Characterization OfAntibodies To Subcellular Fractions Of
Skeletal Muscles In Patients With Myasthenia Gravis And
Autoimmune Rippling Muscle Disease
By
Staci R. Raab B.S.
Submitted in Partial Fulfillment ofthe Requirements
For the Degree of
Master ofScience
in the
Biological Sciences
Program
SCHOOL OF GRADUATE STUDIES
YOUNGSTOWN STATE UNIVERSITY
August, 1999
Characterization OfAntibodies To Subcellular Fractions Of Skeletal
Muscles In Patients With Myasthenia Gravis And Autoimmune Rippling
Muscle Disease
Staci R. Raab B.S.
I hereby release this dissertation to the public. I understand this dissertation will be housed at the
Circulation Desk ofthe University library and will be available for public access. I also authorize
the University or other individuals to make copies ofthis dissertation as needed for scholarly
research.
Signature:
Approvals:
Committee Member Date
Date
111
ABSTRACT
Rippling muscle disease (RMD) is a rare autosomal dominant disease
characterized by muscle weakness after resting, and electrically "silent"
wave-like contractions ofskeletal muscle activated by percussion or stretch.
In 1995, Carl Ansevin M.D. diagnosed a patient with rippling muscle
disease associated with myasthenia gravis (MG) (Ansevin and Agmano1is,
1996). This patient had no family history ofRMD and experienced a
disappearance ofRMD symptoms after a thymectomy suggesting that RMD
associated with MG may have an autoimmune origin. From western blot
examination ofsera from this patient and sera from other MG patients at
varying severity levels (both with and without the RMD component) it was
shown that MG/RMD patients displayed immunoreactivity to a skeletal
muscle protein 66-97 kDa in size as well as immunoreactivity to proteins
with molecular weights of200 kDa and 300-500 kDa. Western blot analysis
ofMG/RMD patients' sera displayed immunoreactivity with T-tubu1ar
membranes ofrat skeletal muscle. However, this same immunoreactivity
was also shown in a patient without the rippling muscle component thus
indicating that these antibodies may be due to the MG, not the rippling
muscle component. Sarcoplasmic reticular fractions ofrat skeletal muscle
did not display any immunoreactivity to MG/RMD patient sera.
IV
ACKNOWLEDGMENTS
I would like to thank Dr. Gary Walker for his guidance, patience, and
overall love oflife. Thanks also to Dr. Paul Peterson, Dr. Mark Womble,
and Dr. Carl Ansevin for allowing me to utilize their expertise to enhance
not only my project, but my overall education as well. To Thomas Watkins
M.S., my appreciation for supplying me with far more help, time,
encouragement and friendship than I had ever expected from one person
over the course ofthis project. I have learned far more than I had
anticipated from each ofyou. I would also like to thank all ofthe members
ofthe Walker Lab; Nader Atway, Laura Shea, Tom Watkins, and Shannon
Durkin (by adoption) for making these years at Youngstown State exciting,
memorable and most ofall, fun. Your friendship is, and will always be,
greatly appreciated. Love and gratitude to my devoted parents, Paul and
Joyce Raab. Thanks also to my brother Paul
II
and my sisters; Lisa, Noelle,
and Candra for all oftheir unconditional love and support in every aspect of
my life.
Table of Contents
Abstract 111
Acknowledgments IV
List ofFigures VI
List ofTables VB
Introduction 1
Study Aims 17
Materials and Methods 17
Experimental Design 17
Vesicle Preparation 19
T-tubule Isolation 20
Sarcoplasmic Reticular and T-tubular Isolation 21
Bradford Protein Assay 23
SDS-PAGE 23
Western Blot Analysis 26
NEpHGE 27
Results 31
Discussion 59
Bibliography 67
v
List of Figures
Figure 1 SEM Muscle Picture VIll
Figure 2 Skeletal Muscle Composition 4
Figure 3 Immunocytochemistry 16
Figure 4 Bradford Assay Cricket Graph 29
Figure 5 NEpHGE 30
Figure 6 Gel ofVesicle Preparation Fractions 40
Figure 7 Immunoreactivity ofFractions with MG/RMD Sera 42
Figure 8 NEpHGE Results 44
Figure 9 Presence ofDHPR in T-tubule Final Fraction 48
Figure lOT-tubule Final Fraction Immunoreactivity with
Commercial Antibodies 50
VI
Figure 11 Immunoreactivity ofT-tubular Fraction with
Patient Sera
Figure 12 Protein Composition ofSR/T-tubule Fractions
Figure 13 Presence ofRyR in SR/T-tubule Fractions
52
55
57
List of Tables
Table 1
Summary ofAuto-antibody Immunoblot Patterns
Table 2
T-tubule Western Blot Results
Table 3
Description ofT-tub/SR Isolation Fractions
Table 4
Immunoreactivity ofSR/T-tubule Fractions
with Different Antibodies and Patients' Sera
14
46
53
58
Vll
Figure 1; SEM Photo ofRat Skeletal Muscle
Vlll
1
Introduction
This goal ofthis study is the identification and characterization ofthe
autoantibodies involved with a rare autoimmune rippling muscle component
sometimes associated with myasthenia gravis (MG). Since this rippling
muscle component affects the skeletal muscles ofpatients, the structural
understanding ofskeletal muscles is necessary in this study. This study will
deal predominantly with patients diagnosed as having myasthenia gravis
with a rippling muscle component.
The muscle cells that form skeletal muscles are termed muscle fibers.
It is the components ofthese muscle fibers, and the unique organization of
these components, that is responsible for the contraction and therefore
movement ofskeletal muscle. Muscle fibers are large cylinder shaped cells
that lie parallel to each other and are bundled together with connective
tissue. These multinucleate fibers are 10-100 Jlm in diameter and vary in
length. Each fiber is surrounded by a plasma membrane, or sarcolemma,
which contains groups ofmyofibrils as well as the fibers' cytoplasm, termed
sarcoplasm. The myofibrils found within the sarcolemma are the specialized
contractile elements ofthe skeletal muscles and extend the length ofthe
muscle fiber in which it is contained.
2
Myofibrils are each 1-2 /-lm in diameter and consist ofan organized
network ofthick and thin filaments which are responsible for the contractile
action ofthe muscles. The thick filaments range in diameter from 12 nm to
18 nm and have a length of 1.6/-lm. Each thick filament is made up of
approximately two hundred molecules ofthe contractile protein myosin.
Myosin consists oftwo identical subunits, each having a globular head
region as well as a tail region. The tail regions are intertwined with each
other and are oriented toward the middle ofthe filament while the head
portions project out in the opposite direction. This results in a bipolar
structure. The head portions, or cross-bridges, ofthe myosin molecules
contain two sites, one actin binding site and one myosin ATPase site. These
cross-bridges extend out at regularly spaced intervals toward the thin
filaments.
Thin filaments ofthe myofibrils are 5-8 nm in diameter and are 1.0
/-lm in length. Thin filaments consist ofthree contractile proteins, the major
one ofwhich is actin. Actin molecules are spherical in shape and join
together to form two twisted strands creating the backbone ofthe thin
filaments. Each molecule ofactin has a myosin-binding site to facilitate
binding ofthe myosin head from thick filaments during muscle contraction.
Thin filaments are precisely organized around thick filaments to allow for
3
actin and myosin interaction during contraction. On a microscopic level, the
arrangement ofthese filaments form a striated appearance consisting oflight
and dark bands. The dark A-bands are regions containing stacks ofthick
filaments which extend the entire length ofthe A-band. A-bands also
contain portions ofthe thin filaments which overlap the thick filaments. The
light I-bands contain the remaining portions ofthe thin filaments but do not
contain any thick filaments. Within each A-band there is a narrow, lighter
area in the center known as the H-zone which contains no thin filaments.
The H-zone is also divided by the M-line which is formed by proteins that
hold the thick filaments together in a vertical manner within each stack.
Also present is a region ofdense material within the I-bands termed the Z 
line which contains cytoskeletal proteins that function to connect the thin
filaments. The area between two Z-lines is referred to as the sarcomere and
is the functional contractile unit ofthe skeletal muscle cells.
The other two proteins present in thin filaments are tropomyosin and
troponin. Tropomyosin exists as a threadlike molecule which serves to
conceal the myosin-binding sites on actin when the muscle is in a relaxed
state. Troponin plays a role in stabilizing tropomyosin in this relaxed state
but changes confirmation after the binding ofcalcium (which is released for
muscle contraction). With calcium present, troponin allows tropomyosin to
4
I-band
~~f
j
=R=HffF. 11-+++++
fffff ' l'
M-line
----=-:-----:--
Z-line
H-zone
A-band
Figme 2: Skeletal Muscle Composition
5
slide away from the myosin-binding sites on actin, thus allowing actin to
bind myosin and initiate muscle contraction.
