Download Expression and Localization of Dystrophin in Human Cardiac

147
Expression and Localization of Dystrophin in
Human Cardiac Purkinje Fibers
Roger D. Bies, MD; David Friedman, PhD; Robert Roberts, MD;
M. Benjamin Perryman, PhD; and C. Thomas Caskey, MD
Downloaded from http://circ.ahajournals.org/ by guest on April 29, 2017
Background. Mutations in the dystrophin gene produce clinical manifestations of disease in heart,
brain, and skeletal muscle in patients with Duchenne and Beckers muscular dystrophy (DMD/BMD).
Conduction disturbances and heart block contribute to cardiac decompensation in these patients, which
suggests an important role for dystrophin in the cardiac conduction system. We therefore examined the
messenger RNA (mRNA) expression and protein localization of dystrophin in normal human cardiac
Purkinije fibers.
Methods and Results. Polymerase chain reaction amplification of isolated Purkinje fiber complementary
DNA identified several alternatively spliced mRNA transcripts encoding for carboxy-terminal isoforms of
the dystrophin protein. The predominant mRNA transcript detected was a splice form previously detected
in the brain. Antipeptide antibodies specific for a carboxy-terminal dystrophin sequence were used for
Western blot analysis and immunocytochemical localization. These antisera detect -400,000-d immunoreactive band or bands on Western blot in normal heart and Purkinje fibers but not in DMD heart.
Immunocytochemical staining showed that dystrophin was localized to the membrane surface of the
Purkinje fiber.
Conclusions. These results suggest that dystrophin may be an important molecule for membrane
function in the Purkinje conduction system of the heart and support the hypothesis that defective
dystrophin expression contributes to the cardiac conduction disturbances seen in DMD/BMD. (Circulation
1992;86:147-153)
KEY WoRDs
*
muscular dystrophy
*
heart block
T he cardiac conduction system frequently is affected in diseases that influence contractile
function in heart and skeletal muscle. Heart
block and arrhythmias are manifested in inherited genetic diseases such as myotonic dystrophy,' KearnsSayre disease,2 hypertrophic cardiomyopathy,3 EmeryDreifuss muscular dystrophy,4'5 facioscapulohumeral
muscular dystrophy,6 and Duchenne and Becker muscular dystrophy (DMD/BMD).7-9 The proposed pathophysiological mechanisms that produce conduction defects in these patients remains descriptive, and the
molecular basis for maintenance of a normal conduction
impulse remains poorly understood. Single-gene defects
that produce abnormal cardiac conduction allow insight
into cellular protein interactions associated with normal
impulse generation and propagation.
DMD/BMD has been characterized by gene mutations that result in defective or absent expression of a
From the Cardiology Division (R.D.B., D.F., R.R., M.B.P.) and
the Institute for Molecular Genetics (R.D.B., C.T.C.), Howard
Hughes Medical Institute (C.T.C.), and Baylor College of Medicine, Houston, Tex.
Supported in part by the American Heart Association (R.R.)
and the Bugher Foundation Center for Molecular Biology (862216; R.R.), the Muscular Dystrophy Association, and the Howard
Hughes Medical Institute (C.T.C.).
Address for correspondence: Roger D. Bies, MD, Institute for
Molecular Genetics, Baylor College of Medicine, One Baylor
Plaza, T809, Houston, TX 77030.
Received August 20, 1991; revision accepted March 18, 1992.
*
arrhythmias
*
Duchenne
membrane-associated protein called dystrophin. In addition to the skeletal and cardiac myopathies observed
in DMD/BMD, these patients often develop atrial
tachycardia and are prone to arrhythmias and complete
heart block.7-9 Histological studies have shown that
cardiac Purkinje fibers display pathological necrosis
similar to the degenerative changes seen in the skeletal
muscle of these patients.'0 Therefore, normal dystrophin expression also appears to be required for the
integrity and function of the conduction system of the
heart.
