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End-plate voltage-gated sodium channels are lost in clinical and experimental myasthenia gravis.

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End-Plate Voltage-Gated Sodium Channels
Are Lost in Clinical and Experimental
Myasthenia Gravis
Robert L. Ruff, MD, PhD,* and Vanda A. Lennon, MD, P h D t
This study examined the loss of voltage-gated Na+ channels as well as acetylcholine receptors (AChRs) from the endplate region in patients with acquired myasthenia gravis (MG) and in rats with experimental autoimmune passively
transferred MG (PTMG). Rats received a monoclonal IgG antibody directed against an extracellular epitope of the
nicotinic acetylcholine receptor of muscle (AChR) to produce PTMG. At the end-plate border we examined miniature
end-plate potentials (MEPPs), sodium current (INa)
amplitude, and action potential (AP)properties; the latter two were
also examined on the extrajunctional membrane. In the normal situation, the safety factor for neuromuscular transmission is ensured by the large ZNaat the end plate, which reduces the AP threshold. Among different fiber types, ZNawas
largest for type IIb fibers and smallest for type I fibers. When end-plate border properties of fibers from 3 MG patients
and 15 PTMG rats were compared with controls, ZNa was reduced, AP thresholds were higher, and rates of AP rise were
reduced. Amplitudes of MEPPs and I,, at the end plate indicated that loss of AChRs was greater than loss of Na+
channels in patients with MG and rats with PTMG; INawas reduced to about 60% of control values, whereas MEPPs
were reduced to less than 30% of control values. On the extrajunctional membrane, ZNaand AP thresholds and rates of
rise were similar for MG patients, PTMG rats, and controls. This evidence for loss of voltage-gated Na+ channels at the
motor end plate in both patients with MG and in rats with PTMG reveals a hitherto unrecognized consequence of the
end-plate damage initiated by the binding of complement-fixing IgG to end-plate AChRs.
Ruff RL, Lennon VA. End-plate voltage-gated sodium channels are lost in clinical and
experimental myasthenia gravis. A n n Neurol 1998;43:370-379
Weakness in the autoimmune disease myasthenia gravis
(MG) is caused by antibodies directed against skeletal
muscle acetylcholine receptors (AChRs) on the muscle
membrane portion of the end plate.'-4 These antibodies reduce the number of AChRs at the end platel-' by
a combination of complement-mediated membrane lysis' and acceleration of AChR catabolism by receptor
cross-linking.
The secondary synaptic folds are
simplified due to loss of end-plate
The serum level of AChR binding antibodies does not
predict the severity of weakness,'-4 but the postsynaptic membrane area correlates with the size of the miniature end-plate potentials (MEPP) and with the patient's clinical signs of weakness6
M G can be induced in rats by sensitizing the animals to foreign or self AChR (experimental autoimmune MG, EAMG) or by passively transferring myasthenogenic AChR binding IgG (PTMG).1-.3212-14
Weakness in PTMG begins about 12 hours after antibody injection and peaks at 48 hours.'3314After an initial period in which macrophage invasion is prominent
From the *Neurology Service, Department of Veterans Affairs Medical Center, and Departments of Neurology and Neurosciences,
Case Western Reserve University School of Medicine, Cleveland,
OH; and ?Departments of Immunology, Neurology, and Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
370
Copyright
0 1998
at the end plates of rats with PTMG, the subsequent
electrophysiological changes and loss of synaptic folds
are similar to those found in patients with acquired
~ ~ . 2 , 1 3 , 1 4
In addition to AChRs, the end-plate membrane has
a high density of voltage-gated Na'
channel^.^,'^-^^
These Nat channels are concentrated in the depths of
the secondary synaptic folds, whereas AChRs are concentrated at the
Figure 1 shows the relative
density of voltage-gated Na+ channels at the end plate
compared with the extrajunctional membrane on a
normal muscle fiber (top panel) and the locations of
voltage-gated Nat channels and AChRs on the endplate membrane (middle panel). A localized depolarization, the end-plate potential, results from activation of
AChRs, which are ligand-gated channels that conduct
cations. For muscle contraction to occur, the end-plate
potential must trigger two action potentials, which are
depolarizing waves that propagate from the end-plate
region to both tendon ends of the muscle fiber. The
rising phase of the skeletal muscle action potential (AP)
Received Jun 24,1997, and in revised form Oct 23. Accepted for
publication Oct 23, 1997.
Address correspondence to Dr Ruff, Neurology Service 127(W),
Cleveland VAMC, 10701 East Boulevard, Cleveland, OH 44106.
by the American Neurological Association
Distributionof Na+ channels ( 0 ) on a muscle fiber membrane
Extrajunctional
membrane
Endplate
Na+ Channels and AChRs
-
Extrajunctional
membrane
(v)on a normal endplate
Na+ Channels and AChRs on a MG endplate
3
Fig 1. Diagrammatic representation of the distribution of
voltage-gated Nat channels (e) and acetylcholine receptors
(AChR) ( v ) on muscle fiber membrane. (Top) the density of
Na + channels is greater at the end plate compared with the
extrajunctional membrane. (Middle) AChB are concentrated
on the tops of the synaptic folds, close to the nerve terminal,
and Naf channels are concentrated in the depths of the synaptic folds. (Bottom) In myasthenia gravis (MG), loss of endplate membrane results in simplijcation of the synaptic folds.
