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Electrophysiology of duchenne dystrophy myotubes in tissue culture.

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Electrophysiology of Duchenne
Dystrophy Myotubes in Tissue Culture
Steven M. Rothman, MD, and kchard Bischoff, PhD
Single myotubes from cultures of 6 patients with Duchenne muscuIar dystrophy and 6 patients whose biopsy specimens
lacked histological abnormality were studied with intracellular physiological recording. Average resting membrane
potentials were 47.8 2 2.3 mV and 47.0
1.5 mV for the control and Duchenne cultures, respectively. Action
potentials elicited at a standard resting potential of -80 mV were 97.3 & 4.0 mV and 98.6 f 8.2 mV for the two
groups. Maximum rate of rise of action potentials was 154.2 2 25.2 V/sec for control and 143.1 & 37.1 Visec for
Duchenne myotubes. Action potentials mediated by an influx of calcium ions were seen only when the myotubes from
both groups were bathed in high concentrations of calcium and 4-aminopyridine (20 mM and 1 mM, respectively).
Thus, the plasma membrane of Duchenne dystrophy myotubes does not have active electrical properties that differ
from those in controls. Previous reports of low resting membrane potentials in biopsies studied acutely may reflect
secondary changes in degenerating fibers.
*
Rothmdn SM, Bischoff R Electrophysiology of Duchenne dystrophy myotubes in tissue culture
Ann Neurol 13 176-179, 1983
Understanding the pathophysiology of the muscuiar
dystrophies remains one of the major goals of neurologists. Despite fairly detailed knowledge of the pathological changes that characterize these diseases, there
are still no coherent explanations for the muscle destruction and resulting weakness. A number of recent
experiments have suggested that a primary defect exists
in the muscle plasma membrane and membranes of
other cell types in patients with a variety of muscle
diseases {12, 15, 181. It might be expected that some
of these tentative membrane abnormalities would result in electrophysiological differences between normal and diseased muscle. The reports of diminished
sarcolemmal sodium, potassium adenosinetriphosphatase (“a+, K+]ATPase) in Duchenne dystrophy (DD)
suggest that muscle resting membrane potential should
be low in DD [131. Indeed, this has been found in two
separate studies [ S , 161.
Abnormal physiological findings from biopsied human muscle are difficult to interpret, however, because
they may represent nonspecific changes present in degenerating muscle fibers rather than the elusive primary abnormality responsible for the degeneration.
Furthermore, it is possible that this primary abnormality resides in tissue other than the muscle, so that even
the most sophisticated examination of muscle will fail
to reveal it. Using cultured myotubes grown from human biopsies partially eliminates these problems. Tissue can be studied prior to the onset of degeneration in
a controlled environment containing only myotubes
and fibroblasts in growth medium. Individual, living
myotubes can be seen with phase-contrast microscopy
so that only healthy-appearing cells are examined [ 11,
17). In addition, since many cultures can be prepared
from one biopsy, material from the same patient can be
repeatedly studied over many days. In an attempt to
identify a membrane abnormality in DD, we studied
muscle cultures from affected patients and controls by
means of intracellular recording.
From the Departments of Anatomy and Neurobiology, Pediatrics,
and Neurology, Washington University School of Medicine, St.
Louis, MO 631 10.
Received Feb 22, 1982, and in revised form May 2 1. Accepted for
publication May 28, 1982.
Materials and Methods
Muscle was obtained from 12 patients undergoing diagnostic
muscle biopsy. Six showed the characterisric changes of DD
and 6 lacked histological abnormalities. The muscle was
cleaned of perimysial connective tissue and fat, and 1 to 2
mm’ fragments of tissue were incubated for 1 hour at 37°C in
Earle’s balanced salt solution containing 0 . 1 9 Pronase (Calbiochem-Behring, La Jolla, CA). The tissue was dissociated
by repeated pipeting in culture medium, and single cells were
isolated by filtration through a 10 pm nylon mesh. The cells
were grown o n collagen-coated dishes (Corning, NY) in
Eagle’s minimal essential medium with 10% selected horse
serum (Grand Island Biological Co, Grand Island, NY) and
10% calf serum collected locally. The medium was changed
daily. Cells were removed from dishes with 0.05F trypsin
before the onset of myoblast fusion and were subcultured at
an initial cell density of 1.5 x 10’icm’. Experiments were
performed on cells that had been ailowed to fuse during the
second subculture period.
