Electrical properties of normal and dysgenic mouse skeletal muscle in culture

Electrical properties of normal and dysgenic mouse skeletal muscle were studied by intracellular recording from embryonic cells developing in vitro. Passive membrane constants were determined from records of transmembrane potential responses to hyperpolarizing pulses of current using two types of analyses, assuming the tubes to be finite cylinders: the off transient and steady state analyses. The following properties of normal and dysgenic fibers were also studied. (a) membrane potentials (b) acetylcholine sensitivity (c) α-Bungarotoxin binding and (d) maximum rate of rise, overshoot and one-half fall time of the action potential. Rare electrotonic coupling between fibroblasts and myotubes was noted. An anomalous type of rectification Was observed in some fibers in which the transmembrane potential responses possessed under and overshoots. These responses may have affected the values of membrane constants as derived by the off transient analysis. In all parameters studied, including membrane constants derived by the steady state analysis, the cultured mouse cells resembled adult denervated mammalian muscle rather than innervated muscle. There were no differences between normal and dysgenic fibers with respect to any of the parameters studied. Dysgenic fibers did not contract although they displayed passive and active membrane properties like those in normal, non-dysgenic fibers.     

Electrical properties of chick skeletal muscle fibers developing in cell culture

The membrane properties of individual skeletal muscle cells were studied with intracellular microelectrodes as the fibers developed, in vitro, from mononucleated precursor cells. Passive membrane constants were determined from analysis of transmembrane potential responses to pulses of current assuming the myotubes could be represented as sealed, finite cylinders. Resting membrane potentials increased from 10–15 mV in the shortest, youngest myotubes to ca. 60 mV in the longest, most mature fibers. The increase in membrane potential was not associated with a change in membrane resistivity. Action potentials occurred spontaneously in the most mature cells and repetitive spikes could be evoked by depolarizing current pulses. Spikes and twitches could be evoked in young myotubes provided the membrane was first hyperpolarized to 60–70 mV. Apparently the membrane potential is the rate limiting factor in the maturation of excitation-contraction mechanisms.     

Charge movement and Ca2+ release in normal and dysgenic foetal myotubes.

Intramembrane charge movement and Ca2+ release from sarcoplasmic reticulum was studied in foetal skeletal muscle cells from normal and mutant mice with 'muscular dysgenesis' (mdg/mdg). It was shown that: 1) unlike normal myotubes, in dysgenic myotubes membrane depolarization did not evoke calcium release from the sarcoplasmic reticulum; 2) when all ionic currents are pharmacologically suppressed, membrane depolarization produced an asymmetric intramembrane charge movement in both normal and dysgenic myotubes. The relationship between the membrane potential and the amount of charge movement in these muscles could be expressed by a two-state Boltzmann equation; 3) the maximum amount of charge movement associated with depolarization (Qon max) in normal and in dysgenic myotubes was 6.3 +/- 1.4 nC/microF (n = 6) and 1.7 +/- 0.3 nC/microF (n = 6) respectively; 4) nifedipine (1-20 microM) applied to the bath reduced Qon max by about 40% in normal muscle cells. In contrast, the drug had no significant effect on the charge movement of dysgenic myotubes; and 5) the amount of nifedipine-resistant charge movement in normal and in dysgenic myotubes was 3.5 nC/microF (n = 3) and 1.7 nC/microF 1 maximum (n = 3), respectively.     

Transmembrane electrical characteristics of cultured human skeletal muscle cells

Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3-4 days, and fusion of myoblasts was observed in 5-10 days. Multinucleated myotubes were impaled under high optical magnification, at 37 degrees C, with conventional glass microelectrodes. The mean resting potential was -44.4 mV +/- 2.4 (n = 399); -33 +/- 2.3 mV at 9 days (n = 10) vs -48 +/- 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M omega (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 +/- 2.1 mV with a mean maximum rate of rise (Vmax) of 72.1 +/- 7.5 V/s. The mean overshoot was 13.9 +/- 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5-100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.     

Electrophysiological Actions of Oxytocin on the Rabbit Myometrium

The electrical activities of myometrial cells of the pregnant rabbit uterus have been studied by means of sucrose-gap and intracellular micro-electrode recording techniques. The resting potential of the myometrial cell was about -50 mv, and it is unaffected by the duration of pregnancy or placental attachment. Action potentials of the myometrium, although dependent on external Na⁺, were not always of the regenerative type; preparations from nonparturient uteri often produce mainly small spikes. The mean spike amplitude was 35 mv, rising at a mean maximum rate of 3 v/sec. Oxytocin, in concentrations less than 500 µU/ml, increased the mean spike amplitude to 48 mv and the mean maximum rate of rise to 7 v/sec, without affecting the resting potential. The relation between membrane potential and dV/dt of the spike was steepened by oxytocin, suggesting that oxytocin increased the number of normally sparse sodium gates in the myometrial membrane. By this action, oxytocin is believed to increase the probability of successful regenerative spikes and thereby initiate electrical activity in quiescent preparations, increase the frequency of burst discharges, the number of spikes in each burst, and the amplitude of spikes in individual cells.     

Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane

The effects of various divalent cations in the external solution upon the Ca spike of the barnacle muscle fiber membrane were studied using intracellular recording and polarizing techniques. Analysis of the maximum rate of rise of the spike potential indicates that different species of divalent cations bind the same membrane sites competitively with different dissociation constants. The overshoot of the spike potential is determined by the density of Ca (Sr) ions in the membrane sites while the threshold membrane potential for spike initiation depends on the total density of divalent cations. The order of binding among different divalent and trivalent cations is the following: La+++, UO2++ > Zn++, Co++, Fe++ > Mn++ > Ni++ > Ca++ > Mg++, Sr++.     

Spike Potentials Recorded from the Insect Photoreceptor

Slow and spike potentials were recorded from single cells in the receptor layer of the compound eye of the drone of the honeybee. From electron microscopic observation of the drone ommatidium, it was concluded that the response had been recorded from the retinula cell. The following hypothesis is suggested for the initiation of spike potentials in the drone compound eye: Photic stimulation results in a decrease in the resistance of all or part of the retinula cell membrane, giving rise to the retinal action potential. The retinal action potential causes outflow of the current through the proximal process of the cell. This depolarizing current initiates spike potentials in the proximal process or axon of the retinula cell which are recorded across the soma membrane of the retinula cell.     

Dynamic relationship between the slow potential and spikes in cockroach ocellar neurons

The relationship between the slow potential and spikes of second-order ocellar neurons of the cockroach, Periplaneta americana, was studied. The stimulus was a sinusoidally modulated light with various mean illuminances. A solitary spike was generated at the depolarizing phase of the modulation response. Analysis of the relationship between the amplitude/frequency of voltage modulation and the rate of spike generation showed that (a) the spike initiation process was bandpass at approximately 0.5-5 Hz, (b) the process contained a dynamic linearity and a static nonlinearity, and (c) the spike threshold at optimal frequencies (0.5-5 Hz) remained unchanged over a mean illuminance range of 3.6 log units, whereas (d) the spike threshold at frequencies of less than 0.5 Hz was lower at a dimmer mean illuminance. The voltage noise in the response was larger and the mean membrane potential level was more positive at a dimmer mean illuminance. Steady or noise current injection during sinusoidal light stimulation showed that (a) the decrease in the spike threshold at a dimmer mean illuminance was due to the increase in the noise variance: the noise had facilitatory effects on the spike initiation; and (b) the change in the mean potential level had little effect on the spike threshold. We conclude that fundamental signal modifications occur during the spike initiation in the cockroach ocellar neuron, a finding that differs from the spike initiation process in other visual systems, including Limulus eye and vertebrate retina, in which it is presumed that little signal modification occurs at the analog-to-digital conversion process.     

The N-Shaped Current-Potential Characteristic in Frog Skin: II. Kinetic Behavior during Ramp Voltage Clamp

Previous step voltage-clamp measurements on frog skin showed the presence of an N-shaped current-potential (I-V) relation in excitable skin. However, the collection and reconstruction of I-V data using discrete step changes of skin potential was tedious because of the long refractory period (up to 1 min) in frog skin. A direct and rapid (5 msec) method for recording the N-shaped I-V characteristic in real time is presented. Ramp functions are used as the command to the clamp system instead of a step function. Consequently the skin potential is forced to change in a linear manner (as commanded) and the skin current can be recorded as a continuous function of the controlled change of skin potential. With the ramp clamp, a low-resistance membrane state ( 10 Omega . cm(2)) resembling a breakdown phenomenon was observed at high skin potential ( 300 mv). Entry into the low resistance state resulted in a collapse of the N-shaped I-V relation to a nearly linear function. The utility of the ramp measurement is demonstrated by predicting (1) that the maximum rate of rise of the spike occurs at a voltage corresponding to the valley (local minimum) in the N-shaped I-V curve, (2) that the rate of rise of the spike increases with increasing clamp currents, (3) the voltage peak of the spike, and (4) the time course of the rising phase of the spike.     

Development of action potential in larval muscle fibers in Drosophila melanogaster.