To provide a basis for understanding the conduction
defect in DMD/BMD, normal human Purkinje fibers
were analyzed for the presence of dystrophin messenger
RNA (mRNA) transcript(s) and protein. We found
several alternatively spliced mRNA transcripts from the
dystrophin gene expressed in Purkinje fibers. These
different transcripts encode for multiple isoforms of the
dystrophin protein, which primarily is localized to the
Purkinje fiber cell membrane.
Methods
Purkinje Fiber Isolation
Human cardiac Purkinje fibers were isolated from 36
explanted hearts at the time of transplantation. The
fibers were clipped as free intraventricular strands that
cross the ventricular cavity, avoiding contamination with
cardiac muscle cells." Purkinje fibers were either immediately frozen in liquid nitrogen or fixed in 4%
148
Circulation Vol 86, No 1 July 1992
Downloaded from http://circ.ahajournals.org/ by guest on April 29, 2017
paraformaldehyde in phosphate-buffered saline (PBS)
at 4°C overnight.
Polymerase Chain Reaction Analysis of
Dystrophin Transcripts
Total RNA from human cardiac Purkinje fibers obtained from 15 explanted hearts was prepared by phenol/chloroform extraction and precipitation with isopropanol.'2 The RNA was dissolved in water followed by a
second ethanol precipitation. RNA 5 ,ug was then
reverse-transcribed into complementary DNA (cDNA)
as described previously13 and resuspended in 50 ,ul H20.
Polymerase chain reaction (PCR) was carried out using
40 ng cDNA, 200 gmol each dNTP and 2U Taq
polymerase in 10 mmol Tris-HCI (pH 8.3), 50 mmol
KCl, and 1.5 mmol MgCl2, with 200 nmol of each primer
in a 50-,ul reaction volume. Reaction conditions were
94°C (30 seconds), 61°C (20 seconds) annealing, and
72°C (3 minutes) extension for 35 cycles. The primers
used were designed to amplify three segments of the 3'
dystrophin mRNA transcript containing regions known
to undergo alternative splicing.14 Oligonucleotides 25
nucleotides in length were prepared on an Applied
Biosystems oligonucleotide synthesizer.
The dystrophin segments analyzed were as follows.
Segment 1 was cDNA sequence 9,938-10,311 amplified
with forward primer 5'-TCCCAAGACAGTTGGGTGAAGTTGC-3' and reverse primer 5'-TCTCTCCTGATGTAGTCGGAGTGC-3'. This segment contains a
167-base-pair (bp) sequence (10,016-10,182) that has
been shown to be alternatively spliced in smooth muscle, resulting in a frame shift encoding for a truncated
protein.14 Segment 2 was dystrophin cDNA human
sequence 10,287-10,948 amplified with forward primer
5 'GCACTCCGACTACATCAGGAGAAGA-3' and
reverse primer 5'-TTCCAGGATTTGCATCCTGGCTTCC-3'. This segment contains a 330-bp sequence
(located between bases 10,432 and 10,761) with four
tandem exons of sizes 39 bp, 66 bp, 66 bp, and 159 bp,
which are alternatively spliced in mouse and human
brain and heart tissue.1415 Segment 3 was human cDNA
sequences 10,945-11,345 amplified with forward primer
5'-GGAAGACCACAATAAACAGCTGGAG-3' and
reverse primer 5'-GCTCCTTCTTCATCTGTCATGACTG-3' containing an alternatively spliced 32-bp
exon (bases 11,223-11,254) that causes a frame shift
that encodes for a unique C-terminal peptide sequence.15 PCR products were analyzed by 10 ,ul of the
reaction on a 3% NuSieve gel and staining the electrophoretically separated DNA with ethidium bromide.
