From the Department of Medicine, Section of
Cardiology, and Department of Human Genetics, ¶ Department of
Molecular Genetics and Cell Biology, and
Department of
Pathology, The University of Chicago, Chicago, Illinois 60637
Received for publication, March 1, 2001, and in revised form, March 30, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The sarcoglycan complex is found normally at the
plasma membrane of muscle. Disruption of the sarcoglycan complex,
through primary gene mutations in dystrophin or sarcoglycan subunits, produces membrane instability and muscular dystrophy. Restoration of
the sarcoglycan complex at the plasma membrane requires reintroduction of the mutant sarcoglycan subunit in a manner that will permit normal
assembly of the entire sarcoglycan complex. To study sarcoglycan gene
replacement, we introduced transgenes expressing murine Muscular dystrophy is a genetically heterogeneous disease, but
loss of function mutations in the genes encoding the
dystrophin-glycoprotein complex
(DGC)1 contribute
significantly to the muscular dystrophy phenotype. Therapy aimed at
restoring the missing proteins in muscle has been initiated in humans
with these disorders (1). Sarcoglycan is a subcomplex within the DGC,
and mutations in Replacement of missing DGC components can be achieved with transgenesis
or viral-based approaches to evaluate the assembly and function of the
DGC. Transgene-mediated expression of dystrophin in the mdx
mouse, a mouse model for Duchenne muscular dystrophy (DMD), eliminates
dystrophic symptoms (11), and transgenic rescue of dystrophin
expression has been used to identify structure-function relationships
within the DGC. In these studies, expression or overexpression of
dystrophin from the tissue-specific muscle creatine kinase (MCK)
promoter was not associated with toxicity (11). However, less is known
about sarcoglycan replacement, and unlike dystrophin, the sarcoglycans
are transmembrane proteins that assemble within the secretory pathway.
Restoration of proper targeting and assembly of the sarcoglycans may
involve additional regulatory aspects than those required for
expression of cytoplasmic proteins.
Adeno- and adeno-associated viruses carrying normal genes have been
successfully used to rescue skeletal muscle defects in mice lacking
dystrophin or sarcoglycans (12-15). These studies describe the
efficiency of protein expression as well as the immune response
generated by the introduction of the transfer viruses and the vectors
carrying the gene. The dose response of treatment, including studies of
the minimally effective dose and the potential for toxicity related to
overexpression, has not yet been explored fully. With the initiation of
phase I clinical trials using gene therapy for limb girdle muscular
dystrophies (1), it is extremely important to determine the risks and
benefits of this potentially very powerful approach.
In this study, we generated transgenic mice expressing different levels
of Generation of MCKgsg Transgenic Mice--
Full-length mouse
Southern, Northern, and Immunoblot Analysis--
The MCKgsg
transgene was detected in transgenic mice by Southern blot analysis of
tail DNA with Histology and Immunocytochemistry--
Muscles (gastrocnemius
together with soleus and quadriceps) and hearts from 6-week-old mice
were isolated and weighed. 10-µm sections were fixed in methanol for
immunostaining or in 10% formalin for hematoxylin/eosin or Masson
trichrome staining. Primary antibodies were the same for immunoblotting
analysis except polyclonal anti- Staining for Ca2+-dependent ATPase
Activity--
Fiber types (fast and slow fibers) were distinguished
using the procedure as described previously (20). An alkaline
preincubation at pH 9.4 was used.
Generation of Transgenic Mice Overexpressing
Marked Overexpression of
The marked muscle wasting was associated with premature death. Two
independent high copy founders from MCKgsg line 2 and MCKgsg line 4 died at day 45 and day 110, respectively. Given the limited survival of
the male founder for MCKgsg line 2, we attempted to propagate this line
through in vitro fertilization but were unsuccessful. The
female founder for MCKgsg line 4 reproduced once. The progeny from
MCKgsg line 4 died prematurely and did not reproduce. Thus, neither
high copy line was successfully maintained because of the lethal nature
of the overexpression of
Histologic analysis of muscles from high copy transgenic mice (MCKgsg
line 4) using hematoxylin/eosin and Masson trichrome staining showed
severe dystrophic changes, including wide variation in fiber size, an
inflammatory infiltrate, increased connective tissue, and adipocyte
replacement of myofibers (Fig. 2, A and B).