Address reprint requests to Dr Rothman, Department of Anatomy
and Neurobiology, Washington University School of Medicine, 660
S Euclid, St. Louis, M O 63 110.
176 0364-5 1~4/83/020176-O4$150 0 1982 by the American Neurological Association
Fig I . (A) PhaJe-rontrastphotomici*ograph of aii elezmen-day rulture obtainedfrom a contml patient. Multinudeated nryotubes ure
present. IB) Fourteen-day culture.from a patieirt with Duchenne
dystrophy, again showing multinui-leatedmj~otubes.(Both
x 100.1
Cultures were used for intracellular recording after approximately one to two weeks, when many myotubes large
enough to tolerate impalement had formed (Fig 1). Culture
medium was replaced with Hank’s balanced salt solution
(HBSS) that had been modified to contain 3 mM K + , 3.5
mM Ca2+, 1 mM Mg*+, 400 mgidlglucose, and 5% calf sera.
The p H of the HBSS was adjusted to 7.3 with 10 mM
HEPES. Some experiments were done i i ! sodium-free balanced salt solution containing 110 mM choline chloride, 3
mM K’ , 10 to 20 mM Ca” , 1 mM Mg”, and 100 mgidl
glucose, buffered to p H 7.3 with 20 mM Tris. The culture
dish was mounted o n the stage of an inverted phase-contrast
microscope and kept at 36°C with a heating coil placed inside
the dish. A layer of light mineral oil was put over the bath to
prevent evaporation. Cells were impaled under direct vision
at a magnification of 400 x .
Electrodes were made with a horizontal puller and filled
with 3 M potassium chloride. Electrode resistances ranged
from 10 to 30 MO with thin-walled Omega Dot tubing (Glass
Company of America, Bargaintown, NJ). Cells were penetrated with either one or two microeiectrodes. In the former
case, stimulation and recording were done with a standard
bridge circuit and high-impedance amplifier (WPI M707).
Bridge balance was verified after electrode withdrawal, and
results discarded if balance was not maintained. When possible, separate current and voltage electrodes were used. In a
few cells, action potentials were recorded with both a voltage
microelectrode and the bridge circuit, and appeared identical.
Action potentials were differentiated electronically to obtain
the maximum rate of rise.
Results
The average resting membrane potentials (RMP) of
control (N = 135) and DD (N = 120) myotubes were
47.8 k 2.3 mV and 47.0 t 1.5 mV, respectively
(Table). The maximum RMP was 60 mV in both control and DD myotubes. Values less than 40 mV were
discarded because of the likelihood of damage from
impalement. Low resting potentials were seen no more
frequently in the DD cultures than in control cultures.
They were found most often in small cells that were
difficult to penetrate. In a few cultures studied in
sodium-free balanced salt solution, resting potentials as
high as 80 mV were found, suggesting some contribution of sodium to the resting potentials measured in
our standard HBSS.
Unlike cultured rat or chick myotubes, the human
myotubes virtually never showed spontaneous mechanical or electrical activity (seen in 1 of 255 cells).
Action potentials were therefore produced by depolarizing the cells with an intracellular electrode. Because the magnitude and maximum rate of rise of the
action potential are functions of RMP (Fig 2C), cells
were hyperpolarized to a standard potential of -80
mV and then depolarized with a 15 msec pulse to produce an action potential. Depolarization sometimes
failed to produce action potentials at the RMP, and in
this situation a long hyperpolarization was effective (Fig
2D). At -80 mV, depolarizing pulses were always
effective .