The sarcolemma, the muscle fiber's plasma membrane, sends deep
invaginations into the muscle fibers at the junctions ofthe A- and the 1 
bands forming transverse tubules (T-tubules). Closely associated with the T 
tubules within the muscle fiber's cytoplasm is a membrane-bound tubular
network called the sarcoplasmic reticulum (SR) which is modified
endoplasmic reticulum. The SR system surrounds each myofibril and serves
to store calcium when the muscle fiber is relaxed. The sarcoplasmic
reticulum is not continuous throughout the entire cell. Each SR segment
ends in a lateral sac which comes into close proximity to the T-tubule
system. When an action potential propagates into the T-tubules, the
membrane permeability ofthe sarcoplasmic reticulum for calcium increases
greatly, therefore allowing calcium to be released into the myofibrils it
surrounds.
Another type offilament present in skeletal muscle are elastic
filaments. These elastic filaments are primarily comprised of the protein
titin. This protein performs a stabilizing function by connecting the thick
filaments to the Z lines ofthe muscle. Titin, also referred to as connectin,
may also function to return the sarcomere to its correct resting length when
the muscle fiber relaxes (Tortora and Grabowski, 1993; Guyton and Hall,
1996).
Contraction ofa skeletal muscle fiber results from a series ofevents
that begins with the release ofacetylcholine from the motor neuron
innervating the muscle fiber. This release induces an action potential to
propagate across the surface ofthe sarcolemma. The action potential also
travels into the center ofthe muscle fiber via the T-tubule system. The
action potential activates dihydropyridine receptor (DHPR), voltage-gated
calcium channels, resulting in calcium release from the SR. The lateral sacs
ofthe SR respond to the action potential by releasing stored Ca++ into the
myofibrils via ryanodine receptor. Once the Ca++ enters the myofibrils, it
binds to the troponin molecules ofthe thin filaments. A change in
conformation occurs which causes the troponin-tropomyosin complex to
move aside, thus allowing the myosin cross-bridges and the myosin binding
sites on actin molecules to come together. Powered by ATP, the myosin
head then undergoes a power stroke that pulls the thin filaments inward
toward the Z-line. This movement ofthe thin filaments causes the
sarcomere to shorten in length and is responsible for muscle contraction.
Shortening continues as long as there is Ca++ present to keep the myosin 
binding sites uncovered.
6
7
Myasthenia gravis (MG) is an autoimmune neuromuscular disorder.
MG is a disease that causes the skeletal muscles ofthe body to become
abnormally weak and afflicts approximately 1 in 20,000 people (Guyton and
Hall, 1996). Symptoms occur in an aSYmmetrical pattern. Some ofthe
sYmptoms include problems with the extraocular muscles ofthe eyes,
difficulty in swallowing or chewing, and slurred speech. Generalized
muscle fatigue and weakness in the face, neck, arms, or legs are also
common sYmptoms (Grob et al., 1986; Aarli et al., 1990). The muscles of
the body are affected because MG patients produce autoantibodies to the
nicotinic acetylcholine receptors at the skeletal muscle neuromuscular
junction (Bartoccioni et al. 1980). These antibodies bind to the postsynaptic
acetylcholine receptor at the motor end plate. This disturbs normal
neuromuscular signal transmission by preventing the binding ofthe
neurotransmitter acetylcholine to the receptor. The signal from the nerves to
the muscle cells is greatly reduced due to these antibodies, therefore leading
to the muscular weakness experienced by the patients. Antibodies to
acetylcholine receptors are not the only antibodies present in MG patients.
Anti-thYroid autoantibodies, as well as autoantibodies against both skeletal
and heart muscle, have also been demonstrated in various studies using sera
from MG patients (Beutner et al., 1962; Strauss and Kemp, 1967; Penn et
8
at., 1986; Aarli et at., 1990; Gautel et at., 1993). Among patients that
have demonstrated additional autoantibodies are those diagnosed with a
thymoma, a slowly growing epithelial tumor ofthe thymus gland, in addition
to the myasthenia gravis. Thymoma seems to accompany the MG in about
15% ofpatients (Williams and Lennon, 1986). These patients show a higher
level ofskeletal muscle antibodies than MG patients without thymoma.
Penn et at. (1986) and Williams et at. (1986) used immunoflourescence to
determine what portion of muscle fibers have autoantibodies against them.
The striational autoantibodies found typically occur in three different
patterns. Immunoreactivity occurs with A-bands only, I-band only, or with
both A and I-bands in the region ofthe Z-line (Penn et at., 1986; Williams
and Lennon, 1986; Vetters, 1967). These antibodies are present in 80-95%
ofMG patients with thymomas and are directed against some ofthe
muscles' main proteins, including titin, actin, myosin, tropomyosin and
alpha-actinin (Aarli et at., 1990; Ohta et at., 1990; Pagala et at., 1990;
Williams and Lennon, 1986). The cause for striational autoantibody
production is unknown.
Another type ofautoantibody found in approximately 50% ofMG
patients with a thymoma are antibodies to the ryanodine receptor. The
amount ofantibodies for ryanodine receptor protein increases as the disease
9
increases in severity (Mygland et al., 1994). These antibodies are not,
however, found in MG patients without a thymoma.
The ryanodine receptor (RyR) is a calcium release channel protein in
the sarcoplasmic reticulum that has a molecular weight between 300 and 400
kDa (Zorzato et al., 1986; Lai and Messiner, 1990; Mygland et al., 1994).
The ryanodine receptor participates in excitation-contraction coupling by
mediating the release ofstored Ca++ from the sarcoplasmic reticulum into the
muscle cell cytoplasm. Within skeletal muscle fibers, RyR Ca++ release
channels consist offour ryanodine receptor subunits arranged in rows.
Ryanodine receptors connect the T-tubules to the SR. The ryanodine
receptor subunits are arranged in rows and are believed to contact the
neighboring ryanodine receptors allowing all the adjacent RyR channels to
open and close together.
Every other ryanodine receptor is associated with a T-tubular surface
dihydropyridine receptors (DHPR). In the leading theory describing
excitation-contraction coupling, depolarization ofthe T-tubule surface
membrane is thought to cause DHPR to trigger Ca++ release through the
associated ryanodine receptors via some type ofmechanical connection
between the two (Bers and Fill, 1998; Marx et aI., 1998; Tanabe et al.,
1990). Those ryanodine receptors not directly associated with a DHPR are
10
possibly then triggered by neighboring ryanodine receptors which are in
contact with the T-tubular surface via a DHPR. This is ofinterest
considering that some studies have demonstrated that many MG with
thymoma patients have abnormalities in the excitation-contraction coupling
event (Nielsen et al., 1982; Coronado et al., 1994; Skeie et al., 1996).
Rippling muscle disease is an inherited muscular disorder first
described by Torbergsen (1975) in a family in which five members (out of
the thirty-two studied) were afflicted with rolling muscle contractions after
stretch or percussion, muscle stiffness, and mounding ofthe muscle after
percussion. These contractions were found to be electrically silent. Other
studies have also been done involving members ofa different family with
the same problem. Other studies have supported Torbergsen in determining
this disease to be an autosomal dominant disorder (Ricker et al., 1989;
Stephan et al., 1994; Kosmorsky et al., 1995; Ansevin and Agmanolis,
1996).
Rippling muscle disease was thought to be only an inherited disorder
until a patient report by Ansevin (1996). This patient was different from
previous cases in that neither his nine siblings nor his family history showed
any signs ofneuromuscular disease, leading to the conclusion that his
rippling muscle disease was not a result ofinheritance. The patient was seen
11
at the age of56 in 1990 with muscle spasms and rippling muscles. He
returned again in 1995 with symptoms such as muscular weakness, fatigue
and autoantibodies for acetylcholine receptors which had not been present in
his prior visit. This led to a MG diagnosis. The patient was treated with the
immunosuppresent drugs, pyridostigmine and prednisone, and also
underwent a thymoma removal afterwhich the rippling muscle and MG
symptoms disappeared. Other cases ofmyasthenia gravis with rippling
muscles have since been reported. Since the contractions involved with
rippling muscle occur after stretch and percussion but are electrically silent,
it has been suggested that the contractions may result from the activation of
stretch-activated or mechanosensitive ion channels (Ansevin and Agmanolis,
1996).
The existence ofmechanosensitive channels in muscle tissue was
demonstrated by Guharay and Sachs (1984) in studies with chick skeletal
muscle. Since then, similar channels have been described in human cardiac
and smooth muscle. (Kirber et al., 1988; Bedard and Morris, 1992;
Ruknudin et al., 1993). Stretch activated channels are opened by the
stretching ofa muscle fiber, therefore allowing ions, including Ca++, to flow
into the muscle fiber. In cases ofrippling muscle, this inflow ofions may be
responsible for initiating the contractions observed upon percussion.
12
Rippling muscle patients may also have some additional abnormality that
would account for their enhanced sensitivity to the effects ofchannel
opening, which may explain why persons without the disorder do not
experience percussion activated contractions.
A variety ofpatients' sera was made available for analysis in the
present study. This has allowed for the comparison ofsera from MG with
rippling muscle (MG/RMD) to those from patients who have other problems
related to MG (such as thymomas). The patients whose sera was used in this
study have varying types ofMG; one patient has MG with rippling
muscles and a thymoma (we also have sera from the same patient when his
symptoms later returned after treatment and a thymoma removal), two
patients have MG with the rippling muscle component and no thymoma, two
patients have MG with thymoma and no rippling muscles, one patient has
MG without rippling muscles or thymoma and two patients have
comparatively mild MG without rippling muscles or thymoma. Also
included: a case ofocular MG, sera from an individual with malignant
hyperthermia and sera from a healthy individual to serve as a negative
control.