Abundant central nuclei were also evident in the dystrophic muscle
(Fig. 2A). Muscle from MCKgsg line 3, a low copy number line, appeared normal (Fig. 2, C and D) when
compared with wild type control muscle (Fig. 2, E and
F). The dystrophic phenotype of muscles from high copy
transgenic mice was very similar to that seen in a Overexpression Inhibits Normal Cellular Targeting of
Lack of
Immunoblot analysis confirmed the up-regulation of
To determine whether up-regulation of Fast Fibers Are Preferentially Affected--
Muscle fibers respond
to damage differently depending on their fiber types. It was shown that
fast muscle fibers are preferentially affected in DMD (21). We examined
how fiber types respond differently in muscular dystrophy mediated by
Disruption of the DGC is responsible for a number of genetically
distinct forms of muscular dystrophy. This includes mutations in the
dystrophin gene that cause DMD and mutations in the sarcoglycan genes
that cause the limb girdle muscular dystrophies (2, 23). Many of these
mutations are thought to produce disruption of the DGC by loss of
function or loss of protein expression. Because mice null for
sarcoglycan genes have been generated (6, 7) and these mice fully
recapitulate the membrane instability and other features of human
muscular dystrophy, we generated mice expressing For these transgenic studies, we used the MCK promoter to drive high
level, striated muscle-specific expression. We selected the MCK
promoter because it has been used extensively in transgenic gene
replacement studies of dystrophin (11). We found a profound, lethal
muscle wasting disorder developed in transgenic mice that had high
level overexpression of murine The high level of Despite the similar histology between Transgenic rescue of dystrophin deficiency in the mdx mouse
has been used to demonstrate that dystrophin replacement is effective (11, 22). This approach has been highly illustrative to delineate structure-function relationships within dystrophin and its interaction with the remainder of the DGC (11). Furthermore, overexpression of
utrophin, the highly related dystrophin homolog, also can fully rescue the dystrophic phenotype in the mdx mouse (29). In
these studies, the same MCK promoter was used but did not result in myofiber toxicity as seen here for These studies highlight the complexity of simple gene replacement
strategies and underscore the importance of animal models for the
treatment of human disease. Because viral replacement strategies
typically lead to high level and often variable levels of expression
throughout the transduced tissue, it is likely that some toxicity is
occurring in those myofibers that have been transduced at high levels.
Moreover, previous viral replacement studies, although critically
important in demonstrating that replacement can be effective, have not
explored fully the dose response of treatment that includes studies of
the minimally effective dose and the potential for toxicity related to
overexpression. Because viral replacement gene therapy trials are being
initiated in humans (1), thorough testing in animal models is required
to document the full range of benefits and the potential risks and toxicity.
-sarcoglycan into muscle of normal mice. Mice expressing high levels of
-sarcoglycan, under the control of the muscle-specific creatine
kinase promoter, developed a severe muscular dystrophy with greatly
reduced muscle mass and early lethality. Marked
-sarcoglycan
overexpression produced cytoplasmic aggregates that interfered with
normal membrane targeting of
-sarcoglycan. Overexpression of
-sarcoglycan lead to the up-regulation of
- and
-sarcoglycan.
These data suggest that increased
-sarcoglycan and/or
mislocalization of
-sarcoglycan to the cytoplasm is sufficient to
induce muscle damage and provides a new model of muscular dystrophy
that highlights the importance of this protein in the assembly,
function, and downstream signaling of the sarcoglycan complex. Most
importantly, gene dosage and promoter strength should be given serious
consideration in replacement gene therapy to ensure safety in human
clinical trials.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-,
-,
-, and
-sarcoglycan genes have been
described (2). Most sarcoglycan gene mutations destabilize the entire
sarcoglycan complex at the plasma membrane of muscle myofibers and
cardiomyocytes (3-7). Expression of dystrophin and sarcoglycans seems
to be tightly regulated. Primary mutations in dystrophin lead to
drastic reduction of dystrophin-associated proteins including
sarcoglycans (8-10).
