on intact heart studies [6]. This was confirmed by passing hyperpolarizing current through the recording micro-electrode via a bridge circuit during TTX suffusion. In several such attempts, vmax never changed by more than a few percent although MDP was increased up to 10 mV.
TABLE
1. Effect of 1 mg/l tetrodotoxin monolayer cultures
on action
Control
92.9 f 13.5 v/s -67.6 f 2.0 mV
CIlax MDP Overshoot Amplitude n = 7 impalements, *P < 0.001.
31.0 f
4.8 mV
98.6 f 4.1 mV *
potential
parameters
in rat
ventricle
TTX
22.6 5 -63.8 f 25.9 & 89.7 f
9.6 V/s* 2.6 mV 2.3 mV 2.5 mVt
S.E.
t P < 0.05.
Also evident in Table 1 is a statistically significant (P < 0.05) decrease in action potential amplitude. Such a decrease, due to a loss of overshoot, has been reported following TTX exposure in newborn rat heart [Z]. The effect of TTX on overshoot was most marked in those cells exhibiting the greatest vm,, (Figure 2).
TETRODOTOXIN
SENSITIVITY
IN RAT
CARDIAC
CULTURES
497
The minimal effect on overshoot in other cells could result from an underlying slow channel automaticity-similar to that seen in TTX insensitive rat cardiac cultures [13]-which sustains activity even after the fast response is substantially blocked. In this regard, TTX ( 1 mg/ 1) re d uced the spontaneous rate by 2 1o/O in these cells, which is roughly comparable to that seen in slow response cardiac cultures [ 241. These results support and extend to a mammalian species the recent conclusion of Lompre et al. [9] that dedifferentiation does not occur in monolayer cardiac cultures. Their suggestion that cell depolarization resulting from fibroblast overgrowth of the cultures causes the appearance of dedifferentiation is consistent with our protocol and electrophysiological data, but is insufficient to explain the absence of other aspects of dedifferentiation, such as loss of myofibrils, in our cultures. It is possible that excessive trypsinization could account for poor differentiation, including morphological differentiation, in some culture preparations. In this respect, however, the limited electrophysiologic data presented here showed no detectable trend in vmax with culture age. The ability to maintain well differentiated cardiac cells in culture, and the determination of the specific culture conditions required, has important implications for biochemical and receptor studies on monolayer cultures in which average properties of all cells in the population are determined. Supposedly dedifferentiated cardiac cultures may, in fact, simply be enzyme damaged or contaminated by non-muscle cells. R. B. ROBINSON AND M. J. LEGATO* Department of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, J?ew 2’ork, New York 10032 U.S.A. * and Department of Medicine, the Roosevelt Hospital, 428 West 59 Street, New York, New York 10019, U.S.A.
REFERENCES 1. ATHIAS, P., PINSON, A., FRELIN, C., PADIEU, P. & KLEPPING, J. Comparative study on the effects of exogenous palmitate and erucate on intracellular electric properties of cultured beating heart cells. 3ou7ml of Molecular and Cellular Cardiology 11, 755-767 (1979). 2. BERNARD, C. & GARGOUIL, Y.-M. Acquisitions successives, chez l’embryon de Rat, des permeabilities specifiques de la membrane myocardique. Comptes Rendus Academic des Sciences, Paris, Series D 270, 1495-1498 (1970). 3. CATIIXRALL, W. A. & NIRENBERG, M. Sodium uptake associated with activation of action potential ionophores of cultured neuroblastoma and muscle cells. Proceedings of the National Academy of Sciences, U.S.A. 70, 3759-3763 (1973). l
Correspondence to Dr R. B. Robinson.
M.C.C.
u
498 4.
9. 10.
11. 12.
13.
14.
15.
16. 17.
18.
- k3. - KWBINSUN -^- ____^__ .__- _. _ _--.-ANU M. J. LLLiATO
K.