Action potentials averaged 97.3 ? 4.0 mV (N =
66) and 98.6 8.2 mV (N = 63) for the control and
DD myotubes, respectively (see the Table). Most cells
in both populations had thresholds of about - 50 mV,
explaining the difficulty in producing an action poten-
*
Summary of Resting Potential and Action Potential Values for all Cultures”
Subjects
Resting Membrane
Potential (mV)
Action Potential
imV)
Controls (6 patients)
Duchenne dystrophy (6 patients)
47.8 2 2.3 (N = 135)
47.0 +- 1.5 (N = 120)
97.3
98.6
~
“Values are means
-t 1 SD
* 4.0 (N = 66)
2
8.2 (N = 63)
Maximum Rate
of Rise (Visec)
154.2 2 25.2 (N = 66)
143.1 & 37.1 (N = 63)
~~~~~~~~
Numbers in parentheses are total number of measurements of the variable for either control or dystrophic patients
Rothman and Bischoff: Physiology of Duchenne Dystrophy
177
A
n---
c
.
__
B
Fig 2. (A)Typical action potential in a control niyotube. demonstrating steep rate of rise from - 80 mV resting potential. (BI
Typical action potentiul in Dur-hrnne&trophy. (Ci Action potential~in Dnchenne mytube pmdui-ed by depolaviwrion at dz'fferent resting potentid\, Note the small ownhoot at lourst restiiig
potential. (Di Action potential1 produced bji long hj~perpolarizing
pulses at resting potential in a Dur6enne rrqotube. For all recordings, top line is zero potential. middle line i.s voltage, and bottom
line is derivatiw of aoltage. Scule: 80 mV, 400 VIA-el-;10 rnsec
A-CJ and 100 m~ec(D).The bottom line u'as retoui-bedin A
through D [or i-larity.
tial at RMP. No qualitative differences in appearance
of action potentials were noted between the two populations (Fig 2A, B). Typically, the action potentials had
a steep rate of rise from threshold, the maximum rate
of rise for control and DD myotubes being 154.2 k
25.2 V/sec and 143.1 k 37.1 V/sec, respectively (see
the Table). The briefest action potentials were 3 msec
at half amplitude, but some exceeded 10 msec. The
slow rate of repolarization was the factor most responsible for the long-duration action potentials. When the
myotubes were hyperpolarized to - 80 mV, the action
potentials had depolarizing afterpotentials. However,
action potentials produced at lower RMP showed hyperpolarizing afterpotentials of up to 15 mV.
Fewer than 10% of the action potentials (2 control, 4
DD) had a different shape, showing either a biphasic
peak or a regenerative response during the afterpotential (Fig 3). These second responses were smaller and
slower than the initial action potential and resembled
calcium-mediated potentials seen in cultured rat and
chick myotubes and some neurons [4, 7). In order to
study these responses further, five cultures were
bathed in sodium-free balanced salt solution containing
20 mM calcium and 0 to 1 mM 4-aminopyridine. This
drug blocks outward potassium currents and therefore
should increase the likelihood of seeing calcium spikes.
Most myotubes failed to produce regenerative responses when depolarized in this solution and instead
showed the delayed decrease in membrane resistance
typical of excitable cells (Fig 4A). However, small regenerative responses were elicited in 2 of 11 myotubes
178 Annals of Neurology Vol 13 No 2
February 1983
-'
3
.... ....
I
L
-
--
Zhsec
F i g 3. iA1, 2 ) Uiphasic action potentials in control rnyotuhes.
demonstrating t w o separate peaks. (Bl-3, Biphasic action potentiah in Duchenne niyotubes. shou'ing a sirnilar phenomenon.
A
~
- -~
-_-____
B
.