Results from a previous study by Watkins (1999) suggest that a
number ofmuscle proteins are recognized by sera from MG patients.
13
Western blot analysis has shown immunoreactivity between muscle proteins
and sera from several MG patients both with and without thymoma and the
rippling muscle component. All patients showed immunoreactivity to a low
molecular weight protein (66-97 kDa). Those MG patients with rippling
muscles also showed reactivity to two other proteins with high (300-500
kDa) and intermediate (200 kDa) molecular weights. These were not seen
on most blots using sera from MG patients without rippling muscles (see
Table 1). Watkins also performed immunocytochemistry and detected
striationallike banding in areas that appeared to be the T-tubule portion of
the myocyte. These results are shown in figure 5.
Table 1
Summary ofAuto-antibody Immunoblot Patterns*
14
Pat. Patient Symptoms Intermediat High Bands in
# e Mol. Wt Stacking
Mol. Wt
1 RM/MG/thymoma-no symptoms after 0
+ +
treatment
1 RM/MG -symptoms returned
++ +++ +++
13 RM/MG +/-
+ +/-
9 RM/MG
++ +++ ++
3 MG/thvmoma
+ +++ ++
10 MG/thymoma 0 0
+/-
7 MGmild 0 0 0
6 MG 0 0 no data
12 MGmild 0 0 0
8 malignant hyperthermia 0 0 no data
2 negative control 0 0 no data
5 OcularMG 0 0 0
*Taken with permission from Thomas Watkins' thesis "Characterization ofSkeletal
Muscle Antibodies in Patients with Autoimmune Rippling Muscles and Myasthenia
Gravis"
MG/thymoma
Figure 3 Immunocytochemistry
Immunofluorescent micrographs ofhuman skeletal muscle showing
striational immunoreactivity (arrows). Images A and C show bright field
micrographs ofhuman skeletal muscle tissue sections. Images Band D
show the same fields under fluorescence.
15
Figure 3 ImmuDocytochemistry*
*Taken with permission from Thomas Watkins' thesis "Characterization ofSkeletal
Muscle Antibodies in Patients with Autoimmune Rippling Muscles and Myasthenia
Gravis"
16
17
The goal ofthis study was to further identify and characterize the
autoantibodies associated with the autoimmune rippling muscle component
that accompanies myasthenia gravis by determining the subcellular
distribution oftheir antigens.
Materials and Methods
Experimental Design
This study was designed to determine ifautoantibodies in MG/RMD
patients' sera react with T-tubular and sarcoplasmic reticular membrane
proteins in fractions ofskeletal muscle. In order to make this determination
subcellular fractionation ofskeletal muscle was performed in three different
isolation procedures. A vesicle preparation was performed first to yield a
fraction rich in vesicle membranes. This type ofisolation did not show the
immunoreactivity between the patients' sera and the high and intermediate
molecular weight proteins as seen in previous data, for this reason a different
isolation procedure was performed to isolate T-tubular membranes ofthe
muscle cells specifically. Since both RyR and DHPR Ca++ channels are
located in this area ofskeletal muscle, it was hypothesized that MG/RMD
patients may have autoantibodies to this portion ofthe muscle thus possibly
playing a role in the electrically silent muscle contractions ofthese patients.
18
A third isolation was performed to isolate sarcoplasmic reticular membrane
as well as T-tubular membrane. This would allow us to further distinguish
whether the autoantibodies were to the SR and/or the T-tubular portion of
the muscle cell. The protein composition ofthe different samples or
fractions produced from all three isolation procedures were analyzed using
SDS-PAGE and western blot analysis. SDS-PAGE was used to determine
the protein concentration and composition ofeach fraction and western
blotting was used to determine which proteins react with antibodies of
MG/RMD patients. NEpHGE was also used in an attempt to further
characterize autoantigens involved.
Normal rat skeletal muscle was used with MG patients' sera for
analysis ofany autoantibodies present. The sera were collected from
patients diagnosed with MG who also demonstrated the rippling muscle
component and, for comparative purposes, from several other MG patients
without rippling muscles. A negative control sera from a healthy individual
without MG was also available for study. Antigen specificity ofthe various
sera were determined by SDS-PAGE or NEpHGE in combination with
western blot analysis. Subcellular localization ofautoantigens was
performed by western blot analysis ofsubcellular fractions.
19
Vesicle Preparation
In order to prepare a membranous fraction ofskeletal muscle
containing T-tubular membrane, vesicles were prepared according to de
Meis et ai. (1971). Approximately 15g ofrat skeletal muscle was prepared
in the following manner. The muscle was minced with a razor blade then
mixed with 45 ml ofa cold solution containing 100 mM KCl, 2 mM EDTA,
2.5 mM KH
Z
P0
4
and 2.5 mM K
z
HP0
4
• This mix was placed into a Waring
blender for 2 minutes. Next, centrifugation was performed in a Beckman
centrifuge at 3,300 x g for 30 minutes to sediment myofibrils. The
supernatant was centrifuged at 3,500 x g for 45 minutes to remove the
mitochondria. The supernatant was then ultracentrifuged at 44,000 x g for 1
hour after which the pellet was suspended in 4.8 ml ofa solution containing
1M sucrose and 50 mM KCl, sonicated to disperse the material, and
centrifuged at 4,500 x g for 15 minutes. After removal ofthe pellet, 3.9 ml
ofa solution containing 2M KCl and 5mM ATP was added to the
supernatant and ultracentrifuged at 80,000 x g for 90 minutes. The resulting
pellet was washed twice with 10 ml ofO.lM KCl. In each washing, the
material was dispersed with a sonicator and centrifuged at 80,000 x g for 60
minutes. The final pellet was suspended in 1 ml ofthe O.IM KCl solution
20
and stored at 5°C. All steps were run at 4°C and the pellets and supernatants
from each step saved for possible use in future procedures.
T-tubule Isolation
T-tubular membranes were prepared from rat skeletal muscle
according to Florio et al. (1992). Back and hind leg rat skeletal muscle (40
grams) was used for the isolation ofT-tubules for analysis. The muscle was
minced with a razor blade, mixed with 50 ml ofbuffer #1 containing
100mM Tris, 0.3 M sucrose and placed into a blender for 30 seconds. More
buffer (50 ml) was added and again blended for 30 seconds followed by the
addition ofbuffer #2 consisting of20mM Tris. The homogenized muscle
mixture was placed on ice for 5 minutes then blended again for 30 seconds.
This step was repeated seven times before centrifugation ofthe mixture at
3600 rpm in a Beckman J6 rotor for 20 minutes. The top layer and pellet
were both removed and saved. The supernatant was filtered through a
double layer ofcheesecloth and centrifuged again at 10,000 x g for 20
minutes. The pellet was removed and powdered KCl was added to the
supernatant to reach a final concentration of0.5M KCl. This solution was
stirred for 30 minutes on ice before being centrifuged at 150,000 x g for 45
minutes to collect a crude T-tubule pellet. The pellet was washed in buffer
#2 and resuspended in this buffer at a concentration of 10 mg/ ml. The
21
membranes were then homogenized six times in a homogenizer and frozen
for analysis in the lab.
Sarcoplasmic Reticulum and T-tubule Isolation
T-tubular and sarcoplasmic reticulum membranes were prepared
according to Sabbadini and Okamoto (1983). Approximately 50g ofrat
skeletal muscle was prepared in the following manner. The muscle was
homogenized in a Waring blender for 15 seconds every 5 minutes for an
hour with 150 ml ofa cold solution containing 10 mM MOPS, pH 6.8, and
10% sucrose. The pH ofthis mix was maintained by the occasional
addition ofa few drops of5% NaOH. Next, centrifugation was performed at
15,000 x g for 20 minutes. The supernatant was then filtered through
cheesecloth and ultracentrifuged at 40,000 x g for 90 minutes after which the
pellet was suspended and incubated for 1 hour in a solution containing 10
mM MOPS, pH 6.8, and 0.6 M KCl. Centrifugation was performed at
15,000 x g for 20 minutes. The supernatant was ultracentrifuged for 90
minutes at 40,000 x g. The pellet was then suspended to a protein
concentration of0.35 mg/ml in an oxalate loading solution containing 20
mM MOPS, pH 6.8, 2 mM CaCh, 2 mM EGTA, 5 mM potassium oxalate,
80 mM KC1, 5 mM MgCh, and 5 mM ATP and incubated at 37°C for 10
minutes. The loading mixture was layered in 17 ml aliquots on top ofa 2
22
layers sucrose gradient. The first layer was a 4 ml solution containing 52%
sucrose and 20 mM MOPS, pH 6.8, and the second layer was a 7 ml
solution containing 30% sucrose and 20 mM MOPS, pH 6.8. The gradient
was then ultracentrifuged at 140,000 x g for 120 minutes. The top T-tubule
layer was drawn offwith a pipet, reincubated in loading solution and
ultracentrifuged in the same manner previously described to further purify
the fraction. The top layer was collected, washed in 50 mM imidazole, pH
7.0 and suspended in 30% sucrose, and 20 mM imidazole, pH 7.0 to final
protein concentration of 1-2 mg/ml. The pellets ofboth centrifugations were
suspended in an oxalate unloading solution of 10 mM MOPS, pH 6.8, 0.6 M
KC1, 1 mM EGTA to a protein concentration of0.2-0.35 mg/ml (determined
by Bradford Assay) and incubated at 5°C for 12-18 hours. The mixture was
layered in 15 ml aliquots on 4 ml of52% sucrose, and 20 mM MOPS, pH
6.8, then ultracentrifuged at 141,000 x g for 1 hour. The top layer and the
pellet were collected and used for analysis. All ultracentrifugations occurred
using a Beckman anglehead rotor unless otherwise stated. All steps were
run at 4°C and the pellets and supernatants from each step saved for possible
use in future procedures.