GALPER, J. B. & CATTERALL, W. A. Developmental changes in the sensitivity of embryonic heart cells to tetrodotoxin and D600. Develo&nental Biology 65, 216-227 (1978). HERMSMEYER, K. & ROBINSON, R. B. High sensitivity of cultured cardiac muscle cells to autonomic agents. American Journal of Physiology 233, Cl 72-C 179 (1977). IIJIMA, T. & PAPPANO, A. J. Ontogenetic increase of the maximal rate of rise of the chick embryonic heart action potential. Circulation Research 44, 358-367 (1979). LAMPIDIS, T. J. & SCHAIBERGER, G. E. Age-related loss of DNA repair synthesis in isolated rat myocardial cells. Exfierimental Cell Research 96, 412-416 (1975). LEGATO, M. J. Ultrastructural characteristics of the rat ventricular cell grown in tissue culture, with special reference to sarcomerogenesis. Journal of Molecular and Cellular Cardiology 4, 299-317 (1972). LOMPRE, A. M., POGGLIOLI, J. & VASSORT, G. Maintenance of fast Na+ channels during primary culture of embryonic chick heart cells. Journal of Molecular and Cellular CardioloQ 11, 813-825 (1979). MARVIN, JR., W. J., ROBINSON, R. B. & HERMSMEYER, K. Correlation of function and morphology of neonatal rat and embryonic chick cardiac and vascular muscle cells. Circulation Research 45, 528-540 ( 1979). POLINGER, I. S. Growth and DNA synthesis in embryonic chick heart cells, in vivo and in vitro. Experimental Cell Research 76, 253-262 (1973). SACHS, H. G., MCDONALD, T. F. & DEHAAN, R. L. Tetrodotoxin sensitivity of cultured embryonic heart cells depends on cell interactions. Journal of Cell Biology 56, 255-258 (1973). SCHANNE, 0. F., RUIZ-CERETTI, E., PAYET, M. D. & DESLAURIERS, Y. Influence of varied {Caz+}a and {Na+}, on electrical activity of clusters of cultured cardiac cells from neonatal rats. Journal of Molecular and Cellular Cardiology 11, 477-484 (1979). SCHANNE, 0. F., RUIZ-CERETTI, E., RIVARD, C. & CHARTIER, D. Determinants of electrical activity in clusters of cultured cardiac cells from neonatal rats. Journal of Molecular and Cellular Cardiology 9, 269-283 (1977). SHIGENOBU, K. & SPERELAKIS, N. Development of sensitivity to tetrodotoxin of chick embryonic hearts with age. Journal of Molecular andcellular Cardiology 3,271-286 (197 1). SPERELAKIS, N. & LEHMKUHL, D. Insensitivity of cultured chick heart cells to autonomic agents and tetrodotoxin. The American Journal of Physiology 209, 6933698 ( 1965). SPERELAKIS, N., SHIGENOBU, K. & MCLEAN, M. J. Membrane cation channels: Changes in developing hearts, in cell culture, and in organ culture. In Developmental and Physiological Correlates of Cardiac Muscle, pp. 209-234. M. Lieberman & T. Sano, Eds. New York: Raven Press (1976). WINAND, R. J, & KOHN, L. D. Thyrotropin effects on thyroid cells in culture. Journal of Biological Chemistry 250, 6534-6540 (1975).
RAPID
COMMUNICATIONS
Maintained Differentiation in Rat Cardiac Monolayer Cultures : Tetrodotoxin Sensitivity and Ultrastructure While cardiac cell culture would seem to be a promising technique for examining the development of the myocardial cell and its modification by the extracellular environment, a continuing concern is the loss in monolayer culture of certain characteristics of the intact source cardiac tissue, often attributed to cell “dedifferentiation” to an embryologically younger stage [17]. The most frequently cited example is loss of the tetrodotoxin (TTX) sensitive fast upstroke to the action potential [16]. It has been determined that the enzymes employed in tissue disaggregation can destroy or inactivate the TTX sensitive membrane channels; recovery of functional channels occurs in aggregate, but not monolayer, cultures and is .dependent on protein synthesis [12]. However, cardiac cells in monolayer culture are capable of performing unscheduled DNA synthesis after radiation induced damage [7] and monolayers of other cell types have been demonstrated to resynthesize certain membrane receptors that were lost upon trypsinization [18]. It is therefore puzzling that cardiac cells fail to repair or resynthesize the TTX sensitive fast channels in monolayer cultures. Several studies have questioned the concept of dedifferentiation in cardiac cultures. Polinger concluded from studies of DNA synthesis that overgrowth by fibroblasts rather than dedifferentiation was occurring in cardiac cultures [II]. Galper and Catterall demonstrated that the Na+ ionophore veratridine-which may act at the rapid Na+ channel [3]- increases Na+ flux across the membrane by a TTX sensitive mechanism [4], suggesting