The identity of the individual bands from each PCR
reaction were verified by Southern blot transfer to
GeneScreen (DuPont) and probed with 5' y-ATP32
(New England Nuclear) end-labeled internal oligonucleotides specific for the dystrophin transcript.1516 The
size of the bands detected on Southern blot were known
alternatively spliced transcripts previously sequenced
from heart and brain tissues.14,15
Antipeptide Antibodies
Synthetic peptides corresponding to the deduced
amino acid sequence for dystrophin16 were prepared by
solid-phase synthesis.17 Two milligrams of a 10-aminoacid C-terminal dystrophin peptide (R-N-T-P-G-K-PM-R-E) was coupled to 2 mg bovine serum albumin
(BSA) with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Pierce) and purified by gel
filtration before injection. The BSA-peptide conjugate
(200 1£g) was then injected subdermally into New
Zealand White rabbits with Freund's complete adjuvant
(Pierce) and subsequent booster injections at 8-week
intervals in Freund's incomplete adjuvant. Rabbit serum containing antidystrophin antibodies was collected
14 days after the last booster injection.
Commercially available mouse monoclonal antidystrophin antibodies (Novocastra, UK) also were used to
confirm our antibody specificity.
Anti-creatine kinase (MCK) antibodies used for control immunofluorescence staining have been characterized previously.'s
Western Blot Analysis
Purkinje fibers and cardiac ventricular tissues from 11
explanted hearts were solubilized in buffer (100 mmol
Tris [pH 8.0], 10% sodium dodecyl sulfate, 10 mmol
EDTA, and 100 mmol ,B-mercaptoethanol) at a ratio of
1:10 (wt/vol) using a Dounce homogenizer. The samples
were boiled for 5 minutes and then cooled briefly on ice.
The supernatant was collected after centrifugation at
3,500g for 15 minutes and stored at -20°C. Proteins
were resolved electrophoretically by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis using 4.0%
polyacrylamide gels with a bis-to-acrylamide ratio of
1:29 and a 3.0% stacking gel. The gels then were blotted
onto a nitrocellulose membrane (0.2 mm, Schleicher
and Schuell) for 2 hours at 220 mA constant current
using a Tris (25 mmol)-glycine (190 mmol) transfer
buffer containing 20% methanol. The membrane was
blocked in Tris (10 mmol [pH 8.0])-buffered saline,
0.05% Tween-20 (TBST) containing 3% BSA for 15
minutes. Serum containing antidystrophin antibodies
was diluted 1:500 in TBST and incubated with the
membrane for 1 hour at room temperature. The membrane then was washed three times for 5 minutes with
TBST and incubated with alkaline phosphatase-labeled
anti-rabbit immunoglobulin (Ig)G antibodies (1:1,000
dilution in TBST; Sigma Chemical, St. Louis, Mo.) for 1
hour. The membrane then was washed three times for 5
minutes with TBST, and the immunoreactive bands
were detected by incubation with nitroblue tetrazolium
(0.2 mg/ml, Sigma Chemical) and bromochloroindolyl
phosphate (0.1 mg/ml, Sigma Chemical) in buffer (0.1
mol/l Tris [pH 9.5], 0.1 mol/l NaCI, 5 mmol MgCl2) for
30 minutes.
Immunocytochemistry
Human cardiac Purkinje fibers from 10 explanted
hearts were fixed in 4% paraformaldehyde and imbedded in paraffin, and 5-,um sections were prepared for
staining. The slides were blocked with 3% BSA in TBST
for 15 minutes and then incubated with antidystrophin
antibody (1:100 dilution) for 2 hours at room temperature, washed three times for 5 minutes with TBST, and
incubated with FITC-coupled anti-rabbit IgG secondary antibody at 1:128 (Sigma Chemical) for 1 hour. The
slides then were washed three times with TBST for 5
minutes, and the fluorescence was visualized under
ultraviolet light with a Zeiss Auxiphot microscope.
Bies et al Dystrophin in Cardiac Purkinje Fibers
segment I.
Segment 2
0X174
Hiae III Seg I
Seg 2
Downloaded from http://circ.ahajournals.org/ by guest on April 29, 2017
44
A
FIGURE 1. Panel A: Polymerase chain reaction
(PCR) amplification of three segments of 3' complementary DNA from human cardiac Purkinje fibers
detects multiple spliced transcrzftts. Top: Line diagram
of exons that undergo alternative splicing. Left panel:
Segment]1 (bases 9,938-10,311) amplification shows
only the full-length 374-bp PCR product (top arrow),
and the spliced product with a 159-bp e-xon excised is
P u
w
detected (lower arrow). Middle panel: Segment 2
~~~~~~not
(bases 10,287-10,948) amplification detects the full___________ ~length 662-bp PCR product (top arrow) and several
smaller bands, the lowest at 332 bp (bottom arrow).