End-plate membrane loss includes the crests of the synaptic
folds (AChR rich) and the depths of the folds (containing
N a f channels).
results from the rapid opening of voltage-gated N a t
channels. N a + current (INa)passing through the open
ampliN a + channels depolarizes the muscle fiber. INa
tude for a region of membrane depends o n the density
of N a t channels in the membrane, how much INaa
single channel conducts (single-channel conductance),
and the fraction of N a + channels that open in response
to membrane depolarization.
T h e safety factor (SF) for neuromuscular transmission can be defined as SF = EPP/(RP - EAp),where
EPP is the end-plate potential amplitude, RP is the
restin membrane potential, and EApis the AP threshold.?, ,21 T h e high concentration of voltage-gated Na’
channels at the end plate increases the safety factor for
neuromuscular transmission by lowering the threshold
of depolarization needed to generate an AP.2’,2G,27
To investigate whether end-plate N a + channels were
lost together with AChRs from the muscle fibers of
patients with MG and rats with PTMG, we compared
myasthenic with normal control subjects’ values for the
at the end-plate border and on the
amplitudes of INa
extrajunctional membrane. Measurements of MEPP amplitudes assessed changes in AChR-induced current, and
AP characteristics assessed the impact of changes in INa.
.f
Patients and Methods
Patients
The protocol for studying human intercostal muscle was approved by the Institutional Review Board of the Department
of Veterans Affairs Medical Center at Cleveland. All biopsies
were obtained with the subjects’ informed consent. Three
male patients with MG (age, 35, 42, and 47 years) donated
intercostal muscle biopsies at the time of thymectomy. All
had moderately severe generalized MG (Osserman class 2B)
and were seropositive for AChR binding antibodies but seronegative for striated muscle antibodies. Before thymectomy, each patient received five courses of plasmapheresis
(2-3 L removed at each session). Seven control male subjects
(age, 35-52 years), lacking evidence of neuromuscular disease, donated control intercostal muscle biopsies at the time
of a thoracotomy for treatment of cardiac or pulmonary disease. Criteria for excluding a muscle biopsy from any subject
were (1) muscle disease other than MG, (2) inability to give
informed consent, (3) elevated serum creatine kinase values,
(4) exposure to human immunodeficiency virus infection,
(5) history of viral hepatitis, or (6) intravenous drug abuse.
PTMG Protocol
Female Lewis rats, weighing 180 to 200 g, were injected intraperitoneally once with a myasthenogenic rat monoclonal
anti-AChR IgG (McAb3; 1 X 10-l’M) or a control rat
monoclonal IgG (McAbl), which recognizes a determinant
specific to Tovpedo AChR and does not induce PTMG.’*
Rats were killed by sodium pentobarbital overdose 24 hours
after injection. Omohyoid and adductor longus muscles were
removed for MEPP and INarecordings. The rat omohyoid
muscle contains mostly type IIb fibers and the adductor longus contains mostly type I fibers.28Selection of these muscles
allowed dissection of bundles one to two fiber layers thick.
This enabled precise definition of the end-plate regions by
light microscopy using Nomarski optics.16
Tyrode solution for human and rat muscle contained
(mM) NaCl 135, KCI 3.5, MgC1, 1, CaC1, 6, HEPES 10,
and glucose 10. Solutions were gassed with 0, and the pH
was adjusted to 7.4. Experiments were performed in vitro at
19 2 1°C.
Tissue Preparation
Superficial connective tissue was gently dissected from rat
and human muscles in Tyrode solution. The muscles were
then incubated for 45 minutes in oxygenated circulated Tyrode solution containing collagenase 2 mg/ml (type I, Sigma
Chemical, St Louis, MO) and bovine albumin 1 mg/ml
(fraction V, Sigma). For further dissection, bathing solution
with 2 mg/ml collagenase and 1 mg/ml albumin was flowed
over the central portion of the muscle, and bathing solution
without collagenase was circulated across the tendon regions.
The muscle regions finally dissected were one to two fibers
thick, permitting visualization of the end plates, using a Nikon Diaphot inverted phase-contrast microscope equipped
with Nomarski optics (Nikon, Instrument Group, Melville,
NY). Before current recordings were made, the collagenase
was diligently washed from the bathing chamber. Additional
details of the dissections are described elsewhere. ‘63’73’9*20
Loose Patch Voltage CLamp
Technical details of the loose patch voltage clamp technique
used to measure I,, were described p r e ~ i o u s l y . ’ ~To
~”~~~~~~
record membrane currents, a micropipette with a fire-
Ruff and Lennon: End-Plate Nat Channel Loss in MG
371
polished tip was pressed against the sarcolemma to form a
resistive seal between the pipette and the membrane. The
seal electrically isolated the patch of membrane under the
pipette. The potential of the membrane patch was controlled
by the potential within the pipette. Currents elicited from
the membrane patch were recorded by the patch pipette. The
seal between the membrane and pipette was 3 to 10 M R ;
consequently, a fraction of the current produced by the ionic
channels within the membrane patch passed across the seal
resistance rather than through the pipette. The fraction of
membrane current lost across the seal was corrected for by
analog and digital compensation. Micropipettes with tip diameters after fire polishing of 5 to 10 p m and resistances of
200 to 300 kR were used. Pipettes of these sizes were chosen
to permit sampling from a sufficiently large patch of menibrane to minimize local variations in ZN1density, and yet not
stimulate too large a current so that voltage control of the
cell was maintained. To reduce capacitive coupling between
the bath and pipette, the pipettes were coated with a double
layer of Sylgard (Dow Corning 184, Midland, MI) to within
100 p m of the tip. Minimal suction was applied to the micropipettes to avoid the formation of membrane blebs.20
Identification of the neuromuscular junction by direct visualization with Nomarski optics enabled the micropipette to
be positioned precisely on the muscle fiber membrane with
respect to the nerve terminal. For recordings of INafrom the
end-plate border, the pipette was positioned on the muscle
fiber sarcolemma so that the pipette was next to the margin
of the nerve terminal. Recordings from the extrajunctional
membrane were made more than200 p m away from the
end-plate border. Inward, ,Z was determined from a group
of six depolarizing pulses (each 4 msec in duration) applied
in steps of 6 mV in the vicinity of the maximal inward INn.