that inactive TTX sensitive fast channels were present. A recent article in this journal reported the presence of active fast channels in cardiac monolayer cultures [9]. While the upstroke values (pm,,) in this latter study were only 65 V/s, and the TTX concentration was high (10 mg/l), the report is significant in providing the first electrophysiologic support for the possibility that cardiac cells in monolayer culture do not revert to a more primitive form. Although the majority of studies have been conducted on cultures derived from embryonic chick heart, the concept of dedifferentiation has been extended to cultures from mammalian sources such as the neonatal rat heart. The developing * This work is supported by USPHS-NHLBI Heart Association. OOZO-2828/80/050493+06
$02.00/O
grant HL 12738 and a grant from the New York 0 1980 Academic Press Inc. (London)
Limited
494
R. B. ROBINSON
AND
M. J. LEGATO
rat heart is qualitatively similar to the chick in that the earliest recorded action potentials in both intact hearts are of a slow response, TTX insensitive form [Z, 151. In both cases, the source tissue has usually developed TTX sensitive fast response activity prior to culturing. Studies of rat heart monolayer cultures, like earlier studies on the chick, report slow upstroke action potentials [I, 141 which are TTX insensitive [14]. We now report culture conditions for rat cardiac monolayers which permit the recording of fast upstroke action potentials which are highly TTX sensitive. These cells are also well differentiated ultrastructurally, so that by both morphological and functional criteria they have not dedifferentiated in culture. The cultures were produced by a modification of an earlier method [lo]. Ventricles from 2- to 3-dayold rats are minced and dispersed by 12 serial exposures (15 min each) to 0.375 mg/ml trypsin (ICN, 1 : 250) in a HEPES buffered Ca2+-Mg2+ free salt solution at 37°C. The last 10 digests are collected, diluted 10% with cold horse serum, pooled and centrifuged at 200 x g for 5 min. The pellet is resuspended in growth medium (MEM in Hank’s Balanced Salt Solution, 12 mM additional NaHCO,, 2 pg/ml hypoxanthine, 20 pg/ml gentamycin and 10% horse serum) and plated at 5 x 105 cells/ml. After 14 to 24 h, when most non-muscle cells have attached, the muscle cell enriched supernatant is transferred to new dishes containing poly-1-lysine coated glass coverslips. The media is changed the next day to remove any unattached cells, and every three days thereafter. For electrophysiologic experiments coverslips from 3 to 7 day cultures are transferred to an open chamber on the stage of an inverted microscope equipped with interferencecontrast optics [S] and continuously suffused at 37°C with a serum-free solution based on the salt and glucose components of the growth medium. Cells are impaled with 30 to 50 MR glass microelectrodes filled with 3 M KC1 and attached to stage-mounted micromanipulators (E. Licht Company). The action potential is electronically differentiated by a circuit which is linear to beyond 200 V/s. For electron microscopic observation coverslips with attached cells were fixed by standard techniques [8] in gluteraldehyde and osmium, alcohol dehydrated and embedded in Epon by inverting the coverslip over an open ended Beam capsule. The coverslips were removed from the hardened blocks by submersion in warm water. The blocks were stained with lead citrate and uranyl acetate and cut at a shallow angle to the plane of cell growth. The monolayer preparation of rat myoblasts is not completely homogeneous ultrastructurally, with cells exhibiting varying degrees of differentiation in the electron microscope. Figure 1 is an electron micrograph of one of the more advanced myocytes. Such cells are filled with orderly rows of myofibrils and are ultrastructurally very similar to those of newborn rat myocardium. They are dominated by large nuclei (not shown) which have prominent nucleoli and an outer membrane covered with ribosomes. Mitochondria are abundant, with well developed internal cristae. Z-bands are wider and somewhat more irregularly
TETRODOTOXIN
SENSITIVITY
IN RAT
CARDIAC
CULTURES
495
FIGURE 1. An electron micrograph ofa portion of a well-differentiated myoblast from a 7-dayold monolayer preparation. Myofibrils fill the body of the cell and are arranged in orderly parallel array. Z-bands, which resemble those of the in uivo fetal heart, are wider and have a more irregular shape than in the adult myocardial cell. x 16 100.