Segment 3
*PCR products in segment 2 determined by Southern
blot to represent splice forms of the dystrophin gene
(see panel B and Reference 15). The identity of the
other two bands has not been confirmed, and they
Seg 3
may represent nondystrophin amplification products. 15
Right panel: Segment 3 (bases 10, 945 -11,345) amplifi cation detects both the full-length form at 401 bp
(top arrow) and the presence of the smaller spliced
form at 369 bp (lower arrow) with a 32-bp exon
excised. Seg, segment; 'IX1 74, (DXJ 74 DNA cut with
HaeIII as a size standard. Panel B: Detadled representation of the multiple splice formis of dystrophin
detected by amplification of segment 2 (see panel A).
Southern blot confirmation of at least five spliced
~~~transcripts is demonstrated using a dystrophin-specific
oligonucleotide internal to the primers used in the
~~~ * ~~~~segment 2 PCR (left inset). These splice forms represent transcripts previously detected in murine and
human brain and heart tissues'4'5 and are shown
schematically by their respective splicing patterns. The
dystrophin complementary DNA coordinates for this
region are along the top of the figure, and the four
tandem alternatively spliced exons are depicted as
rectangles with their base-pair size determined by
sequence analysis.'I5 The size of each PCR fragment
and the corresponding exon deletions (indicated as
"deletion size"'9 are shown to the right of each splice
figure. There is no cross hybridization to the PCR
products derived from segments 1 and 3 (panel A),
which were run in the agarose gel lanes flanking
segment 2 on the Southern blot. The other fainter
bands detected on the Southern blot are other dystrophin splice formsfrom segment 2'15 that do not appear
to be significantly expressed in cardiac Purkinje fibers.
10,287
~~~Segment
10,948
10>7~~~~~~
1
2
10,432
3
--
Y AQZ..39fiM-.'i
a
........
.......
.B
Dlto
C
~ ~ ~ Size
Fragment
~
Segment:. Segment Segment
149
.10,761
RE' 159REMENEEHOO. 3'
662
.
66
596
264
398.
291
371
33.0.:
332:
150
Circulation Vol 86, No 1 July 1992
Results
Dystrophin mRNA and protein expression were analyzed in normal human cardiac Purkinje fibers as a first
step in defining the potential role for dystrophin in the
conduction system of the heart. Multiple alternatively
spliced mRNA transcripts encoding for C-terminal isoforms of the dystrophin protein were detected by PCR
from Purkinje fiber cDNA. Subsequent analysis for
dystrophin protein expression and localization was confirmed by Western blot analysis and immunofluorescent
staining of human cardiac Purkinje fibers. These results
demonstrate the normal expression and localization of
dystrophin isoforms in human cardiac Purkinje fibers.
Downloaded from http://circ.ahajournals.org/ by guest on April 29, 2017
Multiple mRNA Isoforms of Dystrophin Are Detected
in Human Cardiac Purkinje Fibers
Previous studies have analyzed and sequenced alternatively spliced dystrophin mRNA via PCR amplification of selected regions of the transcript from tissues
that express dystrophin.i4,15 PCR amplification of dystrophin cDNA derived from human cardiac Purkinje
fibers detected multiple PCR products (Figure 1A) that
represent a number of alternatively spliced transcripts
previously described in heart and brain.'4,5 For example, Figure 1A shows that PCR amplification of segment
3 detected both a "full length" form (right panel, upper
arrow) and a small amount of the alternatively spliced
form that has a 32-bp exon deleted and is amplified as
a smaller PCR product (right panel, lower arrow). In
contrast, some previously described splice forms were
not detected in cardiac Purkinje fibers. Such is the case
for segment 1, which shows amplification of the fulllength PCR product (left panel, upper arrow) but did
not display amplification of a smaller spliced form
detected previously in smooth muscle from the aorta
(14; left panel, lower arrow).