Each trace was fitted by a third order polynomial to determine the peak inward current during that trace. The six peak
Naf currents obtained in this way were fitted by a parabola.
The minimum of this parabola was taken as the peak inward
Na+ current, I,,,,
and the potential at which the maximal
current occurred was ~ , N e , m n x . T o remove fast and slow in7,19,21,31 the membrane patch was hyperpolarized
by 50 mV for 3 to 5 minutes, until the current from the
obpatch reached a stable level. The stable value of INa,mau
tained at the end of the hyperpolarizing conditioning period
was the maximum value of inward IN:,or maximum I,,,,
and served as a measure of the number of excitable sodium
channels in the membrane patch. Additional details of the
stimulating pulse protocols used to measure I N , were previously d e ~ c r i b e d . ’ ~ . ” ~ ~ ” ~ ~
The steady-state voltage dependence of fast inactivation
was studied by applying 20-msec conditioning pulses that
were immediately followed by 4-msec depolarizing test pulses
to about
Prepulses 20 msec long were sufficient to
study fast inactivation in human and rat muscle fihers. 16,17,19,21,31 The steady-state voltage dependence of fast
inactivation, the “Ax” curve, can be described by a Boltzmann distribution, as follows: h, = ZNa/ZNNa,mz = 1/[1
exp([vn - Vh1,J/Ah)], where ZNa/INa,mz is the relative amplitude of I,, during a test pulse, V, is the membrane potential during a prepulse, V,,,, is the potential of the prepulse that inactivates one-half of the Na+ channels, and A, is
+
372 Annals of Neurology
Vol 43
No 3
March 1998
a parameter determining the steepness of the h,-membrane
potential relation.
MEPPs and Resting Membrane Potentials
MEPPs and resting membrane potentials were measured in
rat and human skeletal muscle fibers at the end plate and on
the extrajunctional membrane by using intracellular microelectrodes filled with 3 M KCI with resistances of 10 to 20
MR. MEPPs were recorded from the end-plate border after
collagenase treatment with the end plate visualized directly
with Nomarski optics. MEPP amplitudes were scaled to a
membrane potential of -80 mV by multiplying the MEPP
amplitudes by -80/RP to adjust for differences in fiber
membrane potentials and to enable comparison with prior
studies of MG.62’3.’4Microelectrode tips were coated with
carbon black to mark an impaled fiber for later histochemical
identification.
AP Thresholds and Maximum Rates of Rise
AP thresholds and maximum rates of rise were measured by
using two microelectrodes, one for passing current and the
other for recording membrane potential. The electrodes were
positioned within 50 p,m of each other and connected to a
voltage clamp amplifier (Axoclamp 2A, Axon Instruments,
Foster City, CA). The microelectrodes and muscle fiber were
visualized with Nomarski optics to enable the microelectrodes to be positioned on the end-plate border or more
than 200 p m away from the end-plate border on the extrajunctional membrane. AP threshold was determined by using
depolarizing current pulses of gradually increasing amplitude
as described p r e ~ i o u s l y . ~ ~ . ~ ~
Histochemical Fiber q p e s
Histochemical fiber types were dererminzd for rat and human by using previously described techniques.17,’”~z”
A muscle bundle containing a fiber marked with carbon black was
placed in skinning solution that containcd (mM) free Mg
2.5, MgATP 10, K 138, creatine phosphate 15, 12ethyleneglycol-bis(P-aminoethylether)-N,N,M ,“-tetraacetic
acid (EGTA) 10, and 3-(N-morpholino)propanesulfonicacid
(MOPS) 15. The marked fiber was identified with a Nikon
Diaphot microscope, and a 4- to 6-mm length of the marked
fiber was dissected free and divided into three segments.
Each segment was placed on an albumin-coated coverglass
and stored at -70°C. ATPase staining with preincubation at
p H 4.3 and 10.3 determined if the fiber was fast or slow
twitch, and NADH-tetrazolium reductase staining determined if the fiber was oxidative or glycolytic. Stain intensity
was determined by examining the slide microscopically and
comparing the fiber with muscle of known fiber type. The
fibers were classified into the following three groups: slow
twitch, oxidative (type I); fast twitch, oxidative-glycolytic
(type IIa); and fast twitch, glycolytic (type Ilb). Only data
from histochemically characterized fibers were analyzed for
studies of INa,MEPP amplitudes, or AP characteristics.
Statistical Analysis Techniques To Be
Used in the Projects
Data were subjected to analysis of variance (ANOVA) testing
by using two-tailed tests with a = 0.05. Subsets of data were
studied by ANOVA to determine significant differences
among groups of fibers. When significant interactions were
present, post hoc comparisons between different groups were
made by using Tukey's HSD (Honestly Significant Difference) test for painvise comparisons, Scheffk's S method
when more than two means were compared, and the MannWhitney U test for comparisons of groups that did not satisfy normality criteria.32333Data are expressed as mean ?