In the presence of tetraethylammonium or barium ions, the larval muscle fibers of Drosophila melanogaster were found to produce an all-or-none action potential operated by the calcium channels. The development of this distinctive membrane property during the maturation of muscle cells was studied by measuring the maximum rate of rise of the action potential in the larval muscle fibers at different stages of development from the sixteenth to ninety-sixth hours after hatching. The value increased significantly with age until a peak was reached at the sixty-fourth hour, although it became lower again as puparium formation neared at about the ninety-sixth hour. This suggests that during larval development the muscle fibers develop the ability to generate an action potential due to an inward current through the calcium channels, although the ability became lower at the later stage of larval development.     

Skeletal myotube integration with planar microelectrode arrays in vitro for spatially selective recording and stimulation: a comparison of neuronal and myotube extracellular action potentials

Microelectrode array (MEA) technology holds tremendous potential in the fields of biodetection, lab-on-a-chip applications, and tissue engineering by facilitating noninvasive electrical interaction with cells in vitro. To date, significant efforts at integrating the cellular component with this detection technology have worked exclusively with neurons or cardiac myocytes. We investigate the feasibility of using MEAs to record from skeletal myotubes derived from primary myoblasts as a way of introducing a third electrogenic cell type and expanding the potential end applications for MEA-based biosensors. We find that the extracellular action potentials (EAPs) produced by spontaneously contractile myotubes have similar amplitudes to neuronal EAPs. It is possible to classify myotube EAPs by biological signal source using a shape-based spike sorting process similar to that used to analyze neural spike trains. Successful spike-sorting is indicated by a low within-unit variability of myotube EAPs. Additionally, myotube activity can cause simultaneous activation of multiple electrodes, in a similar fashion to the activation of electrodes by networks of neurons. The existence of multiple electrode activation patterns indicates the presence of several large, independent myotubes. The ability to identify these patterns suggests that MEAs may provide an electrophysiological basis for examining the process by which myotube independence is maintained despite rapid myoblast fusion during differentiation. Finally, it is possible to use the underlying electrodes to selectively stimulate individual myotubes without stimulating others nearby. Potential uses of skeletal myotubes grown on MEA substrates include lab-on-a-chip applications, tissue engineering, co-cultures with motor neurons, and neural interfaces.     

The Effect of Intracellular Current Pulses in Smooth Muscle Cells of the Guinea Pig Vas Deferens at Rest and during Transmission

The effect of intracellular current pulses on the membrane of smooth muscle cells of the guinea pig vas deferens at rest and during transmission was studied. Two main response types were identified: active response cells, in which a spike was initiated in response to depolarizing currents of sufficient strength and duration; passive response cells, in which depolarizing currents gave only electrotonic potential changes. These cells were three times more numerous than the active response cells. During the crest of the active response the input resistance fell by about 25% of the resting value. Comparison of the active response with the action potential due to stimulating the hypogastric nerve showed that the former was smaller in amplitude and had a slower rate of rise and higher threshold. Electrical coupling occurred between the smooth muscle cells during the propagation of the action potential. Depolarizing current pulses had no effect on the amplitude of the excitatory junction potential (E.J.P.) in passive response cells, but in general did decrease its amplitude in active response cells. These results are discussed with respect to the mechanism of autonomic neuroeffector transmission.     

Electrical Inexcitability of the Frog Neuromuscular Synapse

Frog muscle endplates were explored with an extracellular microelectrode. An intracellular microelectrode nearby simultaneously monitored invasion of the endplate by a spike directly evoked by a third microelectrode placed away from the endplate in the same fiber. External positivities were seen only at sites generating miniature endplate potentials. The external positivity reached a maximum prior to the internally recorded potential and was followed by a small late negativity. Small movements away from active synaptic sites resulted in positive-negative-positive potential sequences characteristic of activity and propagation. Since the external potential is a function of membrane current, the absence of negativity associated with the rising phase of the spike indicates the absence of inward current at synaptic sites. Thus, the synaptic membrane appears not to be excited by a depolarization of the magnitude of an action potential. In an Appendix it is shown that the late negativity and earlier maximum of the external potential can be accounted for by capacitative current through passive membrane.     