-sarcoglycan under the control of the MCK promoter. We found that
mice carrying a high copy number of the
-sarcoglycan gene,
concomitant with marked overexpression of
-sarcoglycan protein,
showed profound muscular dystrophy as revealed by extreme muscle
wasting and premature death. Histopathology showed muscular dystrophy
with variable fiber size, centrally placed nuclei, and increased
fibrosis. In addition, fast fibers seem to be preferentially affected.
In muscle overexpressing
-sarcoglycan, we found that
-sarcoglycan
failed to reach the cell membrane and was associated with intracellular
aggregates. The remaining sarcoglycan subunits were targeted to the
sarcolemma; however,
- and
-sarcoglycans were significantly
up-regulated at the muscle membrane. These findings suggest that
overexpression of
-sarcoglycan and misregulation of
- and
-sarcoglycan can cause muscular dystrophy.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sarcoglycan (nucleotides 152-1027 of GenBankTM
accession number AF282901) was obtained using mouse skeletal muscle cDNA as template and amplified with the following primer pairs: gsgF, 5'-AGAAGCTTGCCATGGTTCGAGAGCAGTAC-3'; gsgR,
5'-TAGGATCCTCAAAGACAGACGTGGCTG-3'. The polymerase chain reaction
product was digested with HindIII and BamHI. The
digested polymerase chain reaction product was then ligated to the
pBluescript II KS (Stratagene, La Jolla, CA) that was treated with
HindIII and BamHI. Polyadenylation and
termination signals of bovine growth hormone from pcDNA3
(Invitrogen, Carlsbad, CA) were added at the XbaI site.
Muscle-specific creatine kinase promoter was amplified with the
following primer pairs: MCK-F, 5'-ATCTCGAGCAGCTGAGGTGCAAAAGGCT-3' and
MCK-R, 5'-ATAAGCTTGGGGGCAGCCCCTGTGCCCC-3' (16). The resultant
1.35-kb fragment was inserted into the XhoI and
HindIII sites. Sequences were verified using cycle
sequencing. After digestion with BssHII, the prokaryotic
vector was separated from the transgene by sucrose gradient (17).
Transgenic mice were generated by microinjection of transgene DNA into
the pronucleus of fertilized single-cell BL6/DBA embryos as previously
described (18, 19).
-32P-labeled DNA probe containing the
full-length
-sarcoglycan coding region (nucleotides 152-1027 of
GenBankTM accession number AF282901). Genomic DNA was
predigested with BamHI and HindIII. Signal
strength was quantified using ImageQuant software and a STORM 860 phosphorimager (Amersham Pharmacia Biotech). Transgene copy number was
determined by comparing the
-sarcoglycan transgene and endogenous
-sarcoglycan gene. MCKgsg mRNA expression was detected with the
same full-length
-sarcoglycan probe as described above. The
following probes to murine sarcoglycan sequences were also used:
-sarcoglycan cDNA coding region (nucleotides 254-1417,
GenBankTM accession number AB024920);
-sarcoglycan
partial cDNA coding region (nucleotides 325-834,
GenBankTM accession number AB024921). Total RNA was
isolated using TRIzol reagent (Life Technologies, Inc.). Fifteen µg
of total RNA was loaded in each lane. For protein analysis, 50 µg of
total protein was resolved on a 4-12% Tris-glycine gradient gel
(Invitrogen BV/NOVEX, Groningen, Netherlands). Immunoblotting was
performed using the following primary antibodies: monoclonal
anti-
- and -
-sarcoglycan antibodies (NovoCastro Laboratories,
Newcastle upon Tyne, UK), polyclonal anti-
- and -
-sarcoglycans
(6), polyclonal anti-dystrophin antibody (7), and monoclonal
anti-myosin heavy chain antibody (Developmental Studies
Hybridoma Bank, University of Iowa, IA). Horseradish
peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary
antibodies (Jackson ImmunoResearch, West Grove, PA) were used.
-sarcoglycan antibody (7) was used.
Fluorescein isothiocyanate- or Cy3-conjugated secondary antibody
(Jackson ImmunoResearch Laboratories) was used. Counterstaining with
4,6-diamidino-2-phenylindole (DAPI) was included in the mounting medium
(Vector Laboratories, Burlingame, CA). Images were acquired on AxioCam
and analyzed with AxioVision 2.0 (Carl Zeiss, Inc., Thornwood, NY).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Sarcoglycan--
The MCKgsg transgene was engineered by placing
the full-length mouse
-sarcoglycan cDNA sequence under the
control of the 1.35-kb MCK promoter (16) followed by bovine growth
hormone termination and polyadenylation signals (Fig.