23
Bradford Protein Assay
Bradford protein assays were used to determine the protein
concentration ofsamples obtained during the T-tubule/ SR isolation
procedure in order to dilute samples to a specific concentration as called for
in the isolation procedure protocol. Five standards and one sample were all
tested in triplicate. A dye solution is made with 24 parts ddHzO and 6 parts
concentrated Bio-Rad protein dye reagent. To each test tube 990J..ll ofthis
solution is added to 10J..ll ofthe sample or standard being used. The five
standards used include immunoglobin or BSA at 0, 0.34, 0.68, 1.02, and
1.36 J..lg/ml. Each sample's optical density was read using a Beckman
spectrophotometer at 595A visible light and the data obtained analyzed on a
Cricket graph in order to obtain information allowing for the calculation of
the unknown sample's protein concentration. Figure 4 is a sample Cricket
graph.
SDS-PAGE
Protein composition was analyzed by sodium dodecyl sulfate
polyacrlylamide gel electrophoresis (SDS-PAGE). This was used to analyze
the various proteins present in samples from each step ofthe previously
described isolation procedures. The purpose ofSDS-PAGE is the separation
ofproteins through an acrylamide gel matrix based on molecular weight.
24
During SDS-PAGE two types ofacrylamide gels are responsible for the
protein separation, resolving gel and stacking gel. The main sieving gel, or
resolving gel, is poured in sets of 10-12 and vary in acrylamide
concentration depending on the degree ofseparation required. Typically in
this study 10%,7.5% and 5% gels were used.
The 10% gels were used to initially analyze the overall protein
concentration ofeach sample in order to best determine the amount of
sample needed to load into gels for western blotting. The 7.5% and 5% gels
were used during western blotting to allow very large proteins normally
caught in the stacking gel portion ofthe gel to diffuse down into the
resolving gel (the lower the acrylamide concentration, the better able
proteins are to move through the gels). When SDS-PAGE is performed, a
layer ofstacking gel is poured overtop ofthe resolving gel and functions to
concentrate the samples before diffusion into the resolving portion ofthe
gel. A comb is then placed in the stacking gel after it is layered over the
resolving gel. The stacking gel polymerizes and the comb removed to form
wells into which the samples can be loaded.
Each sample was prepared for electrophoresis by adding sample
buffer and was then placed into boiling water for 1 minute. Between 5-10 I-tl
ofeach sample (depending on its' protein concentration) was then loaded
25
into the stacking gel and the gels ran for 90 minutes at a constant current of
0.025 amps. The SDS added to the samples serves to impart a negative
charge on the proteins within the sample. A negative electrode is placed at
the top portion ofthe gel and a positive electrode at the bottom. Since the
proteins have a negative charge due to the SDS, they move through the gel
matrix toward the positive electrode, the smaller proteins moving faster than
the larger allowing the separation by size. Gels were then either stained with
coomassie blue or the proteins transferred to nitrocellulose sheets for further
analysis by western blotting.
Proteins were transferred from the polyacrylamide gels to a
nitrocellulose sheet by placing them into a "Genie" tray from Idea Scientific
in the following manner. An electrode and bubble screen were placed first
on the bottom ofthe plexiglass tray followed by a Scotch Brit pad and sheet
offilter paper. Next, the gel is placed over the filter paper followed by a
nitrocellulose sheet, a sheet filter paper, a Scotch Brit pad, bubble screen,
electrode, and another sheet ofplexiglass is added after the chamber is filled
with transfer buffer. The materials are carefully layered into the chamber to
ensure that air bubbles do not get trapped between the gel and transfer
membrane. The transfers were then run for 45 minutes at 25 volts. After the
26
transfers are complete, the transfer membranes are removed from the
chamber for western blotting.
Western Blot Analysis
Immunoreactivity between patients' sera and proteins in subcellular
fractions ofskeletal muscle was determined by western blotting techniques.
Antibodies were also used during western blot analysis to determine the
composition ofsamples. The blots were performed by exposing the
nitrocellulose sheets, obtained after transferring proteins from an SDS 
PAGE gel, to a blocking buffer for 60 minutes to eliminate nonspecific
binding sites. Blocking buffer contains 10% non-fat dry milk in TBS-T.
TBS-T (Tris buffered saline solution containing Tween-20 detergent) is
made with 0.5 M NaCl, 20 mM Tris (hydroxymethylaminomethane), and
2% Tween 20. After several rinses in TBS-T, the blot paper was placed for
60 minutes into a 1% milk solution containing patient's sera at a dilution of
1: 1000. The blotting paper was removed, washed several times with TBS-T
to remove primary antibody, and then placed in a 1% milk solution
containing 3 III ofsecondary horseradish peroxidase conjugated goat anti 
human antibody for an additional 60 minutes. Upon removal, the paper was
rinsed twice with TBS-T for 10 minutes each and then twice with TBS (Tris
buffered saline without the addition ofTween 20). The blots were
27
developed using a chemi1uminescent method (PIERCE Super Signal
ULTRA substrate) with autoradiography. The presence ofspecific marker
proteins in samples was detected in the same manner using commercial
antibodies anti-RyR and anti-dystrophin (Chemicon Inc.) , anti-AchR
(Serotec) and anti-DHPR, specific for the a subunit, (Upstate Biotechnology
Inc.) as primary antibodies and it's corresponding secondary antibody.
NEpHGE
Samples requiring additional analysis were examined using NEpHGE
(non equilibrium pH gel electrophoresis), a two dimensional form ofgel
electrophoresis. NEpHGE separates proteins based on electrical charge and
molecular weight as opposed to SDS-PAGE which separates based on
molecular weight alone. Samples which showed immunoreactivity in
western blot analysis after SDS-PAGE were freeze-dried overnight and
added to lysis buffer to prepare the sample for electrophoresis. Tube gels
were the first dimension ofNEpHGE. After 20 J.l1 ofsample is pipetted into
the tube gels, they were run for 1600-2000 volt/hours and then removed
from the tubes intact. Once removed from the tubes, the gels were referred
to as worms. The second dimension ofthe procedure was performed by the
placement ofone worm across the top ofan SDS-PAGE resolving gel and
anchored into place with a 3% solution ofagarose and run again at .025
voltlhours
28
amps constant current for 90 minutes. These gels were also transferred onto
nitrocellulose paper for further analysis by western blotting, as described
above.
Figure 4 Bradford Protein Assay
9131198 prot ass~orkjng
y =5.144x - 0.036 r~::0.993
L'i.....-~~----_-..:._----"
29
0,5
o
O.D.
'"d
o
~TI)(ml!"JI1l)
~orkjng
~
L'i.....-~~----_-..:._----"
~TI)(ml!"JI1l)
II
Tube Gel
II
30
Figure 5 NEpHGE
First Dimension NEpHGE
-separation ofproteins
by pI
Second Dimension NEpHGE
-separation ofproteins by
molecular weight
31
Results
A vesicle preparation was performed from rat skeletal muscle as
described in the materials and methods in order to isolate and analyze a
membranous fraction. The protein composition ofeach ofthe subcellular
fractions collected during the vesicle preparation was analyzed by
comparing the unknown protein bands within the samples with known
protein standards added during SDS-PAGE. Most ofthe fractions were rich
in protein with multiple bands in each lane. Samples F, H ,I and K show
little or no protein banding in the SDS-PAGE Figure 6 due to the low
concentration ofproteins in these samples. The samples are as follows: F) is
supernatant after centrifugation at 4,500 x g for 15 minutes, H) is
supernatant after the addition ofKCI solution and ATP and ultracentrifuged
at 80,000 x g for 90 minutes, I) is the supernatant after another
centrifugation at 80,000 x g for 60 minutes, and K) the final pellet in KCI
solution. These results and descriptions ofeach sample can be seen in
Figure 6.
Next, western blot analysis ofeach ofthe samples was performed
using sera from a myasthenia gravis patient with rippling muscles.