The effect of Ca2+ ionophores upon the synthesis of proteins in cultured skeletal muscle

The influence of the Ca2+ ionophores, ionomycin and A23187 upon the incorporation of [35S]methionine into proteins of cultured chicken pectoralis muscle was studied during differentiation of myoblasts into multinucleated myotubes. Fusion was reversibly arrested by growing cells in low-calcium media from the time of plating. Exposure of normal and fusion blocked cultures to 10-6-10-5 M ionomycin or A23187 for 2-6 h on the second to fourth day of growth, resulted in a selective increase in the incorporation of [35S]methionine into two proteins of about 100 000 and 80 000 dalton. When 10-5 M ionomycin or A23187 were added to older cultures, all large myotubes contracted and detached from the plate. Only the adhering myoblasts and small myotubes incorporated [35s[methionine into the muscle proteins and showed increased incorporation of label into 100 000 and 80 000 proteins. After ionophore pulse, the adhering cells retained the ability to differentiate and accumulate myosin. The effect of Ca2+ ionophores upon the rate of protein synthesis is presumably related to increased influx of extracellular Ca2+ with a rise in the Ca2+ concentration of the cytoplasm. We conclude that Ca2+ sensitive mechanisms may regulate the synthesis of a select group of muscle proteins.     

Phosphofructokinase isozyme expression during myoblast differentiation

Isozyme expression of phosphofructokinase (PFK), the key regulatory enzyme for glycolysis, was studied during differentiation of mouse C2 myoblasts to myotubes. The total PFK activity increased 20-fold during in vitro myogenesis. The rate of synthesis, relative to the rate of total protein synthesis, measured by pulse labeling and immunoprecipitation was lowest for muscle PFK (PFK-A), 0.008% in myoblasts, while those for liver (PFK-B) and brain (PFK-C) PFK were 0.017 and 0.014%, respectively. The relative rate of PFK-A synthesis increased sharply (5-fold) at an initial period of differentiation (8 h) and reached maximum of 10-fold at 48 h, to make PFK-A the major isoform synthesized in myotubes. The relative rates of synthesis for both PFK-B and PFK-C did not change drastically, decreasing slightly at 8 h, but were restored to 1.5-2-fold of myoblasts. cDNA sequences coding for mouse muscle PFK were cloned and used along with those for mouse liver PFK, which we have previously cloned, to measure by Northern blot analysis under highly stringent conditions the steady-state mRNA concentrations for muscle and liver PFK during C2 differentiation. The hybridizable mRNA level for PFK-A increased gradually, reaching 13-fold at 48 h when 80% of cells was fused to myotubes. The PFK-A mRNA level at 96 h was 90-fold of that for myoblasts. In contrast, the mRNA level for PFK-B increased slightly during differentiation, showing a maximum of 4-fold at 96 h. These results indicate isozyme-specific control of muscle PFK gene expression during C2 myoblast differentiation.     

Activity and regulation of calcium-, phospholipid-dependent protein kinase in differentiating chick myogenic cells

The activity of calcium-, phospholipid-dependent protein kinase (PKc) was measured in (a) total extracts, (b) crude membrane, and (c) cytosolic fractions of chick embryo myogenic cells differentiating in culture. Total PKc activity slowly declines during the course of terminal myogenesis in contrast to the activity of cAMP-dependent protein kinase, which was also measured in the same cells. Myogenic cells at day 1 of culture possess high particulate and low soluble PKc activity. A dramatic decline of particulate PKc activity occurs during myogenic cell differentiation and is accompanied, through day 4, by a striking rise of the soluble activity. The difference in the subcellular distribution of PKc between replicating myoblasts and myotubes is confirmed by phosphorylation studies conducted in intact cells. These studies demonstrate that four polypeptides whose phosphorylation is stimulated by the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate in myotubes, are spontaneously phosphorylated in control myoblasts. Phosphoinositide turnover under basal conditions in [3H]inositol-labeled cells is faster in myoblasts than in myotubes, a finding that may in part explain the different distribution of PKc observed during the course of myogenic differentiation.     

A novel calcium current in dysgenic skeletal muscle

The whole-cell patch-clamp technique was used to study voltage-dependent calcium currents in primary cultures of myotubes and in freshly dissociated skeletal muscle from normal and dysgenic mice. In addition to the transient, dihydropyridine (DHP)-insensitive calcium current previously described, a maintained DHP-sensitive calcium current was found in dysgenic skeletal muscle. This current, here termed ICa-dys, is largest in acutely dissociated fetal or neonatal dysgenic muscle and also in dysgenic myotubes grown on a substrate of killed fibroblasts. In dysgenic myotubes grown on untreated plastic culture dishes, ICa-dys is usually so small that it cannot be detected. In addition, ICa-dys is apparently absent from normal skeletal muscle. From a holding potential of -80 mV. ICa-dys becomes apparent for test pulses to approximately -20 mV and peaks at approximately +20 mV. The current activates rapidly (rise time approximately 5 ms at 20 degrees C) and with 10 mM Ca as charge carrier inactivates little or not at all during a 200-ms test pulse. Thus, ICa-dys activates much faster than the slowly activating calcium current of normal skeletal muscle and does not display Ca-dependent inactivation like the cardiac L-type calcium current. Substituting Ba for Ca as the charge carrier doubles the size of ICa-dys without altering its kinetics. ICa-dys is approximately 75% blocked by 100 nM (+)-PN 200-110 and is increased about threefold by 500 nM racemic Bay K 8644. The very high sensitivity of ICa-dys to these DHP compounds distinguishes it from neuronal L-type calcium current and from the calcium currents of normal skeletal muscle. ICa-dys may represent a calcium channel that is normally not expressed in skeletal muscle, or a mutated form of the skeletal muscle slow calcium channel.     