The most complex splicing pattern is observed within
segment 2, where PCR amplification detected a number
of bands suggesting multiple splice forms in this region
(Figure 1A, middle panel). To elucidate which of the
bands in segment 2 corresponds to products derived
from the dystrophin gene, Southern blot analysis was
performed using a 32P-labeled internal oligonucleotide
as a probe (cDNA sequence 10,391-10,415). Figure 1B
(left inset) shows the Southern blot analysis of a gel
containing the PCR products from segments 1-3 (Figure IA reactions displayed as adjacent lanes on an
agarose gel) and demonstrates hybridization to at least
five bands in only the segment 2 lane, each of which
represent a different splice form. There is no hybridization to the bands derived from segment 1 or 3 of the
dystrophin transcript, illustrating the specificity of the
oligonucleotide hybridization. Previous studies had
demonstrated by direct-sequence analysis the existence
of at least 11 different splice forms in this region of the
dystrophin transcript formed by alternative splicing of
four tandem exons in various combinations.'5 The splicing format of the more predominant splice forms detected in cardiac Purkinje fibers is represented schematically in Figure 1B. The major splice form in cardiac
Purkinje fibers appears as the lowest band on the
ethidium-stained gel and Southern blot, which has 330
bp (all four exons) spliced out (Figures 1A and 1B).
Dystrophin Protein Is Localized at the Purkinje Fiber
Cell Membrane
Isolated human Purkinje fiber homogenates were
resolved by SDS-PAGE size separation followed by
Western blot analysis. Figure 2 shows the pattern of
immunostaining using antidystrophin antibodies. A specific -400,000-d protein band is detected in normal
heart and cardiac Purkinje fibers but not in the heart of
dystrophin-deficient patient with DMD. The size and
specificity of this immunoreactive band were verified
with a commercially available mouse monoclonal antidystrophin antibody (Novocastra Labs). Specific dystrophin immunoreactivity was present in eight normal
human and C57 mouse muscle tissues and was absent in
dystrophin-deficient muscle tissue from four DMD patients and eight mdx mice (data not shown), consistent
with previously reported data.19 Because of the large
size of the dystrophin protein, resolution of the dystrophin isoforms detected in cardiac Purkinje fibers (predicted by the cDNA splice forms to vary by "-'1-10,000
d) was not possible in this gel system (Figures 1 and 2).
Immunocytochemical staining of dystrophin in tissue
sections of isolated human cardiac Purkinje fibers
a
Purkinje
00Fiber
......
...
DMD
Normal
Hea00trt 0:Heat ;i ;
......
...
....
FIGURE 2. Western blot analysis of dystrophin protein exin cardiac Purkinje fibers. Dystrophin is detected in
cardiac Purkinje fibers as immunoreactive band(s) at
400,OOO d, which is consistent with the predicted size of
dystrophin observed in skeletal muscle, brain, and
heart.222529 Control lanes show that a band of the same
pression
molecular size is detected in normal heart tissue but not in
dystrophin-deficient Duchenne heart tissue. DMD, Duchenne
muscular dystrophy.
Bies et al Dystrophin in Cardiac Purkinje Fibers
Downloaded from http://circ.ahajournals.org/ by guest on April 29, 2017
A
C
151
B
D
FIGURE 3. Immunofluorescent staining of human cardiac Purkinjefibers. Panel A: Negative control; absent staining ofPurkinje
fibers with preimmune sera and FITC-coupled anti-rabbit immunoglobulin G. Panel B: Cytosolic control; positive staining using
anti-creatine kinase antibodies shows a granular intracellular immunofluorescence with no background staining of the Purkinje
fiber membrane. Panel C: Antidystrophin antibodies show immunofluorescent localization of dystrophin to the membrane of a
Purkinje fiber cell shown in cross section. Some cytosolic staining also is apparent. Panel D: Low-power view of a cross section
of a Purkinje fiber bundle with antidystrophin antibodies showing immunofluorescent dystrophin membrane staining present in all
Purkinje fibers.