Immunoreactivity was detected between MG/RMD patient's sera and
protein bands in three ofthe subcellular fractions obtained including a whole
32
muscle sample. The samples showing immunoreactivity are A, B, and C
described below (see Figure 6). Each ofthe reactive samples have similar
protein compositions consisting ofmajor bands around the molecular
weights 29, 45 and 66 kDa with multiple lighter bands throughout the entire
range ofthe SDS-PAGE gel. These results can be seen in Figure 7. Lane 1
shows the molecular weight standards added during SDS-PAGE having
molecular weights of29, 45,66,97.5 and 116 kDa. The protein
composition ofthe whole muscle sample (A) as determined by SDS-PAGE
is shown in lane 2 and the immunoreactivity ofthat sample with sera from a
MG/RMD patient is shown in lane 3. The MG/RMD sera reacted with two
protein bands in this sample, one band having a molecular weight around 97
kDa and the another band with a molecular weight between 45 and 65 kDa.
In sample B, the supernatant ofthe whole muscle sample after centrifugation
at 3,300 x g for 30 minutes, two bands were also detected. One band with a
molecular weight between 45 and 66 kDa, and a smaller band around 97
kDa as shown in lane 5. The protein composition ofthis fraction can be
seen in lane 4. Sample C, the supernatant after another centrifugation at
3,500 x g for 45 minutes to pellet mitochondria, showed identical results as
the western blot ofsample A. The protein composition ofsample C is
shown in lane 6 and the western blot ofthis sample with MG/RMD sera is
33
shown in lane 7. It is unclear as to why the 97 kDa band is much fainter in
sample B as compared to the other samples showing immunoreactivity at the
same molecular weight.
In an attempt to further characterize the proteins in the vesicle
preparation samples showing reactivity, non equilibrium pH gel
electrophoresis or NEpHGE was performed. NEpHGE is a two dimensional
form ofgel electrophoresis which further separates proteins based on
electrical charge and molecular weight as opposed to SDS-PAGE which
separates proteins based on molecular weight alone. After six NEpHGE
tube gels were run (2 for each sample showing immunoreactivity), one gel
was stained with coomassie brilliant blue stain while the others were frozen
for future western blotting. The coomassie stained gel showed an atypical
staining pattern that indicated problems with the NEpHGE method used in
the lab led to the distortion ofthe gel pattern. Normal results from a
NEpHGE gel would separate proteins first by pH with the basic proteins on
one end ofthe gel and the acidic proteins at the opposing end with
intermediate pI (isoelectric point) in between. In the second portion ofthe
procedure the proteins were then further separated by molecular weight, the
high molecular weight proteins stay near the top ofthe gel with the
intermediate sized proteins in the middle, and lower weight proteins at the
34
bottom ofthe gel. The gels that were stained after the second dimension with
coomassie blue showed an inconsistent immuno-staining pattern. When
western blotted with patient sera, the NEpHGE gels either showed no
immunoreactivity or showed immunoreactivity patterns not usable for
analysis (see Figure 8).
Overall, the results from the vesicle preparation did not indicate new
information nor did the vesicle preparation appear to be a pure subcellular
fraction. Results from a previous study indicated that there were at least two
proteins at high (300-500 kDa) and intermediate(~200kDa)molecular
weights that were not seen on our vesicle preparation gels or blots. For this
reason we sought out a protocol that would allow us to isolate aT-tubular
membrane in a higher concentration. The T-tubular region is ofinterest
since this is the region ofCa++ entrance into the cell and antigens in this
region may be responsible for rippling muscle symptoms. The T-tubular
membrane proteins may have been present in the vesicle preparation
fractions but possibly not in a large enough concentration to be detected
during the western blot analysis.
A modified T-tubule isolation procedure from Florio et at. (1992) was
used to isolate the T-tubular membranes. The final sample was western
blotted with anti-DHPR as a primary antibody to determine ifthe fraction
(~200kDa)
35
contained T-tubular membrane. Since DHPR is associated only with the T 
tubular membrane, detection ofDHPR in a western blot showed that we did
indeed collect T-tubular membrane.
Four total fractions were collected during the T-tubule isolation and
were western blotted with various sera and antibodies. The four fractions
include the following. Fraction I is the discarded material after the first
centrifugation at 3600 rpm in a Beckman J6 rotor for 20 minutes. Fraction 2
is the pellet after centrifugation at 10,000 x g for 20 minutes. Fraction 3 is
the supernatant after centrifugation at 75,000 x g for 90 minutes and
fraction 4 is the final T-tubule sample. The patient sera used for western
blot analysis included a MG/RMD patient, a patient with MG but without
the rippling muscle component, a MG patient with a thymoma, and a
normal, non-diseased human. Results can be seen in Table 2.
The western blot using anti-DHPR antibody shows DHPR present in a
detectable concentration in the final sample only (see Figure 9), which
indicates that there is T-tubular membrane present in the final fraction. The
blots performed with the sera from a MG/RMD patient detected a great deal
ofimmunoreactivity in several bands ofeach fraction. Many bands were
also detected within the stacking gel indicating many ofthe proteins were
too large to enter into the resolving gel. The final fraction was also blotted
36
for the presence ofAchR, dystrophin, and RyR. There was no
immunoreactivity detected from any ofthese three antibodies. The results of
these western blots can be seen in Figure 10. The sera from a patient with
MG but without the rippling muscle component displayed some banding but
not to the extent found with the MG/RMD patient. The patient with MG and
a thymoma showed no reactivity (just a bit ofbackground noise) as did the
sera from a normal individual. Results illustrated in Figure 11.
In order to further characterize which proteins were displaying
reactivity with patient sera, another isolation procedure was performed to
isolate both T-tubular and sarcoplasmic reticular membranes. Since the
previously attempted isolation for T-tubular membrane may have included
portions ofSR membranes, some ofthe immunoreactivity detected during
western blot analysis ofthe T-tubule final sample may have been from
proteins within the SR membrane. This SR/T-tubule isolation allowed
further isolation ofthese closely associated membranes in order to determine
whether T-tubules and/or SR contain antigens to the MG/RMD patient sera.
This isolation procedure was followed according to Sabbadini et at. (1983).
Fractions from all supernatants and pellets were collected during the
isolation procedure and all 15 fractions were then analyzed by SDS-PAGE
and western blot analysis. The description ofeach fraction is shown in
37
Table 3 and the protein composition ofeach fraction as determined by SDS 
PAGE is shown in Figure 12. The antibodies used to test the fractions from
this isolation procedure include; anti-DHPR, anti-RyR, anti-AchR, anti 
dystrophin, and sera from a MG/RMD patient. RyR presence is shown in
Figure 13. The results ofthese blots are shown in Table 4. All western blots
showed antibody immunoreactivity to one or more ofthe fifteen samples
except the dystrophin antibody. Anti-AchR displayed immunoreactivity
with 3 ofthe fractions and was assumed to have been isolated and removed
since it is not reactive with any ofthe samples taken from the remaining
steps ofthe isolation procedure. Both the anti-DHPR and anti-RyR
antibodies displayed immunoreactivity with the majority ofthe fractions.
Both are absent from several fractions taken at the latter portion ofthe
isolation procedure but do react with the final pellet. Since both DHPR and
RyR are present in the final pellet, it is probable that a good separation ofthe
two membranes was not achieved. The sera from the MG/RMD patient was
extremely reactive with fractions taken from the first part ofthe isolation but
was not reactive with any ofthe fractions taken from the last halfofthe
procedure including the final pellet fraction which displayed reactivity to
both anti-DHPR andanti-I~.yRantibodies.anti-I~.yR
38
One problem arose that impaired the ability to collect data during the
last portion ofthis project. The swinging bucket rotor necessary for the T 
tubule/SR isolation procedure, was unavailable for use due to mechanical
problems. The ultracentrifugation that was to be performed in the swinging
bucket rotor was instead performed using an anglehead rotor with
unsatisfactory results. A complete separation ofT-tubular membrane from
SR membrane was not possible with the type ofrotor used.
39
Figure 6 Gel ofVesicle Preparation Fractions
Lane A
LaneB
LaneC&D
LaneE
LaneF & G
LaneR
Lane I
LaneJ
LaneK
Whole rat muscle sample
Supernatant ofmuscle sample after centrifugation at
3,300 x g for 30 minutes.
After centrifugation again at 3,500 x g for 45 minutes,
samples were taken from both the supernatant (C) and the
mitochondrian pellet (D).
Supernatant after ultracentrifugation at 44,000 x g for 60
minutes.
After centrifugation at 4,500 x g for 15 minutes samples
were taken from the supernatant (F) and the pellet (G).
Supernatant after addition ofKCl solution and ATP and
ultracentrifugation at 80,000 x g for 90 minutes.
Supernatant after another ultracentrifugation at 80,000 x
g for 60 minutes.
Final pellet
Final pellet in KCl solution
Figure 6 Gel ofVesicle Preparation Fractions
40
ABCDEFG H I J K
41
Figure 7 Immunoreactivity of Fractions with MG/RMD Sera
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Molecular weight standards
SDS-PAGE of sample A
Western blot ofsample A showing immunoreactivity to sera
from a MG patient with the rippling muscle component.
SDS-PAGE ofsample B
Western blot ofsample B showing immunoreactivity to sera
from a MG patient with the rippling muscle component.
