Journalof

Molecular

and Cellular Cardiologv

Maintenance

11, 813-825

of Fast Na-Channels During Primary of Embryonic Chick Heart Cells*

A. M. LOMPREt, Luboratoire

(1979)

J. POGGIOLI

AND

Culture

G. VASSORT

de Physiologie Com&arke et de Physiologic Cellulaire associe’ au CNRS, Universitk Paris-XI. F 91405, Orsay, France

(Received

2 1 August

1978, accepted in revisedform

22 December

1978)

A. M. LOMPRE, J. POGGIOLI AND G. VASSORT. Maintenance of Fast Na-Channels During Primary Culture of Embryonic Chick Heart Cells.Jounal of Molecular andCellular Cardiology (1979) 11, 813-825. Cells isolated from 6-day-old chick embryonic heart were cultured for periods up to 5 days. Their electrical activity was recorded and resting membrane potential or maximal diastolic potential, threshold potential and maximal upstroke velocity were analyzed after 4,24 and 48 h and 4 or 5 days of culture. The spontaneously beating cells have a low maximal diastolic potential at 4 and 24 h after the dissociation treatment. Spontaneous activity may be supported by fast Na-channels and/or by slow channels. After 48 h of culture, very few cells remain spontaneously active. The resting membrane potential and maximal upstroke velocity of quiescent cells recover values close to that of the cells in vivo. Later, myocardial cells are gathered in mono or multilayers with many fibroblasts. Although their maximal diastolic potential remains highly negative they contract spontaneously. These cells have a low maximal upstroke velocity and show no or very little tetrodotoxin sensitivity. When a spontaneously active cell is stimulated at a higher rate, the rate and the amplitude of the diastolic depolarization decrease while the maximal upstroke velocity increases. In “pure culture”, myocardial cells form isolated clumps which are generally quiescent and which, when stimulated, develop fast rising, tetrodotoxin-sensitive action potentials. Appearance of automaticity, low maximal upstroke velocity and tetrodotoxin insensitivity have been proposed as criteria for “dedifferentiation” of cultured myoblasts. It is suggested, here, that these changes result from the dissociation process and later from the proliferation of fibroblasts and that the membrane of myocardial cells keeps its excitability properties unchanged. KEY WORDS:

Heart

cell culture;

Electrical

activity;

Fast Na-channel;

Fibroblast.

1. Introduction Heart cells isolated from 6- to 15-day-old chick embryo have been shown to lose their sensitivity to tetrodotoxin during culture [32, 33, 361. They have also been found to partially recover their younger embryonic properties such as low maximal diastolic potential (MDP), low maximal upstroke velocity (MUV) and automaticity in a *This work was supported by DGRST. tPresent address: A. M. LomprC, Groupe de Recherches vaisseaux, Unite 127 INSERM, Hop&al Lariboisiere, 75010 OOZZ-2828/79/080813+

15 $02.00/O

sur le Metabolisme Paris.

@ 1979 Academic

Press Inc.

du coeur (London)

et des Limited

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A. M. LOMPRE

ET AL.

process named “dedifferentiation”. This process is supposedly a reversion of the described evolution of chick myocardial cells during the course of normal embryonic development [18, 301. The slowing of the upstroke and the lack of effect of tetrodotoxin (TTX), a well-known specific inhibitor of the Na-conductance [Z, II] have been attributed to the disappearance of fast Na-channels. However, from morphological and biochemical evidence, Polinger [2.5] has suggested that cardiac myocytes, in vitro, maintain their differentiated state. Furthermore, the tetrodotoxin receptor is always present in the plasma membrane of cultured cells [7]. In the present work, the decrease of the rate of the action potential upstroke and of the TTX-sensitivity at different times of culture is tentatively explained mainly by a decrease of the availability of the Na-conductance rather than by the disappearance of Na-channels. It is suggested that the dissociation process at the beginning of the culture and later on, the presence of fibroblasts which have a very low membrane potential, may modify the electrical activity of the myoblasts. 2. Materials