152
Circulation Vol 86, No 1 July 1992
showed intense immunoreactive staining of the Purkinje
membrane and, to a lesser extent, staining within the
cytosol (Figures 3C and 3D). Two types of controls were
used to assess the specificity of the immunostaining.
First, the use of preimmune serum at an identical
dilution showed no significant reaction product (Figure
3A). Second, a different antipeptide antisera directed
against an unrelated protein (M-creatine kinase
[M-CK])18 shows a different pattern of staining, only
within the cytosol of the Purkinje fiber (Figure 3B).
Downloaded from http://circ.ahajournals.org/ by guest on April 29, 2017
Discussion
Patients with DMD/BMD have a spectrum of clinical
phenotypes that include skeletal myopathy, cardiomyopathy, and mental retardation,20,21 and they have been
characterized by absent or diminished expression of the
dystrophin protein in skeletal muscle, heart, and
brain.22-25 Histological Purkinje fiber necrosis and conduction abnormalities in patients with DMD/BMD suggest that dystrophin is important for the function and
integrity of cardiac Purkinje fiber.9'10 Although the
absence of dystrophin may not be the sole mechanism
for cardiac conduction disturbances in these patients, it
probably plays a major role. This is the first study
defining the normal mRNA expression and protein
localization of dystrophin in human cardiac Purkinje
fibers, a subset of a variety of cell types that comprise
the conduction system in the heart.26
Cardiac Purkinje fibers display a number of differences in membrane anatomy, intracellular proteins, and
cellular morphology that distinguish them from the
surrounding myocardium. They are multinucleated cells
that lack a T-tubule system,1"27 and they express an
isoform of myosin heavy chain not found in the surrounding ventricular myocardial cells.28 It is unclear
whether they are developmentally derived from mesodermal or ectodermal (neural crest) origin. Analysis of
creatine kinase isoforms in cardiac Purkinje fibers shows
that the differential expression of this enzyme displays
some brainlike properties (M.D. Cortez, D.L. Friedman, T.S. Ma, H.T. Shih, P.R. Puleo, R. Roberts, M.B.
Perryman; manuscript in preparation). Previous studies
have shown that dystrophin mRNA is alternatively
spliced at the 3' end encoding multiple protein isoforms,
and that some of these isoforms are expressed in a
tissue-specific and developmentally regulated pattern.15
Human cardiac Purkinje fibers express a splice form of
dystrophin mRNA that has been shown to predominate
in brain tissue.14'5 Brain dystrophin is found only at
cortical postsynaptic neuronal junctions,25 in contrast
with the ubiquitous membrane staining in skeletal muscle and heart tissues.22,23 It is tempting to speculate that
dystrophin splice forms represent a variation in dystrophin function or a diversification in a region of the
protein that associates with other cytoskeletal or integral membrane proteins. The expression of multiple
isoforms of dystrophin in cardiac Purkinje fibers may
reflect the specialized functional properties of this
tissue.
The exact function of dystrophin is not understood. It
is believed to be structural protein that is associated
with a large integral membrane glycoprotein complex at
the inner surface of the cell membrane.29 When dystrophin is absent in skeletal muscle, at least one of these
glycoproteins is lost,29 and intracellular calcium levels
are increased.30 These observations suggest that dystrophin may be linked to receptors or ion channels in the
cell membrane. At least some of the elevated cytosolic
calcium is known to be due to an increase in calcium
leak channel activity at the membrane surface.30 An
increase in stretch-inactivated calcium channels in dystrophic muscle also has been described.31 It is believed
that elevated intracellular calcium activates proteolytic
enzymes and phospholipases that degrade cellular
phospholipids and produce lysophosphoglycerides,
compounds that have significant electrophysiological
toxicity.32 Lysophosphoglyceride production via elevated intracellular calcium in ischemic myocardium has
been shown to cause membrane depolarization, automaticity, and changes in action potential configuration.33 This suggests that the conduction disturbances in
DMD/BMD may be a combination of calcium ion
disequilibrium and cellular necrosis. In the absence of
dystrophin, expression of other dystrophin-like molecules34 may play a role in these abnormal physiological
mechanisms.