SEM values.
and omohyoid, -95.4 ? 0.8 mV (n = 139; range,
-103 ro -88 mV).
The resting membrane potentials of human or rat
muscle fibers did not change appreciably during the
experiments, which lasted up to 8 hours. The mean
resting potentials of fibers from rat or human muscle
bundles varied by less than 2 mV between the beginning and the end of an experiment.
Results
Fibers f i o m Patients with MG and fiom PTMG
Rats Had Smaller MEPPs Compared with Controls
Table 1 shows that the amplitudes of MEPPs from patients with MG were reduced to 27% of the control
value. MEPP amplitudes from different fiber types,
scaled to a membrane potential of -80 mV, were
grouped together because MEPP amplitudes did not
vary based on fiber type. The 31 control fibers consisted of 14 type IIb, 12 type Ila, and 5 type I fibers.
Resting potentials of the human control fibers were as
follows: type I, -84.6 t 2.2 mV; type IIa, -94.5 -t
1.2 mV; and type IIb, -94.3 2 1.0 mV. MEPP amplitudes for control human fibers were as follows: type
I, 0.901 2 0.024 mV; type IIa, 0.910 2 0.024 mV;
and type IIb, 0.907 -t 0.022 mV. The 28 fibers from
patients with MG consisted of 13 type IIb, 11 type IIa,
and 4 type I fibers. Resting potentials of the MG fibers
were as follows: type I, -85.5 t 2.7 mV; type IIa,
-93.8 2 1.4 mV, and type IIb, -95.4 t 0.9 mV.
MEPP amplitudes of the M G fibers were as follows:
type I, 0.245 -+ 0.027 mV; type Ha, 0.250 t 0.019
mV; and type IIb, 0.243 -+ 0.015 mV.
MEPP amplitudes of adductor longus or omohyoid
muscles from PTMG rats were reduced to 23% of the
control values (see Table 1). For control rats, the
MEPP amplitudes of type IIb omohyoid muscle fibers
were similar to those of type I adductor longus fibers.
Omohyoid and adductor longus fibers from PTMG
rats also had similar MEPP amplitudes. Control fiber
resting potentials were as follows: adductor longus,
-84.6 t 2.2 mV; and omohyoid, -95.3 ? 2.1 mV.
Resting Potentials a j e r Collagenase Treatment Were
Similar in Fibers from MG Patients, PTMG Rats,
and Control Subjects
For control human or rat muscles, collagenase treatment lysed less than 4% of the fibers in a bundle. In
contrast, collagenase treatment lysed 19% to 23% of
the intercostal fibers from the 3 MG patients, and 31%
to 37% of adductor longus fibers and 32% to 39% of
omohyoid fibers from PTMG tats. Resting potentials
in fibers that survived collagenase treatment and were
used to record I,, and MEPPs were similar for controls, MG patients, and PTMG rats. Human external
intercostal muscle contains a mixture of muscle fiber
types, ie, I, IIa, and IIb. After collagenase treatment,
the resting potentials of fibers from control patients
were -90.5 ? 0.6 mV (n = 167; range, -104 to
-79 mV) and fibers from MG patients had resting potentials of -90.9 2 0.6 mV (n = 117; range, - 102 to
-79 mV). In contrast to the mixed population of fiber
types in external intercostal muscles, the rat omohyoid
muscle contains mostly type IIb fibers and the adductor longus contains mostly type I fibers2' After collagenase treatment, the resting potentials of fibers from
control rat adductor longus muscles were -85.9 2 0.7
mV (n = 126; range, -94 to -77 mV) and fibers
from control omohyoid muscles were -95.9 -+ 0.8
mV (n = 137; range, - 102 to -89 mV). The resting
potentials of muscle fibers from PTMG rats after collagenase treatment were as follows: adductor longus,
-85.4 2 0.9 mV (n = 131; range, -95 to -77 mV);
Table 1. MEPP Sizes (mean ? SEM) Recordedfiom the External Intercostal Muscles of Three Patients with M G and Seven
Control Patients andfiom the Omohyoid Muscles of Five Female Lewis Rats with PTMG and Five Control Rats 24 Hours
afier Injection of a Myasthenogenic or Control Monoclonal IgG
~~
Human Intercostal Muscles
MEPP
(mVa
No. of fibers
Rat Omohyoid Muscles
~
Rat Adductor Longus
MG
Control
PTMG
Control
PTMG
Control
0.251 2 0.015
28
p < O.OOlb
0.910 ? 0.016
31
0.204 2 0.017
20
p < O.OOlh
0.905 ? 0.020
21
0.211 2 0.019
22
p < O.OOlb
0.901 ? 0.024
21
"The MEPP amplitudes were corrected to a resting potential of -80 mV, to adjust for differences in fiber resting potentials.
'Significant differences are for comparisons between PTMG and control fibers.
MEPP = miniature end-plate potential; MG
=
myasthenia gravis; PTMG
=
passive transfer MG.
Ruff and Lennon: End-Plate Nat Channel Loss in MG
373
Resting potentials of fibers from PTMG rats were as
follows: adductor longus, -85.3 5 2.1 mV; and omohyoid, -95.4
2.0 mV.
