Development of electrophysiological and biochemical membrane ...

Proc. Natl. Acad. Sci. USA

Vol. 74, No. 11, pp. 5166-5170, November 1977 Neurobiology

Development of electrophysiological and biochemical membrane properties during differentiation of embryonic skeletal muscle in culture (resting potential/electrical excitability/acetylcholine receptor/acetylcholinesterase)

ILAN SPECTOR AND JOAV M. PRIVES Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel

Communicated by Michael Sela, August 22,1977

ABSTRACT Newly fused chick myotubes undergo simultaneous and rapid changes in cell membrane properties during synchronous differentiation in culture. These changes are coordinately regulated and include increases in acetylcholine receptor, acetylcholinesterase, and resting potential, as well as the appearance of action potentials in discrete membrane areas upon stimulation. Subsequently, the acetylcholine receptor reaches maximal levels, whereas the development of electrical properties is marked by a further increase in resting potential, changes in the characteristics of the elicited action potential, and the recruitment of additional membrane areas for action potential generation. Maturation of electrical excitability, marked by the acquisition of the ability to fire repetitively and to conduct action potentials along the membrane, occurs well after resting potential has reached a maximum. During postmaturational development, myotubes exhibit spontaneous electrical and contractile activity, and levels of acetylcholine receptor aecessible to externally applied 125I-labeled a-bungarotoxin decrease markedly. It is suggested that electrophysiological membrane maturation is autonomously regulated with no requirement for neuronal intervention and involves the coordinated biosynthesis of discrete membrane components and their subsequent organization in the myotube membrane. Skeletal muscle cells differentiating in culture are highly suitable for studying the development of the distinctive biochemical and electrophysiological properties of excitable membranes. Fusion of myoblasts in culture to form multinucleated myotubes is accompanied by biosynthesis of contractile proteins, cytoplasmic enzymes, and specialized membrane components and by changes in electrophysiological properties (1-4). Like adult skeletal muscle, mature cultured myotubes are cross-striated, maintain a large resting membrane potential (RP), generate action potentials (APs), and contract rapidly (5). The sequence of development of these characteristics has been obscured by the lack of sufficient synchrony in fusion and myotube maturation in previous electrophysiological studies. Furthermore, while the expression of individual properties has been studied extensively, the sizeable variations in rate and extent of differentiation under different conditions make it difficult to correlate electrophysiological and biochemical aspects of membrane differentiation. In order to determine the developmental sequence of electrophysiological properties and to relate this development to changes in specialized membrane components assayed by biochemical methods in the same cultures, we have used chick embryonic muscle cultures showing rapid and synchronous differentiation kinetics. Our findings show that electrophysiological membrane differentiation is initiated shortly after

fusion and consists of an increase in RP and the sequential acquisition of the capacities for generation and propagation of APs. The initial stage of electrophysiological differentiation and the rapid elaboration of the cholinergic membrane components proceed simultaneously and appear to be regulated through a common developmental pathway. The maturation of electrical and contractile activity is, however, correlated with a progressive decrease in acetylcholine receptor (AcChR) levels, as detected by the specific binding of 25I-labeled a-bungarotoxin, to the surface of myotubes. METHODS Primary cultures of muscle cells were prepared from breast of 12-day chick embryos, as described earlier (6). To obtain rapid differentiation kinetics, cells were plated into collagen-coated 60-mm culture dishes at a density of 2 X 106 cells per dish and grown in Dulbecco's modified Eagle's Medium (88% vol/vol), horse serum (Gibco) (10% vol/vol), and chick embryo extract (2% vol/vol). Cultures were maintained at 370 in an atmosphere of 10% CO2 in air. The degree of cell fusion was determined as described (6). Cytosine arabinonucleoside (10 MAM) was added to cultures 2-3 days after plating for a 2-day period, to minimize fibroblast proliferation (7). Replicate plates were used for biochemical and electrophysiological determinations. AcChR was measured by the specific binding of 125I-abungarotoxin to muscle cultures as described previously (6). Acetylcholinesterase was assayed by measuring the hydrolysis of [1-14C]acetylcholine (Amersham) by intact cells using a modification of the method of Reed et al. (8) as described

(9).