and Methods Culture

Fertilized chick eggs(white Leghorn) were incubated at 38°C. The whole hearts of 6-day-old embryos were used throughout all the experiments. In most experiments, the dissociation and culture processeswere taken from Renaud and Le Douarin [27], i.e., trypsin 0.05% Ca, Mg-free Tyrode for 35 min. Isolated cells and clumps, small groups of cells which escapedfrom complete cell to cell dissociation, were plated. After plating at a density of 2.5 x 105cells per dish (250 mma), cells were incubated in a warm (37°C) saturated atmosphere (95% air, 5% COs). The culture medium consisted of 90% Eagle’s medium (M.E.M. TC 46 Wellcome) containing 200 4 penicillin G, 100 pg streptomycin and 250 pg amphotericin B/ml and 10% of heat-inactivated foetal calfserum. The medium wasrenewed every 48 h. In someexperiments, drugs were added to the standard medium as a small volume of sterile concentrated solution to give a final concentration of IO to 12 mEq for potassium (instead of 7 mEq in standard medium; K+ was measured by flamephotometry after the culture just before electrophysiological investigation) or 10 pg/ml for TTX. The method of Hyde et al. [IO] was used to obtain culture with very few fibroblasts (“pure culture”). After 2.5 h, cells still in suspension,essentially myoblasts, were gathered, centrifuged (3 min) and plated (7.5 x 104cells/dish). Myoblasts

andjibrobht-like

cells

Cell identification in non-confluent culture was basedon morphological criteria [3]. Myoblasts are thick, round or spindle-shapedcells; they are refractile with a dense

FAST

Na-CHANNELS

IN HEART

CELL

CULTURE

815

and granular cytoplasm. Fibroblast-like cells are thin, well spread with an irregular shape; they are non-refractile with few granules in the cytoplasm (Plates 1 and 2).

Beating activity

Twenty microscopic fields per plate were observed at random. About 70 clumps were scored as contracting or non-contracting after a 20 s observation period. Electrical

activity

Intracellular recordings were achieved with microelectrodes (20 to 60 MQ) connected via an Ag-AgCl electrode to a Medistor cathode-follower. The reference electrode was an Ag-AgCl electrode connected to the tissue bath via an agarRinger salt bridge and was earthed. The action potential and its first derivative obtained by a passive RC differentiator (linear from 0 to 150 V/s) were displayed on a Tektronix oscilloscope 502 A. Intracellular stimulation was carried out by another glasscapillary microelectrode connected by an Ag-AgCl electrode to a Tektronix stimulator (series160) and shielded with silver paint to decreasecapacitance. It was impaled at 100 to 200 pm from the recording electrode. The intensity of the applied current varied from 10 to 50 nA. Each selectedvalue of membrane potential was the best of two to four impalements in the same clump; n refers to the number of selected impalements; the statistical results are expressedas mean f

S.E.

3. Results

In order to investigate the properties of heart cells during the course of culture, the electrical activity and TTX sensitivity were checked and compared in beating as well as in quiescent cells, isolated from similar batches of 6-day-old hearts.

General observations (Plates 1 and 2)

After 4 h of culture, clumps isolated from 6-day-old hearts are about 50 to 150 pm in diameter. They are more extended and better attached to the bottom of the culture dish after 24 h. Within 48 h of culture, most of the isolated cells have developed protuberances and are interconnected in small monolayers. The fibroblasts are already numerous. Only some clumps remain isolated. After 4 to 5 days clumps and small monolayers are interconnected to form a confluent sheetof monolayered or of multilayered cells. Many more isolated clumps may be obtained during “pure culture” (see Materials and Methods). Microscopic controls have confirmed that there are very few fibroblasts after 4 or 5 days of culture, even less

816

A. M. LOMPRE

ET AL.