*
Is Increased at the End-Plate Border of
Control Fibers Compared with the
Extrajunctional Membrane
Figure 2 shows maximum I,,,,
the maximum value
of inward IN,, recorded on the end-plate border and
on the extrajunctional membrane of human external
intercostal muscle fibers when slow and fast inactivations were removed. O n the extrajunctional membrane
of control fibers, I,, was larger for type IIb compared
with type IIa fibers ( p < 0.005) and I,, on type IIa
fibers was larger than on type I fibers ( p < 0.001). I,,
on type IIb fibers was 35% larger than on type IIa
fibers and 2.9-fold larger than on type I fibers. For
each fiber type, I,, was larger on the end-plate border
than on the extrajunctional membrane. The ratios of
I,, on the end-plate border compared with I, on the
extrajunctional membrane were as follows: type I, 3.27;
type IIa, 5.37; and type IIb, 5.08. I,, on the end-plate
border of type IIb fibers was 28% larger than on type
IIa fibers ( p < 0.001) and 3.64-fold larger on type IIa
fibers compared with type I fibers ( p < 0.001).
For control rat muscles, I,, on type IIb omohyoid
fibers was larger than type I adductor longus fibers on
the end-plate border and on the extrajunctional membrane (Fig 3). I,, on the extrajunctional membrane of
type IIb fibers was 1.81-fold larger than on type I fibers ( p < O.O01), and I
, on the end-plate border of
type IIb fibers was 4.08-fold larger than on type I fibers ( p < 0.001). The ratios of I,, on the end-plate
border compared with I,, on the extrajunctional membrane were type I, 3.29, and type IIb, 7.42.
I,
Voltage Dependencies of N a t Channel Opening and
Inactivation Were the Same f o r Recordings on the
End-Plate Border a n d the Extrajunctional Membrane
The voltage dependencies of Na+ activation and fast
inactivation varied with fiber type. Table 2 shows the
values of &Na,mm, Vh1,2,and Ah for the human fibers
shown in Figure 2 and the rat fibers shown in
Figure 3. The values of V,
, Vhl,2, and Ah for recordings on the end-plate !%yder of human or rat fibers were not significantly different from the values
found on the extrajunctional membrane.
Increased I, a t the End Plate Reduces the Threshold
f o r Triggering an AP
For human (Table 3) and rat (Table 4) control muscle
fibers, the threshold depolarization for initiating an AP
was lower and the AP rate of rise was greater on the
end-plate border compared with the extrajunctional
membrane. The reduction in the AP threshold on the
end-plate border for control human muscle fibers was
374 Annals of Neurology Vol 43 No 3
March 1998
;I
8
-i
p<0.001
p<0.001
Fig 2. The peak inward Na+ current, INn,mnx,recorded on
the extrajunctional membrane (EJ) and the end-plate border
(EB) of external intercostal muscle jbers fram 3 patients with
myasthenia gravis (MG) and 7 control patients (C). The results were segregated on the basis of the muscle j b e r i histochemical type, as type I, type Ila, or type IIb. Signi9cant d$
ferences in
between jbers fYDm C and MG patients,
for a specific region and j b e r type, are indicated on the
graph.
was reduced at the end-plate border for a l l j ber types from MG patients. Values of INa,mlor on the extrajunctional membrane were not diferent between MG and C
patients. The vertical lines indicate 2 1 SD. The longer horizontal lines bisecting each vertical line indicate the means and
the shorter horizontal lines indicate 2 1 SEM.
12.3 mV for type IIb fibers, 12.4 mV for type IIa fibers, and 7.1 mV for type I fibers (see Table 3). O n
the end-plate border of control human muscle fibers,
the rate of rise of the AP was greatest for type IIb fibers
and least for type I fibers. Table 4 shows that the reductions in AP thresholds at the end-plate border for
control rats were 10.7 mV for type IIb fibers and
7.9 mV for type I fibers. For control human and rat
muscles, each fiber type had similar resting potentials
at the end-plate border and on the extrajunctional
membrane.
from rats with PTMG also had reduced I,, at the endplate border compared with the same type fibers from
control rat fibers (see Fig 3). End-plate border I,, was
reduced 36.3% for type IIb omohyoid fibers and
33.2% for type I adductor longus fibers. I N , on the
extrajunctional membrane was similar to control values
for fibers from patients with M G or from PTMG rats.
KNa,,:Ax, and A, on the end-plate border or the
extrajunctional membrane of fibers from patients with
MG and from PTMG rats were similar to the control
values (see Table 2).
140-
t 30-
100
-
-
-
I .
AP Threshold at the End PLate Is Increased in
MG and PTMG
Table 3 shows that the threshold for initiating an AP
. .
.
..
.
5
3 80E
t
v
.
P
I
60X
a
I
40t
*
p<0.001
pz0.005
OL
- -
PTMG
EJ
-
WMG TE
T E B p T ~ ~FEJ
-
TYPE I
-
_
_
~
M
.
TYPE Ilb
Fig 3. The peak inward Na+ current, INd,,,&, recorded on
the extrajunctional membrane (EJ and the end-plate border
(EB) of adductor longus (type I) and omohyoid (type IIb)
fiom 7fernale Lewis rats with passive transfer myasthenia
gravis (PTMG) or 7 control rats (C). The results were segregated, on the bdtis of the muscle fiber? histochemical type, as
type I OT IIb. SignaJ5cant differences in INa,maw between C and
PTMG fibers for a specajk region and fiber type are indicated
on the graph. INa,mnx was reduced at the end-plate border $3.
both j b e r types fiom PTMG rats. The values of INa,max on
the extrajunctional membrane were not different between
PTMG and C rats. See legend to Figure 2 $3. additional
details.