To our knowledge, this is the first demonstration of a
protein molecule normally expressed in the Purkinje
system of the heart that is directly associated with
conduction disease caused by a genetic defect. Further
studies of the molecular complex associated with dystrophin at the Purkinje fiber cell membrane may provide
insight into the physiological mechanisms responsible for
normal membrane potentials and impulse propagation.
Acknowledgment
We thank M. Dolores Cortez, MD, for her generous contribution of a portion of the Purkinje fiber materials used in this
study and Jeffrey Towbin, MD, for DMD heart tissue.
References
1. Perloff JK, Stevenson WG, Roberts NK, Cabeen W, Weiss J:
Cardiac involvement in myotonic muscular dystrophy (Steinert's
disease): A prospective study of 25 patients. Am J Cardiol 1984;54:
1074-1081
2. Charles R, Holt S, Kay JM, Epstein EJ, Rees JR: Myocardial
ultrastructure and the development of atrioventricular block in
Kearns-Sayre syndrome. Circulation 1981;63:214-219
3. Maron BJ, Roberts WC, Epstein SE: Sudden death in hypertrophic
cardiomyopathy: A profile of 78 patients. Circulation 1982;65:
1388-1394
4. Bailer MG, McDaniel ND, Kelly TE: Progression of cardiac disease in Emery-Dreifuss muscular dystrophy. Clin Cardiol 1991;14:
411-416
5. Rowland LP, Fetell M, Alarte M, Hays A, Singh N, Wanat FE:
Emery-Dreifuss muscular dystrophy. Ann Neurol 1979;5:111-117
6. Stevenson WG, Perloff JK, Weiss JN, Anderson TL: Facioscapulohumeral muscular dystrophy: Evidence for selective, genetic
electrophysiologic cardiac involvement. JAm Coil Cardiol 1990;15:
292-299
7. Perloff JK, deLeon AC, O'Doherty D: The cardiomyopathy of
progressive muscular dystrophy. Circulation 1966;33:625-648
8. Perloff JK: Cardiac rhythm and conduction in Duchenne's muscular dystrophy. JAm Coll Cardiol 1984;3:1263-1268
9. Perloff JK: Neurological disorders and heart disease, in Braunwald
E (ed): Heart Disease. Philadelphia, WB Saunders, 1988,
pp 1782-1786
10. Nomura H, Hizawa K: Histopathological study of the conduction
system of the heart in Duchenne progressive muscular dystrophy.
Acta Pathol Jpn 1982;32:1027-1023
11. Sommer JR, Johnson EA: Purkinje fibers of the heart examined
with the peroxidase reaction. J Cell Biol 1968;37:570-574
12. Chomczynski P, Sacchi N: Single-step method of RNA isolation by
acid guanidinium thiocyanate-phenol-chloroform extraction. Anal
Biochem 1987;162:156-159
13.
Gibbs RA, Chamberlain JS, Caskey CT: Diagnosis of new muta-
tion diseases
using the polymerase chain reaction,
in Erlich HA
Bies et al Dystrophin in Cardiac Purkinje Fibers
(ed): PCR Technology: Principles and Applications of DNA Amplification. New York, Stockton Press, 1989, pp 171-191
14. Feener CA, Koenig M, Kunkel LM: Alternative splicing of human
dystrophin mRNA generates isoforms at the carboxy terminus.
Downloaded from http://circ.ahajournals.org/ by guest on April 29, 2017
Nature 1989;338:509-511
15. Bies RB, Phelps S, Cortez MD, Roberts R, Caskey CT, Chamberlain J: Human and murine dystrophin mRNA transcripts are differentially spliced during development. Nucl Acids Res (in press)
16. Koenig M, Monaco AP, Kunkel LM: The complete sequence of
dystrophin predicts a rod shaped cytoskeletal protein. CeUl 1988;
53:219-288
17. Sarin VK, Kent SBM, Jam JP, Merrifield RB: Quantitative monitoring of solid phase peptide synthesis by the ninhydrin reaction.