contoured than in adult myocardium and centers of new myofilament production, with clusters of polyribosomes and accumulated Z-substance, are prominent. Rudimentary intercalated discs and other simpler specialized intercellular connections are abundant. A sarcoplasmic reticular network can be identified and rough endoplasmic reticulum, also a feature of in viuo fetal myocardium, is often seen. The T system is absent; it is a late post-natal development. Electrophysiologic recordings from these cultures demonstrate a high degree of functional differentiation in most impaled cells, with action potential upstroke velocities as great as 150 V/s. The high control pmax values were confirmed by a total of 28 impalements in cells from three separate culture preparations: 90.1 + 4.7 (S.E.) V/s; maximum diastolic potential (MDP) = -69.1 f Pm* = 1.5 (s.E.) mV. The effect of TTX (1 mg/l) on such a cell is illustrated in Figure 2. decreased more than 50% within 1 min. At the same time there was a ulna* marked decrease in overshoot. The TTX sensitivity was tested in a total of 7 impalements that were maintained during drug exposure (Table 1). The average decrease
496
R. B. ROBINSON
(a ) Control
AND
M. J. LEGATO
(b) TTX
Img/L
(c) Return
control
FIGURE 2. Effect of Tetrodotoxin (I mg/l) on rat cardiac cell culture action potential. (a) Control; (b) same impalement, 105 s after beginning suffusion with tetrodptoxin; (c) same impalement, 100 s after returning to control solution. Upper trace: dk’/dt, with I’,,,., indicated by peak of downward deflection (arrows) ; Lower trace: spontaneous transmembrane action potential.
in vm,, was 75.7%, and was highly significant by the t-test (P < 0.001). There was a non-significant (P > 0.1) decrease in MDP, possibly due to partial loss of the impalement during some experiments. This small decrease in MDP would not be expected to directly result in such a marked decrease in vmax, based on intact heart studies [6]. This was confirmed by passing hyperpolarizing current through the recording micro-electrode via a bridge circuit during TTX suffusion. In several such attempts, vmax never changed by more than a few percent although MDP was increased up to 10 mV.
TABLE
1. Effect of 1 mg/l tetrodotoxin monolayer cultures
on action
Control
92.9 f 13.5 v/s -67.6 f 2.0 mV
CIlax MDP Overshoot Amplitude n = 7 impalements, *P < 0.001.
31.0 f
4.8 mV
98.6 f 4.1 mV *
potential
parameters
in rat
ventricle
TTX
22.6 5 -63.8 f 25.9 & 89.7 f
9.6 V/s* 2.6 mV 2.3 mV 2.5 mVt
S.E.
t P < 0.05.
Also evident in Table 1 is a statistically significant (P < 0.05) decrease in action potential amplitude. Such a decrease, due to a loss of overshoot, has been reported following TTX exposure in newborn rat heart [Z]. The effect of TTX on overshoot was most marked in those cells exhibiting the greatest vm,, (Figure 2).
TETRODOTOXIN
SENSITIVITY
IN RAT
CARDIAC
CULTURES
497
The minimal effect on overshoot in other cells could result from an underlying slow channel automaticity-similar to that seen in TTX insensitive rat cardiac cultures [13]-which sustains activity even after the fast response is substantially blocked. In this regard, TTX ( 1 mg/ 1) re d uced the spontaneous rate by 2 1o/O in these cells, which is roughly comparable to that seen in slow response cardiac cultures [ 241. These results support and extend to a mammalian species the recent conclusion of Lompre et al. [9] that dedifferentiation does not occur in monolayer cardiac cultures. Their suggestion that cell depolarization resulting from fibroblast overgrowth of the cultures causes the appearance of dedifferentiation is consistent with our protocol and electrophysiological data, but is insufficient to explain the absence of other aspects of dedifferentiation, such as loss of myofibrils, in our cultures. It is possible that excessive trypsinization could account for poor differentiation, including morphological differentiation, in some culture preparations. In this respect, however, the limited electrophysiologic data presented here showed no detectable trend in vmax with culture age. The ability to maintain well differentiated cardiac cells in culture, and the determination of the specific culture conditions required, has important implications for biochemical and receptor studies on monolayer cultures in which average properties of all cells in the population are determined. Supposedly dedifferentiated cardiac cultures may, in fact, simply be enzyme damaged or contaminated by non-muscle cells. R. B. ROBINSON AND M. J. LEGATO* Department of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, J?ew 2’ork, New York 10032 U.S.A. * and Department of Medicine, the Roosevelt Hospital, 428 West 59 Street, New York, New York 10019, U.S.A.