I, at the End-Plate Border, but Not the
Extrajunctional Membrane, Was Reduced in
MG and PTMG
Fibers from patients with MG (see Fig 2 ) had lower
I,, on the end-plate border of each type of muscle
fiber compared with controls. The reductions in endplate border IN, for MG fibers compared with control
fibers were 38.6% for type I fibers, 34.2% for type IIa
fibers, and 36.4% for type IIb fibers. Muscle fibers
G
at the end-plate border was increased and the maximum rate of rise of the AP was decreased for all types
of fibers from patients with M G compared with controls. The AP thresholds at the end-plate borders were
increased by 9.4 mV for type IIa fibers, 8.9 mV for
type IIb fibers, and 4.5 mV for type I fibers. AP
thresholds and rates of rise on the extrajunctional
membrane were similar for each type of fiber from patients with MG compared with controls. Membrane
potentials on the end-plate border and on the extrajunctional membrane were similar for the fibers used to
study AP properties. The fibers used to study AP properties from patients with M G and from controls had
similar membrane potentials.
The AP threshold was larger and the maximum AP
rate of rise was slower on the end-plate border for type
IIb and type I fibers from PTMG rats compared with
controls (see Table 4). The end-plate border AP
threshold was increased 8.9 mV for type IIb fibers and
5.1 mV for type I fibers. In contrast, the AP rate of rise
and the AP threshold on the extrajunctional membrane
were similar for type IIb or type I fibers from PTMG
rats compared with controls. End-plate border and the
extrajunctional membrane potentials were similar for
the rat fibers used to study AP properties. Fibers from
PTMG and control rats used to study AP properties
had similar membrane potentials (see Table 4).
Discussion
This study has demonstrated a loss of I,, that is selective for the end-plate region of muscle fibers in patients
with MG (see Fig 2) and in rats in which EAMG was
induced by the passive transfer of a complement-fixing
AChR-specific monoclonal antibody (see Fig 3). The
selective involvement of INa at the end plate makes it
unlikely that antibodies in patients with MG or rats
with PTMG reduced I,, by directly blocking Na+
channels. The muscle fibers from which we recorded
I,, were not depolarized. The voltage dependencies of
Nat channel opening and inactivation of fibers from
patients with MG and rats with PTMG were the same
Ruff and Lemon: End-Plate Naf Channel Loss in MG
375
Table 2. Sodium Channel Voltage-Dependent Gating Properties (mean 2 SEM) on Extrajunctional Membrane of Intercostal
Muscle Fibers from Seven Control Patients and Three Patients with MG" and Omohyoid Muscles from Five Female Lewis Rats
with PTMG and Five Control Rats 24 Hours ajer Antibody Injectionb
Human muscle fibers'
Type IIb, control (n = 10 fibers)
Type IIb, MG (n = 7 fibers)
Type IIa, control (n = 9 fibers)
Type IIa, MG (n = 6 fibers)
Type I, control (n = 17 fibers)
Type I, MG (n = 9 fibers)
Rat muscle fibers"
Type IIb, control (n = 9 fibers)
Type IIb, PTMG (n = 9 fibers)
Type I, control (n = 9 fibers)
Type I, PTMG (n = 9 fibers)
-29.5
-30.1
-28.4
-28.6
-19.5
-19.3
C 1.4
2 1.7
2 1.3
2 2.1
2 1.2
i 1.4
-74.1
-73.5
-68.1
-69.0
-60.8
-60.7
2 0.7
2 1.1
i 0.7
2 1.2
2 0.8
2 0.9
6.22 i 0.29
6.40 2 0.36
6.30 ? 0.22
6.35 2 0.41
7.82 t 0.20
7.91 2 0.28
-24.2
-26.6
-18.5
-18.3
2 1.1
2 1.4
? 1.0
2 1.1
-76.1
-76.2
-68.3
-68.2
-t 0.7
i 0.8
2 0.7
2 0.8
6.22 2 0.29
6.29 i 0.31
8.23 -C 0.32
8.27 ? 0.36
'INu-,," data shown in Fizure 2.
' & ~ , ~ ~ ~
data shown in Figure 3.
'For each fiber tvue. there were no significant
differences between orooerties of fibers from uatients with M G and from controls. For both
"
control subjects and patienrs with MG, the following properties were different for type I fibers compared with type I1 fibers: FNa,max
(type IIa
or IIb, p < 0.01), Kl1,, (type IIa, p < 0.01; or type IIb p < 0.001), and A, (type IIa or IIb, p < 0.001).
dFor cach fibcr type, there were no significant differences between properties of fibers from PTMG and control rats. For both control and
( p < 0.005, for control; p < 0.001,
PTMG rats, the following properties were different for type I fibers compared with type IIb fibers: V,Na,max
for PTMG), y,,,, ( p < 0.001, for control and PTMG fibers), and A,, ( p < 0.001, for control and PTMG).
J l
L
I
MG = myasthenia gravis; PTMG = passive transfer MG; INa,n,au
= peak inward Na+ current; V,Na,max= potential at which maximal current
occurred; V,,,, = potential of prepulse inactivating one-half of Na+ channels; A, = parameter determining steepness of h,-membrane
potential relation.