1A). To assess the potential
toxicity of high level
-sarcoglycan expression, four lines of
transgenic mice were selected for this study. Two lines carried low
copy numbers of the transgene, and two lines carried high copy numbers
of the transgene. Using a full-length
-sarcoglycan cDNA
sequence as a probe, MCKgsg lines 1 and 3 carried approximately three
and four copies, respectively, when compared with the endogenous
-sarcoglycan gene (Fig. 1B) and are considered low copy
transgenic mice. MCKgsg lines 2 and 4, considered as high copy
transgenic mice, carried ~57 and 36 copies of the
-sarcoglycan
gene, respectively, relative to the endogenous gene (Fig.
1B, asterisk). Northern blot analysis showed the
transgenic
-sarcoglycan mRNA at the predicted size of 1.3 kb
(Fig. 1C, arrow). MCKgsg line 2 (high copy)
exhibited more mRNA expression than MCKgsg line 1 (low copy), and
both showed considerably more mRNA than the endogenous 1.6-kb
-sarcoglycan mRNA (Fig. 1C, asterisk). The
MCK promoter expressed
-sarcoglycan mRNA in both cardiac and
skeletal muscle, although lower levels of mRNA expression were seen
in cardiac muscle (Fig. 1C). Immunoblot analysis showed that
-sarcoglycan protein expression correlated with gene copy number in
that high copy number transgenic lines expressed substantially greater
amounts of
-sarcoglycan protein (Fig. 1D, lanes
6 and 9 for MCKgsg lines 2 and 4, respectively). A
serial dilution of protein extract from transgenic muscle showed that
MCKgsg lines 2 and 4 expressed a 150- and 200-fold increase in the
amount of
-sarcoglycan protein, respectively, when compared with wild type, whereas lines 1 and 3 expressed ~3- and 5-fold more,
respectively (Fig. 1D).
View larger version (38K):
[in a new window]
Fig. 1.
Overexpression of
-sarcoglycan in transgenic mice. A,
the construct for the MCKgsg transgene. A 1.35-kb MCK promoter was used
to drive expression of the full-length murine
-sarcoglycan
(gsg) sequence. Bovine growth hormone (BGH)
termination and polyadenylation (poly(A)) sites were added
at the 3' end. Restriction sites used to engineer the construct are
also shown. B, Southern blot analysis of BamHI-
and HindIII-double digested DNA from transgenic mice
carrying low and high copy numbers of the MCKgsg transgene. Both
transgene (arrow) and endogenous
-sarcoglycan gene
(asterisk) are indicated. C, Northern blot
analysis shows mRNA expression from the endogenous
-sarcoglycan
locus (asterisk) and from the transgene (arrow).
Lines 2 and 3 were used in this analysis where line 3 represents a low
copy number transgenic line and line 2 represents a high
copy number line. Expression is shown from quadriceps (Q),
gastrocnemius (G), and heart (H). The total
amount of RNA in each lane was determined to be comparable by ethidium
bromide staining of the 28 S and 18 S ribosomal RNA (data not shown).
D, immunoblot analysis of
-sarcoglycan overexpression in
MCKgsg transgenic lines. Lines 2 and 4 are the high copy number lines,
and lines 1 and 3 are the low copy number lines. Expression of
-sarcoglycan is the expected 35-kDa size. For lines 2 and 4, the
extracts were diluted to compare relative amounts. hi, high
copy; low, low copy; wt, wild type.
-Sarcoglycan Produces Severe and
Rapidly Progressive, Lethal Muscular Dystrophy--
Of the four
independent founder lines and their wild type littermate controls, the
two high copy MCKgsg transgenic mice were found to be significantly
smaller than their wild type littermates and the low copy number MCKgsg
transgenic lines. A representative comparison of a high copy transgenic
(MCKgsg line 4) and a normal littermate control mouse is shown in Fig.