REFERENCES 1. ATHIAS, P., PINSON, A., FRELIN, C., PADIEU, P. & KLEPPING, J. Comparative study on the effects of exogenous palmitate and erucate on intracellular electric properties of cultured beating heart cells. 3ou7ml of Molecular and Cellular Cardiology 11, 755-767 (1979). 2. BERNARD, C. & GARGOUIL, Y.-M. Acquisitions successives, chez l’embryon de Rat, des permeabilities specifiques de la membrane myocardique. Comptes Rendus Academic des Sciences, Paris, Series D 270, 1495-1498 (1970). 3. CATIIXRALL, W. A. & NIRENBERG, M. Sodium uptake associated with activation of action potential ionophores of cultured neuroblastoma and muscle cells. Proceedings of the National Academy of Sciences, U.S.A. 70, 3759-3763 (1973). l
Correspondence to Dr R. B. Robinson.
M.C.C.
u
498 4.
9. 10.
11. 12.
13.
14.
15.
16. 17.
18.
- k3. - KWBINSUN -^- ____^__ .__- _. _ _--.-ANU M. J. LLLiATO
K.
GALPER, J. B. & CATTERALL, W. A. Developmental changes in the sensitivity of embryonic heart cells to tetrodotoxin and D600. Develo&nental Biology 65, 216-227 (1978). HERMSMEYER, K. & ROBINSON, R. B. High sensitivity of cultured cardiac muscle cells to autonomic agents. American Journal of Physiology 233, Cl 72-C 179 (1977). IIJIMA, T. & PAPPANO, A. J. Ontogenetic increase of the maximal rate of rise of the chick embryonic heart action potential. Circulation Research 44, 358-367 (1979). LAMPIDIS, T. J. & SCHAIBERGER, G. E. Age-related loss of DNA repair synthesis in isolated rat myocardial cells. Exfierimental Cell Research 96, 412-416 (1975). LEGATO, M. J. Ultrastructural characteristics of the rat ventricular cell grown in tissue culture, with special reference to sarcomerogenesis. Journal of Molecular and Cellular Cardiology 4, 299-317 (1972). LOMPRE, A. M., POGGLIOLI, J. & VASSORT, G. Maintenance of fast Na+ channels during primary culture of embryonic chick heart cells. Journal of Molecular and Cellular CardioloQ 11, 813-825 (1979). MARVIN, JR., W. J., ROBINSON, R. B. & HERMSMEYER, K. Correlation of function and morphology of neonatal rat and embryonic chick cardiac and vascular muscle cells. Circulation Research 45, 528-540 ( 1979). POLINGER, I. S. Growth and DNA synthesis in embryonic chick heart cells, in vivo and in vitro. Experimental Cell Research 76, 253-262 (1973). SACHS, H. G., MCDONALD, T. F. & DEHAAN, R. L. Tetrodotoxin sensitivity of cultured embryonic heart cells depends on cell interactions. Journal of Cell Biology 56, 255-258 (1973). SCHANNE, 0. F., RUIZ-CERETTI, E., PAYET, M. D. & DESLAURIERS, Y. Influence of varied {Caz+}a and {Na+}, on electrical activity of clusters of cultured cardiac cells from neonatal rats. Journal of Molecular and Cellular Cardiology 11, 477-484 (1979). SCHANNE, 0. F., RUIZ-CERETTI, E., RIVARD, C. & CHARTIER, D. Determinants of electrical activity in clusters of cultured cardiac cells from neonatal rats. Journal of Molecular and Cellular Cardiology 9, 269-283 (1977). SHIGENOBU, K. & SPERELAKIS, N. Development of sensitivity to tetrodotoxin of chick embryonic hearts with age. Journal of Molecular andcellular Cardiology 3,271-286 (197 1). SPERELAKIS, N. & LEHMKUHL, D. Insensitivity of cultured chick heart cells to autonomic agents and tetrodotoxin. The American Journal of Physiology 209, 6933698 ( 1965). SPERELAKIS, N., SHIGENOBU, K. & MCLEAN, M. J. Membrane cation channels: Changes in developing hearts, in cell culture, and in organ culture. In Developmental and Physiological Correlates of Cardiac Muscle, pp. 209-234. M. Lieberman & T. Sano, Eds. New York: Raven Press (1976). WINAND, R. J, & KOHN, L. D. Thyrotropin effects on thyroid cells in culture. Journal of Biological Chemistry 250, 6534-6540 (1975).