Table 3. Resting Membrane Potential and Action Potential Properties (mean ? SEMI on the End-Plate Border Compared with
Extrajunctional Membrane of Intercostal Muscle Fibers JFom Seven Control Subjects and Three Patients with MG
RP (mV)
AP Threshold (mV)
AP dWdt (Vs-')
End-plate border
Type IIb, control (n = 36 fibers)
Type Ilb, MG (n = 21 fibers)
-95.2 2 0.9
-94.0 i 1.6
Type IIa, control (n = 27 fibers)
Type IIa, MG (n = 20 fibers)
-94.1 L 0.7
-93.0 2 1.5
Type I, control (n = 22 fibers)
Type I, M G (n = 15 fibers)
-85.1 2 0.9
-84.3 2 1.5
-71.2 2 2.0
-62.3 t 2.9
p < 0.001"
-69.3 2 1.6
-59.9 +- 2.4
p < 0.001"
-58.6 2 1.7
-54.1 2 2.2
p < 0.02"
611 2 21
412 ? 18
p < 0.001"
579 2 22
371 2 20
p < 0.001"
364 2 17
258 2 18
p < 0.001"
Extrajunctional membrane
Type IIb, control (n = 36 fibers)
Type IIb, M G (n = 21 fibers)
Type IIa, control (n = 27 fibers)
Type IIa, MG (n = 20 fibers)
Type I, control (n = 22 fibers)
Type I, MG (n = 15 fibers)
-95.3
-95.1
-93.8
-94.1
-85.3
-84.8
2 1.0
? 1.1
i 1.2
2 1.0
2 1.2
? 1.1
-58.9
-58.7
-56.9
-56.7
-51.5
-51.0
2
2
2
2
2
2
1.3
1.5
1.5
1.4
1.7
1.5
3 6 7 2 17
359 -C 13
327 i 16
329 2 15
226 i 14
233 i 18
"Significant differences are for comparisons between M G and control fibers.
MG
=
myasthenia gravis; RP = resting membrane potential; AP
=
as for controls (see Table 2), and INa
did not recover
after prolonged conditioning hyperpolarization. We
therefore concluded rhat the most likely cause of diwas loss of end-plate border Nat chanminished INa
nels rather than block of Na+ channels by antibody,
Na+ channel inactivation, or altered Na+ channel
activation.
376
Annals of Neurology
Vol 43
No 3
March 1998
action potential; dV/dt
=
maximum rate of. rise.
Pagala and colleague^^^^^^ found, in intercostal muscle from patients with MG, that APs triggered by nerve
stimulation were compromised, whereas APs initiated
by stimulation of the extrajunctional muscle membrane
were not altered compared with controls. This observation is consistent with a selective loss of end-plate
Nat channels.
Table 4. Resting Membrane Potential and Action Potential Properties (mean
Extrajunctional Membrane f i r Muscle Fibers porn Control and PTMG Rats
?
SEM) on the End-Plate Border Compared with
RP (mV)
AP Threshold (mV)
AP dWdt (Vs-‘)
End-plate border
Type IIb, control (n = 33 fibers)
Type IIb, PTMG (n = 35 fibers)
-95.2 t 1.1
-94.3 ? 1.4
Type I, control (n = 29 fibers)
Type I, PTMG (n = 32 fibers)
-85.7 ? 1.1
-83.7 t 1.4
-70.1 ? 1.9
-61.2 ? 1.7
p < 0.001”
-59.4 -c 2.1
-54.3 t 2.0
p < 0.05”
598 t 21
391 2 17
p < 0.001”
345 5 19
246 -C 20
-59.4 t 1.9
-59.2 t 1.8
-51.5 ? 1.7
-51.0 ? 1.8
356 k
339 ?
210 t
213 -C
Extrajunctional membrane
Type Ilb, control (n = 33 fibers)
Type IIb, PTMC (n = 35 fibers)
Type I, control (n = 29 fibers)
Type I, PTMG (n = 32 fibers)
-95.4
-95.5
-85.3
-86.0
i 1.5
2 1.3
i 1.2
t 1.3
p < 0.001~
12
10
17
16
“Significant differences are for comparisons between PTMG and control fibers.
PTMG = passive transfer myasthenia gravis; RP
=
resting membrane potential; AP = action potential; dVldt = maximum rate of rise.
The reduced IN,at the end-plate border in MG was
associated with an increase in the AP threshold. Prior
studies had demonstrated that IN, was larger on fast
twitch than on slow twitch
and that
ZNa was larger on type IIb fibers than on type IIa fiOur study confirmed, on the end-plate border
and on the extrajunctional membrane of normal fibers,
that I,, was greater for fast twitch fibers than for slow
twitch fiber^,'^-'^^^^ and that type IIb fibers had larger
I,, than type IIa fibers2’ We also confirmed that IN,
was larger at the end plate compared with the extrajunctional membrane.16-19,36 The rate of rise of the
AP depends on I,, amplitude, which depends on the
density of Na+ channel^.^' Although prior studies indicated that the higher INa
at the end plate reduced the
AP threshold,21326327
our study demonstrated that the
AP threshold and the AP rate of rise varied in concert
with ZNa when the characteristics of different histochemical types of rat or human muscle fibers were
compared. For all types of rat 01-human muscle fibers
studied, the AP threshold was lower and the rate of rise
higher on the end-plate border than on the extrajunctional membrane. The AP rate of rise was faster and
the AP threshold was lower for fast twitch than for
slow twitch fibers (see Tables 3 and 4 ) and for type IIb
human fibers compared with type IIa fibers.
Our findings overall suggest that voltage-sensitive
Na+ channels as well as AChRs are lost from the end
plate in patients with MG and in rats with PTMG.