-_--
-
4hV
20nA
L
~
4hsec
F ig 4. (A)Intracellular record from Duchenne myotube in
sodium-free bathingjuid containing 1 mM 4-aminopyridine
and 20 mM calcium. Depolarizing current failed to elicit an action potential, and onh a decrease in resistance t o the current
p d s e is seen. IB) One of the few Duchenne myotubes to show an
action potential when depolarized by current passage. Note the
point of injection on the rising phase of the second curve, indicating a regenerative response. Top line is injected current, bottom
line is voltage.
from control cultures and 5 of 13 myotubes from DD
cultures (Fig 4B). These responses were much smaller
than the action potentials seen in standard HBSS and
had a slower time course. They were never present
when the concentration of 4-aminopyridine was less
than 1 mM.
Discussion
By our measurements, no significant differences in
RMP, action potentials, and maximum rate of rise of
action potentials of control and DD myotubes in tissue
culture were found. These experiments provide no
positive evidence supporting the existence of a primary
membrane abnormality in DD. The most likely explanation for the finding of abnormally low RMP in biopsied DD muscle is that degenerating muscle is unable
to maintain normal ionic gradients [ 5 , 161. The presence of abnormally high concentrations of sodium and
low concentrations of potassium in DD biopsies is
certainly compatible with this hypothesis [Gf. The
nonspecificity of low RMP is further supported by the
low RMP found in muscle biopsies from dystrophies
other than DD [ti].
A primary membrane defect that does not alter resting and voltage-sensitive membrane channels could still
exist. In fact, “a+, K+}ATPase could be diminished
but still present in high enough concentration to allow
maintenance of a normal RMP. Another possibility is
that a specific physiological membrane abnormality appears later in development, at a time when the muscle
cultures are no longer viable. Innervation o r activity,
which each have a role in normal muscle maturation,
could be necessary for the manifestation of this abnormality. Finally, membrane defects in DD could be
mediated by humoral factors not present in these cultures. The recent finding that sera from DD patients
failed to cause necrosis of their own cultured myotubes
provides no support for this last possibility [141.
We recorded in sodium-free balanced salt solution
with high calcium concentrations to try to identify a
difference in the putative calcium action potentials of
normal and DD myotubes. Calcium action potentials
have previously been identified in cultured chick and
rat myotubes 14, 71.The rationale for this experiment
was the finding of excess calcium in biopsied D D muscle, which could conceivably have entered the cell by
passing through voltage-sensitive calcium channels [ 1,
2). The few action potentials found in the sodium-free
balanced salt solution were almost certainly mediated
by calcium, because no other cations present in the
solution produce regenerative potentials. These action
potentials resembled those seen in chick and rat myotubes and were similar in control and D D cultures [ 4 ,
71. These putative calcium action potentials are probably a normal finding in developing muscle. The fact
that no tetrodotoxin-resistant action potentials were
found in another study of normal and DD muscle examined immediately after biopsy is consistent with the
idea that calcium action potentials disappear with muscle maturation [ 5 ] . While a primary abnormality in calcium entry or intracellular calcium distribution may be
present in DD, other methods will be required to detect it [ 3 , 191.
Although no physiological abnormalities have yet
been found in cultures of DD myotubes, cultured human muscle has proved useful in elucidating physiological defects in myotonic dystrophy. Cultured myotubes
from patients with myotonic dystrophy have recently
been reported to have low resting potentials, depolarizing afterpotentials, and a tendency to fire action potentials repeatedly [9, 101. In at least one muscle disease,
therefore, a membrane defect may be expressed in
culture.
Supported by Grants IK07 NS 00568-01 from rhe National Institute of Neurological and Communicative Disorders and Stroke and
the Muscular Dystrophy Association.
Presented ar the Tenth Annual Meeting of the Child Neurology
Society, Minneapolis, MN, Oct 15-17, 1981.
We thank Jan Hoffmann and Sharon Musgrove for typing this paper.
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179
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