SDS-PAGE ofsample C
Western blot ofsample C showing immunoreactivity to sera
from a MG patient with the rippling muscle component.
42
Figure 7 Immunoreactiyity of Fractions with MG/RMD Sera
Figure 8 NEpHGE Results
This figure illustrates the inconsistent banding patterns obtained from the
NEpHGE analysis. Picture A shows a NEpHGE coomassie stained gel.
Pictures Band C both show the immunoreactive banding pattern obtained
during western blot analysis.
43
Figure 8 NEpHGE Results
44
A
B
c
45
Table 2 T-tubule Western Blot Results
This figure illustrates the immunoreactivity ofall the samples obtained from
the T-tubule isolation procedure with commercial antibodies DHPR, RyR,
AchR, and dystrophin. Sera from different patients including a MG/RMD
patient with thymoma, a MG/RMD patient, a MG patient without RMD, a
MG patient with thymoma, and a normal individual were also used. The T 
tubule final fraction reacted with DHPR, one patient with MG, and the
patient with MG/RMD with thymoma.
Table 2 T-tubule Western Blot Results
46
Antibodies/Patient Fraction 1 Fraction 2 Fraction 3 Fraction 4
Sera
DHPR - - - +
MG/RMD/ thymoma ++ ++ ++ ++
MG alone - - - +/--
MG/RMD no thymoma - - - -
MG/thymoma - - - -
Normal Individual - - - -
RyR No data No data No data -
AchR No data No data No data -
Dystrophin No data No data No data -
47
Figure 9 Presence ofDHPR in T-tubule Final Fraction
The presence ofDHPR was found in the final T-tubule sample (4) but is
absent in the fractions 1, 2, and 3. Fraction 1 is the discarded material after
the first centrifugation at 3,600 rpm in a Beckman J6 rotor for 20 minutes.
Fraction 2 is the pellet after the next centrifugation at 10,000 x g for 20
minutes and fraction 3 is the supernatant after centrifugation at 75,000 x g
for 90 minutes.
Figure 9 Presence of DHPR in T-tubule Final Fraction
48
Fractions:
1 2 3 4
49
Figure 10 T-tllbule Final Fraction Immunoreactivity with
Commercial Antibodies
The final T-tubule fraction was analyzed by western blot to determine the
purity ofthe sample and the effectiveness ofthe isolation procedure. DHPR
was detected in the sample (lane 1) while AchR, dystrophin, and RyR (lanes
2, 3, and 4 respectively) did not display any immunoreactivity with the final
T-tubule sample.
Figure 10 T-tubule Final Fraction Immunoreactiyity with
Commercial Antibodies
50
DHPR AchR Dys
,; .,,)..
'" ,y';"
'.
RyR
ImmunoreactiYity
51
Figure 11 Immunoreactiyity of T-tlIbular Fraction with
Patient Sera
All sera was tested against the final T-tubule sample. Lanes A Band Care
all patients with MG. Lane D is MG/T. Lane E is MG/RMD. Lane F is
MG/RMD/T. Only lanes C and F show immunoreactivity which is not
expected since lane C is MG only---no RMD. (May be an extreme case with
a thymoma undiagnosed or may be experiencing the beginnings ofRMD.)
Figure 11 Immunoreactivity of T-tubular Fraction with
Patient Sera
A B C D E
I~
52
5
12 10 RMD RMD/
THY~'10MA
I~
THY~'10MA
Table 3 Description of T-tub/SR Isolation Fractions
53
Fractions; Fraction Descriptions:
1 Whole muscle after homogenized in a Waring blender
2 Pellet after centrifugation at 15,000 x g for 20 minutes
3 Supernatant after centrifugation at 15,000 x g for 20 minutes
4 Fraction 3 after filtered through gauze
5 Pellet after centrifugation at 40,000 x g for 90 minutes
6 Supernatant after centrifugation at 40,000 x g for 90 minutes
7 Pellet after centrifugation at 15,000 x g for 20 minutes
8 Supernatant after centrifugation at 15,000 x g for 20 minutes
9 Supernatant after centrifugation at 40,000 x g for 90 minutes
10 Loaded sample before sucrose gradient
11 T-tubule sample after first sucrose gradient centrifugation
12 T-tubule final sample from tube #1
13 T-tubule final sample from tube #2
14 Final SR top layer
15 Final SR pellet
54
Figure 12 Protein Composition of SR/T-tubule Fractions
This figure shows the protein composition ofthe 15 fractions from the SR/T 
tubule isolation procedure. The S lanes are the protein standards used during
SDS-PAGE. The numbered lanes correlate with the descriptions ofthe
fractions listed in table 3.
Figure 12 Protein Composition of SR/T-tubule Fractions
55
>¥-.¥
S 1 2 5 7 9 10 11 S S S 121314 15
S S
Figure 13 Presence ofRyR in SRff-tubule Fractions
The presence ofRyR in the fractions ofthe SRff-tubule isolation can be
seen in this figure. Immunoreactivity can be seen in both the stacking and
resolving gels. The immunoreactivity detected at the bottom ofthe
resolving gel may be the result ofRyR breakdown.
56
Figure 13 Presence of RyR in SR/T-tubule Fractions
57
Table 4 Immunoreactivity of SRff-tubule Fractions with
Different Antibodies and Patients' Sera
Fractions: DHPR RyR AchR Dystrophin MG/RMD
1 + +
- -
+
2 - + -
-
+
3 + + + - +
4 + + +
-
+
5 + + + - +
6 + +
- -
+
7 + +
- - +
8 + +
- - -
9 +
-
- - -
10 + - - - -
11
-
- - - -
12
- - - - -
13
- - - - -
14
- - - - -
15 + + - - -
58
59
Discussion
The goal ofthis study was to further characterize autoantibodies found
in MG/RMD patients' sera through analysis ofsubcellular fractions of
skeletal muscle. One hypothesis ofthis study was that MG/RMD patients
have antibodies to proteins in the sarcoplasmic reticular and T-tubular
portions ofthe skeletal muscle cell. It is already well established that MG
patients have a great deal ofautoantibodies due to their condition, however
autoantibodies associated with MG/RMD patients have not yet been
thoroughly investigated. Autoantibodies present in MG patients include
those against nicotinic acetylcholine receptors (290 kDa), thyroid, and
skeletal muscle proteins including titin, actin, myosin, tropomyosin, alpha 
actinin and portions ofryanodine receptor (RyR) (300-400 kDa) (Aarli,
1990; Gautel, 1993; Penn, 1986; Ohta, 1990; Pagala, 1990; Williams, 1986).
In a previous study performed by Thomas Watkins M.S., sera ofa
MG/RMD patient was found to have immunoreactivity with whole muscle
proteins when analyzed using western blot techniques. Immunoreactivity to
a high molecular weight protein(~400kDa) was found as was reactivity to a
molecular weight protein approximately 200 kDa in size (an intermediate
molecular weight protein) and a relatively low molecular weight protein
(~77kDa in size). This reactivity was also found in sera from other
(~400
(~77
60
MG/RMD patients. MG patients without the rippling muscle component did
display some reactivity around 40-70 kDa which can probably be explained
as reactivity to acetylcholine receptor (AchR) subunits. These subunits
range in molecular weight from 39 to 64 kDa (Watkins, 1999).
Sarcoplasmic reticulum and T-tubular regions ofthe skeletal muscle
cell were targeted in this study because Ca++ enters into the cell at this
location via ryanodine receptor Ca++ channels. These have a molecular
weight ofbetween 300 and 400 kDa and may explain the immunoreactivity
found in this range. Immunocytochemistry performed during the previous
study displaying immunoreactivity in a banding pattern in the T-tubular
region ofa myocyte suggests this area may contain autoantigens to
MG/RMD patient sera. See Figure 3. This location also correlates to the
possible presence ofmechanosensitive channels (MSC's) or channels
activated or inactivated by stretch ofthe muscle cell since Ca++ enters the
cell in this region. A conformational change ofMCS's may occur due to
the presence ofautoantibodies found in MG/RMD patients and may account
for the rippling muscle component by simply allowing these channels to leak
Ca++ into the myocyte.
The fITst step taken in this study was the preparation ofvesicular
membranes from the myocytes according to de Meis et al. (1971). Fractions
61
were collected at all stages ofthe vesicle preparation and each fraction was
then analyzed by western blot techniques. The fmal fraction did not show
any reactivity to the MG/RMD sera. This may be because the proteins of
interest were not isolated in the final fraction in a significant concentration
to react with the autoantibodies ofthe sera. Other than the whole muscle
homogenate sample (A), only two ofthe fractions, samples B and C, reacted
with the patient sera. Fraction B was the supernatant after the whole muscle
homogenate was centrifuged at 3,300 x g for 30 minutes. Fraction C was the
supernatant after another centrifugation at 3,500 x g for 45 minutes.
Descriptions and results are shown in Figures 6 and 7. Immunoreactivity
was detected in all three samples at~97kDa and again between 45 and 66
kDa. These bands could possibly be acetylcholine receptor subunits.