than in the usual 24 h culture. Only 23.5 & 3.5% of clumps are spontaneously beating after 48 h compared to 46.6 f after 4 and 24 h respectively while synchronous contractions areas of the cell sheet after 4 to 5 days. Nearly all the clumps quiescent. Electro@hysiological

and small monolayers 2.8 and 56.9 f 2.6% are observed in large in “pure culture” are

characteristics

The presenceof fast Na-channels was first checked on the original 6-day-old hearts since their appearance is still controversial and varies from 2 days [26] to at least 5 days [30]. The resting membrane potential was -70 f 2.7 mV and action potentials elicited on atria and ventricles had fast upstroke (MUV = 79.5 + 5.8 V/s, n = 19). Generally, TTX suppressedboth electrical and mechanical activities which occurred spontaneously. The resting membrane potential (RMP) is then stable and is equal to the previous RMP. In a very small area of the atrium which showed action potentials with slow upstroke, TTX was ineffective (three cases). Beating clumps and monolayers (Table 1)

Cells cultured for 4 and 24 h have a low MDP while the MDP of 5-day cultured cells is similar to the RMP of the whole heart. Nevertheless for both types of beating cells the threshold potential, ES, and the MUV are low so that there is no simple correlation between MDP and MUV of beating cells. Tetrodotoxin doesnot noticeably modify the electrical and mechanical activities of 48-h and 5-day cultured cells. However, following a 30 min period in the presence of TTX, the MUV and I& of 4- and 24-h cultured cells are significantly decreased. These action potentials (APs) are slow responses.The clumps which are beating in the presence of TTX include not only those whose electrical properties were not modified by the toxin, but alsothose which were sensitive to it and in which “TTXdesensitization” [19] occurred very rapidly. Such behaviour is illustrated in Figure 1 which evidences two types of electrical activities associated with contractions. A spontaneously contracting cell which had a rather fast upstroke (61 V/s) is blocked by the toxin, However, 22 min later it resumescontracting, at first irregularly, then with about the samefrequency. Its MDP is unchanged while its Es is -52 mV instead of -58 mV before TTX application. The rate of rise of the AP is markedly decreased (22 V/s). It remains constant for at least 32 min. A second aliquot of TTX, added several minutes after the first one, has no further effect. The recovery of spontaneous activity in the presence of TTX (which was interpreted as TTX-desensitization when counting the cell beats) is very frequent for 4-h cultured cells. This accounts for the apparent low sensitivity to TTX of contracting cells when only their mechanical activity is taken into account.

PLATE 1, Micrographs illustrating 4-h and 24-h stages 01’ chicken heart cells in culture. Fibroblasts and rnyoblasts are differentiated by thrir shape and refraction. F. fibroblast; X5, myoblast.

PLATE 2. Micrographs illustrating 48-h and 5-day stages of chicken heart cells in culture. C, clump; F, fibrobht; myoblast.

M,

FAST

TABLE

&-CHANNELS

IN HEART

1. Electrophysiological characteristics (10-s g/ml) of beating myocardial MDP

(mV)

CELL

817

CULTURE

before or after 30 min cells cultured for periods

Es (mV)

of TTX application of 4 h up to 5 days

MUV

(V/s)

4h clumps “in TTX”

(25) (25)

-67.2-&1.6 -62.8f1.4

-52.6*1.4 -42.6*1.1

(65) (17)

-61.5h1.2 -60.6-&1.5

-51.1*1.4 -44.5&Z

(20) ( 17) (4)

-66.3-&2.0 -69.3&2.2 -74.Ort3.8

-55.2&3.0 -57.21-3.0 -57.5k6.5

(27) ( 18) (18)

-71.1&1.6 -71.7k1.7 -66.0f2.1

-52.5h1.4 -56.3 h2.3 -57.5k1.5

45.9*3.3 20.6kl.2

24h clumps “in TTX”

(1) (2)

32.3k1.5 23.3k1.8

48-65 h clumps monolayers “in TTX” 5

27.8hl.8 35.Oh2.4 40.5*5.5*

days multilayers monolayers both in TTX

(3) (4)

25.6*1.2 31.8&1.3 32.2 f 1.2

In this and the following table: results are expressed as mean f s.E.; MDP: maximal diastolic potential; Es: threshold potential; MUV: maximal upstroke velocity. Numbers in parentheses denote the number of clumps studied. Statistical comparison for MUV: (l)-(Z): 0.001