Anal Biochem 1981;117:147-157
18. Friedman DL, Perryman MB: Compartmentation of multiple
forms of creatine kinase in the distal nephron of the rat kidney.
JBiol Chem 1991;266:22404-22410
19. Hoffman EP, Brown RH, Kunkel LM: Dystrophin: The protein
product of the Duchenne muscular dystrophy locus. Cell 1987;51:
919-928
20. Emery AEH: Duchenne Muscular Dystrophy. Oxford, Oxford University Press, 1987
21. Chamberlain JS, Caskey CT: Duchenne muscular dystrophy, in
Appel SH (ed): Current Neurology. Chicago, Yearbook Medical
Publishers, 1990, pp 65-103
22. Bonilla E, Samitt CE, Miranda AF, Hayes AP, Salviati G,
DiMauro S, Kunkel LM, Hoffman EP, Rowland LP: Duchenne
muscular dystrophy: Deficiency of dystrophin at the muscle cell
surface. Cell 1988;54:447-452
23. Arahata K, Ishiura S, Ishiguro T, Tsukahara T, Suhara Y, Eguchi
C, Ishihara T, Nonaka I, Ozawa E, Sugita H: Immunostaining of
skeletal and cardiac muscle surface membrane with antibody
against Duchenne muscular dystrophy peptide. Nature 1988;333:
861-863
153
24. Hoffman EP, Kunkel LM, Angelini C, Clark A, Johnson M, Harris
JB: Improved diagnosis of Becker muscular dystrophy via dystrophin testing. Neurology 1989;39:1011-1017
25. Lidov HGW, Byers TJ, Walkins SC, Kunkel LM: Localization of
dystrophin to postsynaptic regions of central nervous system cortical neurons. Nature 1990;348:725-728
26. Zipes DP: Genesis of cardiac arrythmias: Electrophysiologic considerations, in Braunwald E (ed): Heart Disease. Philadelphia, WB
Saunders, 1988, pp 581-616
27. Glomset DJ, Glomset ATA: A morphologic study of the cardiac
conduction system in ungulates, dog, and man. Am Heart J 1940;
20:677-701
28. Kuro-O MH, Tsuchimochi H, Ueda S, Takaku F, Yazaki Y: Distribution of cardiac myosin isozymes in human conduction system.
J Clin Invest 1986;77:340-347
29. Ervasti JM, Ohlendieck K, Kahl S, Gaver M, Campbell KP: Deficiency of a glycoprotein component of the dystrophin complex in
dystrophic muscle. Nature 1990;345:315-319
30. Fong P, Turner PR, Denetclaw WF, Steinhardt RA: Increased
activity of calcium leak channels in myotubes of Duchenne human
and mdx mouse origin. Science 1990;250:673-676
31. Franco A, Langsman JB: Calcium entry through stretchinactivated ion channels in mdx myotubes. Nature 1990;344:
670-673
32. Weiss JN, Nademanee K, Stevenson WG, Singh B: Venticular
arrhythmias in ischemic heart disease. Ann Intern Med 1991;114:
784-797
33. Corr PB, Gross RW, Sobel BE: Amphipathic metabolites and
membrane dysfunction in ischemic myocardium. Circ Res 1984;55:
135-154
34. Love DR, Morris GE, Ellis JM, Fairbrother U, Marsden RF,
Bloomfield JF, Edwards YH, Slater CP, Parry DJ, Davies KE:
Tissue distribution of the dystrophin-related gene product and
expression in the mdx and dy mouse. Proc Natl Acad Sci U S A
1991;88:3243-3247
Expression and localization of dystrophin in human cardiac Purkinje fibers.
R D Bies, D Friedman, R Roberts, M B Perryman and C T Caskey
Downloaded from http://circ.ahajournals.org/ by guest on April 29, 2017
Circulation. 1992;86:147-153
doi: 10.1161/01.CIR.86.1.147
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1992 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on
the World Wide Web at:
http://circ.ahajournals.org/content/86/1/147
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally
published in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further
information about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation is online at:
http://circ.ahajournals.org//subscriptions/