However, the intermediate and high molecular weight proteins reactive in
the previous study were not seen in this isolation procedure therefore another
isolation procedure, specific for T-tubular membrane, was performed.
T-tubular membranes were isolated according to a modified procedure
from Florio et al. (1992). Four fractions were collected over the course of
the isolation procedure, including the fmal T-tubular sample, and tested by
SDS-PAGE and western blot analysis techniques. Table 2 shows the
immunoreactivity detected during western blots ofthe fractions with several
~97
62
different antibodies and patients' sera. Fraction 4 is ofmain interest since it
should contain the isolated T-tubular membranes. This fraction was
analyzed by western blot to determine the contents. The [mal fraction
contains DHPR. Since DHPR is aT-tubule associated channel protein, its
reactivity with the [mal sample indicates that T-tubule membrane is present
in the sample. DHPR was not detected in any ofthe other three fractions
possibly because it is not present in a large enough concentration. The [mal
sample was also blotted against AchR, dystrophin, and RyR. No
immunoreactivity was detected. This indicates that there is little or no
contamination from plasma membrane associated with AchR which is
present at the neuromuscular junction. This also indicates that the sample
may be isolated T-tubular membrane since RyR is not present. RyR is a
channel protein associated with the sarcoplasmic reticulum. Since the final
sample showed no reactivity with RyR it may be assumed that sarcoplasmic
reticular membrane is not present in the sample. The T-tubular membrane
and SR are normally closely associated, and therefore the absence ofSR in
the final fraction may be indicative ofa fairly pure T-tubular fraction.
The T-tubule fraction did react with sera from two patients: 1) a
MG/RMD/T patient and 2) a patient with MG only. Although sera from
other types ofpatients (MG/T, MG/RMD, and a normal healthy individual)
63
were tested, there was no immunoreactivity detected. This could possibly
illustrate that the antigens reactive in the [mal fraction respond to antibodies
found in some MG patients without the RMD component but is not found in
all MG patients. Banding patterns were in the very high and high molecular
weight ranges which may be consistent with the results obtained from a
previous study (Watkins, 1999).
In order to further characterize autoantigens, another isolation
procedure was performed to isolate both T-tubular and sarcoplasmic
reticular membranes. This isolation procedure was followed according to
Sabbadini et ai. (1983). In the SR/T-tubular membrane isolation, 15
fractions were collected and examined by SDS-PAGE and western blot
analysis. Fraction descriptions are shown in table 3. Western blot results
are shown in table 4. All were blotted with MG/RMD sera and four
commercial antibodies against DHPR, AchR, RyR, and dystrophin.
Dystrophin did not appear in any ofthe 15 fractions. Thus far, the lab has
not received a positive blot with the dystrophin antibody indicating that there
may be a problem with the antibody. A possible reason may be that we were
using the wrong form ofthe antibody (it may not be specific for the type of
muscle contained in the samples). AchR was present in three ofthe 15
fractions. It can probably be assumed that the AchR was removed from the
64
isolation in the pellet referred to as fraction 5 since this pellet is normally
discarded and is the last fraction displaying reactivity with the AchR
antibody. DHPR and RyR are both highly reactive to the fractions from the
fIrst portion ofthe isolation procedure but are undetected in most ofthe last
fractions except the fmal pellet. DHPR is present in all samples (except a
pellet removed and discarded after the fIrst centrifugation) before the
sucrose gradient portion ofthe isolation. RyR is also present in all fractions
before a fmal centrifugation preceeding the sucrose gradient portion ofthe
procedure. However, both DHPR and RyR are detected in the fInal pellet.
The fmal pellet should be isolated SR that had been separated from T 
tubular membranes. A good separation was unfortunately not achieved since
there is T-tubule associated DHPR present in the fraction.
The sera from the MG/RMD patient was reactive to fractions I
through 7 but did not react with any fractions from the remainder ofthe
isolation procedure including the fInal pellet containing SR and T-tubular
membrane. The MG/RMD patient sera should have shown reactivity with
the final pellet since the T-tubular membrane containing fmal fraction ofthe
previous T-tubule isolation procedure showed immunoreactivity to the sera.
Since the fmal fractions ofboth isolation procedures contained T-tubules,
the MG/RMD sera should display immunoreactivity with both fractions.
65
Recent work performed in the lab has yielded a positive immunoreactivity
detection between the [mal fraction ofthe SRff-tubule isolation and the
MG/RMD sera. The recent work performed has not however been able to
achieve a good separation between the SR and T-tubular membrane. DHPR
and RyR are again both present in the [mal pellet which should be SR only
and therefore not be reactive to DHPR.
Inconsistent immunoreactivity results from the data described may be
due to the unavailability ofa necessary rotor required for the SRI T-tubule
isolation. The sucrose gradient portion ofthe isolation was performed with
an anglehead rotor instead ofa swinging-bucket rotor normally utilized for
this type of procedure. The recent work mentioned above was performed
on the correct type ofrotor. This corrected the problem ofnegative
immunoreactivity between the [mal sample and MG/RMD sera but still did
not permit an adequate separation ofthe SR and T-tubular membranes as
expected.
To conclude, three isolation procedures were performed. A vesicle
preparation to isolate vesicle membranes from myocytes, aT-tubular
isolation to isolate T-tubular membranes, and an SRff-tubule isolation for
the purpose ofcollecting isolated sarcoplasmic reticular and T-tubular
membranes. The vesicle preparation provided no new data. The T-tubular
66
isolation procedure illustrated that the final T-tubule containing pellet
displayed immunoreactivity with some MG sera and with sera from some
MG/RMD patients. Therefore, T-tubule antibodies may be manufactured by
some MG patients but do not appear to be produced by only those MG
patients with a rippling muscle component. The SRff-tubular isolation final
fraction does not show immunoreactivity with MG/RMD sera suggesting
that the MGIRMD patients have no antibodies to SR membrane proteins.
Continuing work in the lab may indicate otherwise (perhaps a different
isolation procedure is required). In this continuing work, the reactivity
between the MG/RMD sera and the SR/T-tubule containing [mal pellet is
located in the same molecular weight region as reactivity displayed by
DHPR and RyR antibodies suggesting a possibility that MG/RMD patients
may have antibodies to SR and T-tubular proteins.
67
Bibliography
Aarli, lA, Stefanson, K., Marton, L., Wollmann, R (1990). Patients with myasthenia
gravis have in their sera IgG autoantibodies against titin. Clin. Exp. Immunol. 82,
284-288.
Ansevin. C.F., Agmanolis, D.P. (1996). Rippling muscles and myasthenia gravis with
rippling muscles. Arch. ofNeurol. 53(2), 197-199.
Bartoccioni, E, Scuderi, F., Scoppetta, C., Evoli, A, Tonali, P., Guidi, L., Bartoloni, C.,
Terranova, T. (1980). Myasthenia Gravis, Thymectomy, and Antiacetylcholine
Receptor Antibody. J. Neurol. 224,9-15.
Bedard, E, Morris, C.E (1992). Channels activated by stretch in neurons ofa helix
snail. Can. J. ofPhysiol. Pharmacol. 709(2),207-13.
Bers, D.M., Fill, M. (1998). Coordinated feet and the dance ofryanodine receptors.
Science790~791.
Beutner, E.H., Witebsky, E., Ricken, D., Adler, RH. (1962) Studies on autoantibodies
in myasthenia gravis. JAMA. 182,46.
Coronado, R, Morrissette, l, Sukhareva, M., Vaughan, D. (1994). Structure and
function ofryanodine receptors. Am. J. ofPhysiol. 266, CI485-1504.
de Meis, L., Hasselbach, W. (1971) Acetyl Phosphate as Substrate for Ca++ Uptake in
Skeletal Muscle Microsomes. J. ofBioi. Chem. 246,4759-4763.
Florio, v., Striessnig, J., Catterall, W. A (1992). Purification and Reconstitution of
Skeletal Muscle Calcium Channels. Methods in Enzymology 207,529-546.
Gautel, M., Lakey, A, Barlow, D., Holmes, Z., Scales, S., Leonard, K., Labeit, S.,
Mygland, A, Gilhus, N., Aarli, lA (1993). Titin antibodies in myasthenia gravis:
identification ofa major immunogenic region oftitin. Neurology 43, 1581-85.
Grob, D., Arsura, EL., Brunner, N.G., Namba, T. (1986). The course ofmyasthenia
gravis and therapies affecting outcome. Annals ofN.Y. Acad. ofSciences. 472 
499.
Guharay, F., Sachs, F. (1984). Stretch activated single ion channel currents in tissue 
cultured embryonic chick skeletal muscle. J. ofPhysiol. 352,685-710.
Guyton, AC., Hall, J.E, Textbook ofMedical Physiology. (Philadelphia): W.B.
Saunders Co.; (1996). p. 73-91.
790~791.
68
Hamill, O. P., McBride, D. (1994). The cloning ofa mechano-gated membrane ion
channel. TINS. 17,439-443.
