Development of electrophysiological and biochemical membrane properties during differentiation of embryonic skeletal muscle in culture.

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 afterThe 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 sole...

Development of excitability during the in vitro differentiation of a newly established myogenic cell line.

“…So, it is likely that the action potential-generating mechanism in the M l3 myogenic cell line develops soon after the formation of myotubes and accompanies the increase in Em. These results are quite in agreement with those reported in cultured chick myotubes (KANO et al, 1972(KANO et al, , 1977SPECTOR and PRIVES, 1977) in which the electrophysiological development was ascertained to be initiated shortly after cell fusion. On the other hand, KIDOKORO (1975) reported that mononucleated, undifferentiated L6 cells exhibit a large Em of -71 mV that does not change after cell fusion and also that they have the capacity to generate, though they are abortive, Na+ action potentials by anodal break stimuli.…”

“…In most of the electrophysiological studies on cultured skeletal myotubes, besides the fast action potential, a prolonged action potential has also been reported (KANO et al, 1972;FUKUDA,1974;SPECTOR and PRIVES, 1977) and was also demonstrated in situ by using chick muscle in embryonic and early postnatal development (KANO, 1975). This property was explained as due to a voltage-dependent increase in membrane permeability to Ca2+ (KANO, 1975) or Cl-(FUKUDA, 1974.…”

“…et a!., 1971; KANO et al, 1972KANO et al, , 1977RITCHIE and FAMBROUGH, 1975;SPECTOR and PRIvEs, 1977) and the culture of an established myogenic cell line (LAND et al, 1973;KIDOKORO, 1973KIDOKORO, , 1975. Of these, the clonal cell line would offer an advantageous system for these studies because of its homogeneous cell population and synchronism of differentiation.…”

Communications

“…This is in agreement with the observations of others (Kano & Shimada, 1973;Fukuda, 1974;Spector & Prives, 1977). The development of regenerative potentials is compromised by prior exposure of the culture to 10-Mcytosine-l-/J-D-arabinofuranoside.…”

Communications

“…Membrane potential (Em) rises during myogenesis and post-fusion development of muscle fibres over a 2-to 3-day period in culture to attain levels found in adult muscle (-60 to -70 mV) (Spector & Prives, 1977). We have examined both Em and resting membrane conductance (Gm) in chick embryo breast muscle in culture.…”