The "pacemaker" function of the transient outward current in the rabbit myocardium

The single sucrose gap technique was employed to study the electrically induced automaticity in rabbit papillary muscles. When the potential was clamped at the level of the "maximum diastolic potential" following the first spike of automaticity an initial decline of the outward ionic current with subsequent activation of the delayed potassium current was observed. The initial decline was potential-sensitive with a maximum at approximately -2 mV; it diminished when the rate of stimulation increased and was abolished with 4-aminopyridine plus Sr2+. It is suggested that the transient outward current determines the development of the "pacemaker potential" after the first spike of electrically induced automaticity in rabbit papillary muscles.     

The relationship of polyoma virus-induced tumor (T) antigen to activation of DNA synthesis in rat myotubes

Rat myotubes infected with polyoma virus (PV) introduced into the multinucleated cells by virus-bearing myoblasts at the time of cell fusion incorporate ³H-TdR and exhibit mitotic-type figures. The infected myotubes also produce a viral-specific nuclear antigen, tumor (T) antigen, which appears in groups of adjacent nuclei or in all nuclei of the myotubes. The proportion of myotubes which synthesize DNA, T-antigen and exhibit mitotic-type figures is related to the multiplicity of virus infection.Intact myotubes which are resistant to infection with PV by virus absorption can be infected by microinjection of the virus into the cells. Myotubes thus infected produce T-antigen which appears in multiple nuclei, but do not incorporate ³H-TdR or contain mitotic-type figures. The data suggest that the resistance of myotubes to infection with PV might be due to a change in the cell surface membrane during differentiation so that virus cannot penetrate the cell. The T-antigen apparently has no bearing on the activation of the DNA-synthesizing apparatus in multinucleated muscle cells.     

Calcium transients associated with the T type calcium current in myotubes

Immature skeletal muscle cells, both in vivo and in vitro, express a high density of T type calcium current and a relatively low density of the dihydropyridine receptor, the protein thought to function as the Islow calcium channel and as the voltage sensor for excitation- contraction coupling. Although the role of the voltage sensor in eliciting elevations of myoplasmic, free calcium (calcium transients) has been examined, the role of the T type current has not. In this study we examined calcium transients associated with the T type current in cultured myotubes from normal and dysgenic mice, using the whole cell configuration of the patch clamp technique in conjunction with the calcium indicator dye Fluo-3. In both normal and dysgenic myotubes, the T type current was activated by weak depolarizations and was maximal for test pulses to approximately -20 mV. In normal myotubes that displayed T type calcium current, the calcium transient followed the amplitude and the integral of the current at low membrane potentials (- 40 to -20 mV) but not at high potentials, where the calcium transient is caused by SR calcium release. The amplitude of the calcium transient for a pulse to -20 mV measured at 15 ms after depolarization represented, on average, 4.26 +/- 0.68% (n = 19) of the maximum amplitude of the calcium transient elicited by strong, 15-ms test depolarizations. In dysgenic myotubes, the calcium transient followed the integral of the calcium current at all test potentials, in cells expressing only T type current as well as in cells possessing both T type current and the L type current Idys. Moreover, the calcium transient also followed the amplitude and time course of current in dysgenic myotubes expressing the cardiac, DHP-sensitive calcium channel. Thus, in those cases where the transient appears to be a consequence of calcium entry, it has the same time course as the integral of the calcium current. Inactivation of the T type calcium current with 1-s prepulses, or block of the current by the addition of amiloride (0.3-1.0 mM) caused a reduction in the calcium transient which was similar in normal and dysgenic myotubes. To allow calculation of expected changes of intracellular calcium in response to influx, myotubes were converted to a roughly spherical shape (myoballs) by adding 0.5 microM colchicine to culture dishes of normal cells. Calcium currents and calcium transients recorded from myoballs were similar to those in normal myotubes.(ABSTRACT TRUNCATED AT 250 WORDS)