Electrophysiological measurements were made in the culture dish held in a chamber on the stage of an inverted phase-contrast microscope with temperature maintained at 35-38'. The pH was kept at approximately 7.2 by passing 10% CO2 in air over the dish. The cultures were bathed in the growth medium which contained the following salt concentrations (mM): NaCl, 109.5; KCI, 5.4; CaCl2, 1.8; MgSO4, 0.8; NaHCO3, 44; NaH2PO4, 1. Micropipettes, filled with 3 M KCl and having resistances of 20-100 MQ, were arranged in the bridge circuit of a Bioelectric P system to allow stimulation of the cells through the recording pipette. The transmembrane voltage and stimulating currents as well as the electronically derived time derivative of the transmembrane voltage (dv/dt) were displayed on an oscilloscope and photographed. Passive and active membrane properties were measured as described (10).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviations: RP, resting membrane potential; AP, action potential; AcChR, acetylcholine receptor; dv/dt, time derivative of the transmembrane potential.

5166

Neurobiology: Spector and Prives

Proc. Natl. Acad. Sci. USA 74 (1977)

5167

mV801

A

0

.50

60-

-

40

-

-c 20

X CN

-6

6L.

A

-2

o I

1

.5>

_

-4

II#!

U

ft.£

2 --20

' orc

4

6 nA

O

0

-40

- -60 (D

--80

0

2

4 6 Days in culture

8

10

FIG. 1. Changes in membrane properties during differentiation of chick skeletal muscle in culture. Biochemical and electrophysiological measurements were carried out using replicate culture dishes. Myoblasts were plated at an initial density of 2 X 106 cells per 60-mm dish. (A) Kinetics of cell fusion (X) and of appearance of acetylcholine receptor (0) and acetylcholinesterase (0). Fusion is expressed as the percentage of total nuclei found in multinucleated cells. Acetylcholine receptor is expressed as pmol of 1251-a-bungarotoxin bound per dish. Acetylcholinesterase is expressed as nmol of acetylcholine hydrolyzed/2 min per dish. (B) Changes in RP (@) and maximum rate of rise of the AP (maximum dv/dt) (0). RPs are presented as mean i SD with the number of cells examined shown. RESULTS

The synchronous nature of muscle differentiation obtained under the culture conditions used was reflected by the rapid rate of myoblast fusion and by the simultaneous and rapid appearance of cell membrane components. As shown in Fig. 1A, onset of fusion occurred approximately 30 hr after plating, and by 48 hr the proportion of multinucleated myotubes had reached maximal values of 60-70%. At this point, AcChR and acetylcholinesterase started to increase rapidly and at similar rates. The myotubes showed a high rate of growth between days 3 and 5, and continued to fuse, forming syncytial muscle fibers. During this period AcChR, as assayed by the specific binding of 125I-a-bungarotoxin, reached maximal levels, which were maintained until day 6 and then started to decline, whereas acetylcholinesterase activity continued to increase. As shown in Fig. IB, mononucleated myoblasts had a mean RP of -9 mV and similar values were measured in newly fused myotubes. Shortly after the main period of cell fusion, however, the RP increased sharply, concomitantly with the coordinated appearance of the membrane components, to reach a mean value of -41 mV by day 3. A further rise in RP occurred between days 3 and 5 until maximal mean values of approximately -60 mV were reached. To determine the temporal relationship between the increase in RP and the development of electrical excitability, responses

FIG. 2. (A) Current-voltage relations obtained from n yotubes developing in culture: 55 hr after plating (0) and 96 hr after plating (0) and (A). Ordinate, membrane potential; abscissa, injected current. The points represent the potential change at 150 ms after onset of current pulse. Zero on the voltage axis corresponds to the RP of each cell [20 mV (0); 45 mV (0) and (A)]. Outward-going rectification is present in both myotubes from 96-hr cultures. Note that myotube (A) also shows a small region of inward-going rectification (lower left quadrant). (B and C) Responses to equal pulses of depolarizing and hyperpolarizing current elicited from the resting potential of the 96-hr myotubes. Time variant depolarization in B illustrates delayed rectification while inward-going rectification is shown in C. Myotube in C exhibited only electrotonic potential changes at the point of stimulation. Upper traces in B and C represent injected current (inward current shown as a downward deflection). Lower traces represent the membrane potential. A 10-ms, 20-mV calibration pulse is seen at the left of the lower traces. Calibration bars in B apply to C also.