Figure 1 (bottom panel) illustrates loss of both AChRs
and end-plate Na+ channels in the simplified end-plate
membrane that is characteristic of MG. The low
MEPP amplitudes found in our study were similar
to values reported previously for MG patients6’10’3s
and PTMG rats.13214The magnitude of reduction
in MEPP amplitude indicated that end-plate losses
of AChRs were about 72% for fibers from MG pa-
tients and 77% for fibers from PTMG rats. The fractional loss of Na+ channels was smaller than the
fractional loss of AChRs. Depending on fiber type, the
percent loss of end-plate border Na+ channels on myasthenic fibers compared with controls ranged from 34
to 39% for MG patients (see Fig 2) and 34 to 36% for
PTMG rats (see Fig 3). Loss of end-plate border I,,
was associated with an increase in AP threshold and a
reduction in the rate of AP rise (see Tables 3 and 4 ) . In
, , , , , ,Z
AP thresholds, and AP
contrast, maximum
rates of rise on the extrajunctional membrane were the
same in myasthenic and control fibers, in patients and
in rats. These data are consistent with a loss of voltagegated Na+ channels that is restricted to the end plates
of myasthenic fibers.
The high Na’ channel density at normal end plates
contributes to the safety factor for neuromuscular
transmission by reducing the AP threshold at the end
plate. Loss of Na+ channels from end plates of M G
patients and PTMG rats compromises the safety factor
for neuromuscular transmission by increasing the AP
threshold.
The data presented here do not address the mechanism of loss of voltage-gated Na+ channels at the end
plate in myasthenics. Two points challenge that Nat
channel loss is the result of antibodies binding directly
to Na+ channels. First, only the end-plate Na+ channels were lost. End-plate AChRs are particularly vulnerable to the pathogenic consequences of antibody interaction, because the high concentration of end-plate
AChRs amplifies the extent of end-plate membrane lysis resulting from complement activation. l S 2 AChR
density is greater than Na+ channel density in the endplate membrane and is more than I ,000-fold greater
than the extrajunctional membrane AChR density.* In
contrast, the density of voltage-gated Na+ channels in
the end-plate membrane is only three- to sevenfold
Ruff and Lennon: End-Plate Naf Channel Loss in MG
377
greater than in the extrajunctional membrane (see Figs
2 and 3). If antibodies in MG were specifically directed
at Na+ channels, one might anticipate compromise of
both the end-plate and the extrajunctional Na+ channels. The normal size of extrajunctional ,,/ that we
observed in muscle fibers of both MG patients and
PTMG rats suggests that the reduction in end-plate
border I,, was not due to direct binding of antibodies
to Na+ channels.
Losavio and associates3’ studied AP properties on
the extrajunctional membrane of fast twitch muscle fibers obtained from mice injected intraperitoneally with
sera from patients with MG or control sera. MEPPs
from the mice treated with MG sera were smaller than
those from controls, but the extrajunctional AP amplitudes and rates of rise were not changed. After denervation, muscle fibers expressed Nat channels and had
extrajunctional action potentials that were resistant to
tetrodotoxin. The denervated fibers also had an increased density of extrajunctional AChRs. Denervated
fibers from mice treated with MG sera had smaller AP
amplitudes and AP rates of rise compared with denervated fibers from control mice. The authors suggested
that in denervated fibers, MG sera triggered loss of extrajunctional AChRs and that membrane containing
tetrodotoxin-resistant Na+ channels may also have
been affected by the MG sera. Presumably, the MG
sera had a greater effect on the extrajunctional membrane of denervated muscle fibers because of the increased concentration of extrajunctional AChRs after
denervation.”*
The possibility that antibodies against voltage-gated
Nai channels might exist in patients with MG has not
been investigated. In clinical MG, antibodies can react
with muscle proteins other than the AChR.40 However, challenging that the reduction of end-plate I,, in
fibers of MG patients is caused by antibodies directed
against Na+ channels is that end-plate ,,/ was reduced
in rats by using a high-affinity monoclonal antibody
that reacts specifically with the skeletal muscle
AChR.I4 It is unlikely that this antibody binds to Na+
channels.
If only AChR-containing end-plate membranes were
lost in MG and PTMG, the concentration of Na+
channels at the end plate should have increased. The
reduction in end-plate border,,/ indicates loss of Na+
channels, presumably due to destruction of end-plate
membrane containing both AChRs and Na+ channels.
The larger relative reduction of MEPP amplitudes
compared with IN, indicates that more AChRs were
lost than Na+ channels. This would be consistent with
more extensive damage of end-plate membrane containing AChRs than of end-plate membrane containing
Naf channels.
Our observation that collagenase treatment lysed
more myasthenic muscle fibers than control fibers
378
Annals of Neurology
Vol 43
No 3
March 1998
is consistent with myasthenic fibers being more susceptible to coilagenase-induced injury due to preexisting complement lesions of end-plate membrane.
This would predict injury-induced depolarization
of the end-plate membrane, which in turn would reduce the fraction of excitable Na+ channels due to
voltage-dependent slow and fast inactivation of Nat
Thus, two factors compromise the safety factor for
neuromuscular transmission in clinical MG and in its
IgG-mediated experimental rat model. First, loss of
AChRs reduces the end-plate sensitiviry to acetylcholine, resulting in a diminished EPP.’-4 Second, loss of
end-plate voltage-gated Na+ channels reduces the endplate,,/ and this, in turn, increases the AP threshold.
This study was supported by the Office of Research and Development, Medical Research Service of the Department of Veterans Affairs (R.L.R.), and the Admadjaja Thymoma Research Program
(V.A.L.).
Presented in part at the 1 2 1 s Annual Meeting of the American
Neurological Association, October 1996.
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Ruff and Lennon: End-Plate Naf Channel Loss in MG
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