Hamill, O. P., McBride, D. (1993). Molecular Clues to Mechanosensitivity. Biophysical
J. 65, 17-18.
Hidalgo, C., Gonzalez, M. E., Lagos, R. (1983). Characterization ofthe Ca
2
+_ or
Mg
2
+-ATPase ofTransverse Tubule Membranes Isolated from Rabbit Skeletal
Muscle. J. ofBiological Chem. 258, 13937-13945.
Kim, D.H., Ohnishi, T., Ikemoto, N. (1983). Kinetic Studies ofCalcium Release from
Sarcoplasmic Reticulum in Vitro. J. ofBiological Chem. 258,9662-9668.
Kirber, M.T., Walsh, lV., Singer, lJ. (1988). Stretch-activated ion channels in
smooth muscle: a mechanism for the initiation ofstretch induced contraction.
Pflugers. Arch. 412,339-345.
Kosmorsky, G., Mehta, N., Mitsumoto, H., Prayson, R. (1995). Intermittent esotropia
associated with rippling muscle disease. J. ofNeuro-Ophthalmology 15(3), 147 
151.
Lai, F.A, Messiner, G., (1990). Structure ofthe calcium release channel ofskeletal
muscle sarcoplasmic reticulum and its regulation ofcalcium. Adv. Exp. Med.
BioI.269, 73-77.
Lai, F. A, Erickson, H. P., Rousseau, E., Lui, Q., Meissner, G. (1988). Purification and
reconstitution ofthe calcium release channel from skeletal muscle. Nature. 331,
315-319.
Marty, I., Villaz, M., Arlaud, G., Bally, I., Ronjat, M. (1994). Transmembrane orientation
ofthe N-terminal and C-terminal ends ofthe ryanodine receptor in the
sarcoplasmic reticulum ofrabbit skeletal muscle. Biochem. J. 298, 743-749.
Marx, S.O., Ondrias, K., Marks, AR. (1998). Coupled gating between individual
skeletal muscle Ca
2
+ release channels (Ryanodine Receptors). Science. 818 
821.
Menegazzi, P., Larini, F., Treves, S., Guerrini, R., Quadroni, M., Zorzato, F. (1994).
Identification and Characterization ofThree Calmodulin Binding Sites ofthe
Skeletal Muscle Ryanodine Receptor. Biochemistry 33, 9078-9084.
Mygland, A, Aarli, lA, Matre, R., Gilhus, N.E (1994). Ryanodine receptor
antibodies related to severity ofthymoma associated myasthenia gravis. J. of
Neurology, Neurosurgery, andPsychology 57, 843-46.
69
Mygland, A, Tysnes, 0., Matre, R, Volpe, P., Aarli, J., Gilhus, N. E (1992). Ryanodine
Receptor Autoantibodies in Myasthenia Gravis Patients with a Thymoma. Annals
ofNeurology 32, 589-591.
Mygland, A, Tynses, O.B., Aarli, JA, Flood, P.R, Gilhus, N.E (1991). Myasthenia
gravis patients with a thymoma have antibodies against a high molecular weight
protein in the sarcoplasmic reticulum. J. ofNeuroimmunology 37, 1-7.
Nakai, J., Ogura, T, Feliciano, P., Franzini-Armstrong, C., Allens, P. D., Beam, K G.
(1997). Functional nonequality ofthe cardiac and skeletal ryanodine receptor.
Natl. Acad. Sci. 94, 1019-1022.
Nakai, J, Dirksen, R T, Nguyen, H. T, Pessah, 1. N., Beam, KG., Allens, P. D. (1996).
Enhanced dihydropyridine receptor channel activity in the presence ofryanodine
receptor. Nature 380, 72-75.
Nielsen, YK, Paulson, O.B., Rosenkvist, J., Holsoe, E, Lefvert, AK (1982). Rapid
improvement ofmyasthenia gravis after plasma exchange. Annals ofNeurology.
11, 160-9.
Ohta, M., Ohta, K, Hoh, N., Kurobe, M., Hayashi, K, Nishitani, H, (1990). Anti 
skeletal muscle antibodies in the sera from myasthenic patients with thymoma:
identification ofanti-myosin, actomyosin, actin, alpha-actinin antibodies by solid 
phase radioimmunoassay and a western blotting analysis. Clinica Chimica Acta
187,255-64.
Pagala, M. KD., Nandakumar, N.V., Venkatachari, S.AT, Ravindran, K, Namba,
T, Grob, D. (1990). Responses ofintercostal muscle biopsies from normal
subjects and patients with myasthenia gravis. Muscle & Nerve 13, 1012-22.
Penn, A, Schotland, D.L., Lamme, S., (1986). Antimuscle and antiacetylcholine
receptor antibodies in myasthenia gravis. Muscle andNerve 9, 407-15.
Ricker, K, Moxley, RT, Rohkamm R (1989). Rippling Muscle Disease. Arch. of
Neurol. 46, 405-408.
Ruknudin A, Sachs, F., Bustamante, J.O. (1993). Stretch-activated ion channels in
tissue-cultured chick heart. Am J. Physiol. 264, H960-72.
Sabbadini, R, Okamoto, Y (1983). The Distribution ofATPase Activities in Purified
Transverse Tubular Membranes. Archives ofBiochem. and Biophysics. 223, 107 
119.
Salvatori, Damiani, E, Barhanin, J, Furlan, S., Salviati, G., Margreth, A (1990). Co 
localization ofthe dihydropyridine receptor and the cyclic AMP-binding subunit
ofan intrinsic protein kinase to the functional membrane ofthe transverse tubules
ofskeletal muscle. Biochem. J. 267, 679-687.
70
Skeie, G.O., Bartoccioni, E., Evoli, A, Aarli, l.A, Gilhus, N.E., (1996). Ryanodine
receptor antibodies are associated with severe myasthenia gravis. European J. 0/
Neurology 3, 136-40.
Stephan, D.A, Buist, N., Chittenden, A, Ricker, K., Zhou, l., Hoffman, E.P. (1994).
A rippling muscle disease gene is localized to lq41: evidence for multiple genes.
Neurology 44(10), 1915-20.
Strauss, A, Kemp, P. (1967). Serum autoantibodies in myasthenia gravis and
thymoma: selective affinity for I-bands ofstriated muscle as a guide to
identification of antigens. J. o/Immunology. 99,945-53.
Tanabe, 1., Beam, K. G., Adams, B. A, Niidome, 1., Numa, S. (1990). Regions ofthe
skeletal muscle dihydropyridine receptor critical for excitation-contraction
coupling. Nature. 346, 567-569.
Takeshima, H., Iino, M., Takekura, H., Nishi, M., Kuno, l, Minowa, 0., Takano, H.,
Noda,1. (1994). Excitation-contraction uncoupling and muscular degeneration in
mice lacking functional skeletal muscle ryanodine-receptor gene. Nature 369,
556-559.
Takeshima, H., Nishimura, S., Matsumoto, 1., Ishida, H., Kangawa, K., Minamino, N.,
Matsuo, H., Veda, M., Hanaoka, M., Hirose, 1., Numa, S. (1989). Primary
structure and expression from complementary DNA ofskeletal muscle ryanodine
receptor. Nature 339, 439-445.
Tarroni, P., Rossi, D., Conti, A, Sorrentino, V. (1997). Expression ofthe Ryanodine
Receptor Type 3 Calcium Release Channel during Development and
Differentiation ofMammalian Skeletal Muscle Cells. J. o/Biological Chem. 272,
19808-19813.
Torbergsen,1. (1975). A family with dominant hereditary myotonia, muscular
hypertrophy and increased muscular irratibility, distinct from myotonia congenita
Thomsen. Acta. Neurol. Scand. 51,225-232.
Tortora, G.l, Grabowski, S.R., Principles ofAnatomy and Physiology. (New York):
Harper Collins College Publishers; (1993), p. 241-250.
Vetters, lM., (1967). Muscle antibodies in myasthenia gravis. Immunology. 13,275 
80.
Watkins, 1., (1999). Characterization ofSkeletal Muscle Antibodies in Patients with
Autoimmune Rippling Muscles and Myasthenia Gravis.
Williams, c.L., Lennon, V.A., (1986). Thymic B lymphocyte clones from patients
with myasthenia gravis secrete monoclonal striational autoantibodies
reactingwith myosin, a actinin, or actin. J. txp. Med. 164, 1043-59.
Zorzato, F., Magreth, A., Volpe, P. (1986). Direct photoaffinity labeling ofjunctional
sarcoplasmic reticulum. J. ofBiochem. 261, 13252-57.
71
Youngstown State University / One University Plaza / Youngstown, Ohio 44555-0001
............ , , .
INTEROFFICE MEMORANDUM
...................................................................................................................................................................................................................................
TO: DR GARY WA.lKER
FROM: CHERYL COY
SUBJECT: IACUC PROTOCOL MODIFICATIONS
DATE: 06/03/99
CC: L\Cl'C FILE
Dr. Stephen Flora, I:\CCC Chair has reviewed the modifications you submitted to your protocol
#01-99 and determined they met the conditions outlined by the L\CUC committee with the
exception of the signature of your co-investigator on the declaration page. I have enclosed that page
for Thomas \X,·atkins to sign. Please have him sign and return it to me and then you will have met all
conditions.
Thank you.
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j