elicited from the RP were compared in growing myotubes. The emergence of electrical excitability was evident from the appearance of rectification during development. Myotubes in 2-day cultures exhibited passive, symmetrical responses to equal pulses of depolarizing and hyperpolarizing current. Increments in the intensity of the applied current gave rise to linear steady-state current-voltage (I-V) relationships in both directions, indicating the absence of rectification in newly fused myotubes (Fig. 2A). Rectification, as reflected in nonlinear I-V relations, appeared concomitantly with the rapid increase in RP, between days 2 and 3 in culture. In most cases, cells showed outward-going rectification, that is nonlinear I-V relations in

the depolarizing direction (Fig. 2A). In such cells, the voltage response to strong depolarizing pulses first rose to a peak and then declined to a steady level (Fig. 2B). Inward-going rectification, that is the inverse I-V relation, was often observed at a certain range of membrane potentials when current intensity was low (Fig. 2 A and C). By 5 days in culture, depolarization of the RP resulted in fast APs (Fig. 3A). In addition, prolonged APs that outlasted the duration of the depolarizing step could be elicited, but only if RPs were more negative than -60 mV (Fig. 3B). The fast and prolonged APs were correlated with two types of mechanical responses of the myotube, a twitch and a contracture. However, in long (>1 mm) or branched myotubes both fast APs and twitches were observed only in discrete membrane patches intercalated with large areas showing passive electrical responses and no twitches. That fast AP generation was restricted to discrete regions at this stage was confirmed in several fibers by recording responses to depolarizing current pulses with a second intracellular microelectrode at various distances from the site of stimulation. In contrast, the prolonged AP and the contracture could be elicited from the entire myotube irrespective of the stimulation site or the myotube size. Furthermore, while a contracture was invariably associated with the

5168

Neurobiology: Spector and Prives

Proc. Natl. Acad. Sci. USA 74 (1977)

- Ea" FIG. 3. Electrical activity during muscle differentiation in culture. In all cases, recordings are from RP. (A and B) Voltage responses to depolarizing current pulses of myotubes from a 5-day culture. A fast AP is elicited in myotube A (RP -55 mV) and a prolonged AP associated with a contracture in myotube B (RP -65 mV). Arrows below each trace represent onset and offset of current pulse. Note slower time base in B. (C) Spontaneous discharge of fast APs upon microelectrode penetration of a myotube from a 10-day culture. The AP discharge was associated with twitches. Upper broken line represents zero potential level. Lower trace represents the dv/dt. (D) A fast AP elicited from a spontaneously active myotube in a 7-day culture (RP -60 mV). Two successive traces are superimposed to show the spontaneous and evoked APs. The uppermost trace represents the injected current; the middle trace represents the membrane potential; the lowest trace represents the du/dt. (E) Rapid contractions in the absence of APs in a myotube from a 7-day culture. A subthreshold current pulse applied at the'RP (-60 mV) elicited a passive response that was followed by a train of rapid contractions recorded on a dual time base oscilloscope as voltage fluctuations. Upper trace, fast sweep speed; lower trace, slow sweep speed. Voltage calibration bar in B applies to A and E also.

presence of the prolonged AP, local twitches were noted in response to sufficiently large membrane depolarizations at the site of stimulation or even at distances of 200-300 Atm on the same or a different branch of a myotube with no accompanying fast APs. By 7 days in culture, fast APs and twitches could be elicited from the entire myotube. Myotubes that had reached this stage were uniformly cross-striated and exhibited spontaneous electrical and contractile activity. During the second week in culture high-frequency discharges often accompanied microelectrode penetration of mechanically active myotubes (Fig. 3C). These myotubes did not exhibit prolonged APs when stimulated (Fig. 3D). In all cases fast APs were associated with twitches through the entire myotube; however, as illustrated in Fig. 3E, twitches were not always associated with fast APs. The observation that the RP reaches a maximum before the full expression of electrical excitability and contractility suggests that.changes in excitable properties that are independent of the rise in RP occur during development. In order to examine these changes, we eliminated variations in the initial RP by adjusting the membrane potential of growing myotubes to a standard level of -90 ± 5 mV with a steady inward current. As shown in Fig. 4A, we have found that depolarizing stimuli could produce fast and prolonged APs between days 2 and 3 in culture. Fast APs did not overshoot the zero potential level and had a peak amplitude of about -15 mV, and maximum dv/dt values ranged from 10 to 20 V/s. The threshold potential for AP initiation averaged -45 mV and the amplitude of the rising phase averaged 30 mV. The falling phase, in particular, was poorly developed: it levelled off 5-10 mV below the peak amplitude and had a maximum dv/dt of