2, top panel. Mice from MCKgsg
lines 2 and 4 (high copy) were less active and displayed an abnormal
gait that was characterized by widened hind limb spacing. We examined
skeletal muscles from MCKgsg high copy mice, MCKgsg low copy mice, and
wild type littermate controls. Hind limb from MCKgsg line 4 was visibly
dystrophic with marked muscle wasting and gross fibrofatty replacement
when compared with wild type mice (Fig. 2, top panel,
right side). Muscle mass of individual quadriceps in MCKgsg
lines 2 and 4 (0.07 and 0.05 g, respectively) was significantly
lower when compared with two control littermates (0.16 and 0.14 g). Similar findings were observed in fore limbs of high copy MCKgsg
lines 2 and 4 (data not shown).
View larger version (80K):
[in a new window]
Fig. 2.
Transgenic mice overexpressing
-sarcoglycan and histopathology in MCKgsg
transgenic mice. Shown in the top panel is a mouse from
MCKgsg line 4 next to a wild type littermate control. Dissected lower
limbs from the same MCKgsg line 4 and the littermate control are
shown on the right. hi, high copy; wt, wild type.
Representative pathology is shown with hematoxylin/eosin staining in
the left panels (A, C, E,
and G) and Masson trichrome staining in the right
panels (B, D, F, and
H). A and B, quadriceps muscle from
high copy MCKgsg line 4. C and D, similar
sections from a low copy number MCKgsg line 1. E and
F, normal control muscle sections. G and
H, muscle sections from a
-sarcoglycan-null mutant.
-sarcoglycan. We generated an intermediate
high copy number line, MCKgsg line 5, with a copy number of ~29.
These mice also displayed small size and abnormal gait similar to the
MCKgsg lines 2 and 4. Propagation of MCKgsg line 5 was similarly limited.
-sarcoglycan-null
mutant (7) (Fig. 2, G and H).
-Sarcoglycan to the Plasma Membrane--
-Sarcoglycan is an
integral part of the membrane-associated sarcoglycan complex. Because
of the similar histologic appearance of high copy MCKgsg transgenic
muscle and
-sarcoglycan-null mutant muscle (7), we examined
-sarcoglycan localization in gastrocnemius muscle from normal mice
and MCKgsg line 2. Despite a high level of
-sarcoglycan protein
expression,
-sarcoglycan protein failed to target appropriately to
the cell membrane (Fig. 3A).
The majority of immunoreactive
-sarcoglycan protein was detected as
punctate staining throughout the cytoplasm of the myofibers and was
excluded from nuclei (arrowhead). Similar findings were
observed in gastrocnemius muscle from MCKgsg line 4 (data not shown).
Muscles from low copy MCKgsg transgenic mice were similar to wild type
control muscle with normal
-sarcoglycan localization at the plasma
membrane (Fig. 3, B and C). Although a small
amount of cytoplasmic
-sarcoglycan protein was seen, this was not
toxic to muscle given the normal histology (Figs. 2C and
3B).
View larger version (71K):
[in a new window]
Fig. 3.
Expression of
-sarcoglycan in MCKgsg transgenic mice.
A, expression of
-sarcoglycan in quadriceps muscle from
high copy number MCKgsg mice (line 4). The arrow indicates
that
-sarcoglycan immunostaining is not found in nuclei.
B,
-sarcoglycan staining in low copy number MCKgsg muscle
(line 3). C, normal control.
-sarcoglycan expression at the cell membrane and increased
intracellular
-sarcoglycan altered the expression of the remaining
sarcoglycans in a manner different from loss of function mutations. The
genetic loss of
-sarcoglycan is accompanied by reduced protein
expression at the plasma membrane of the residual sarcoglycans despite
their normal mRNA levels (4, 7). Because high level
-sarcoglycan
expression similarly results in loss of
-sarcoglycan at the cell
membrane, we expected that reduced levels of the other sarcoglycan
proteins may be present. Instead, the increased cytoplasmic expression
of
-sarcoglycan from MCKgsg line 2 (high copy) resulted in
up-regulation of
- and
-sarcoglycans (Fig.
4, A and C) when
compared with wild type (Fig. 4, B and D). Of
note, some
-sarcoglycan was found in a punctate pattern in the
cytoplasm (Fig. 4C) suggesting that it may be aggregated with
-sarcoglycan. Normal levels of
-sarcoglycan and dystrophin were present in MCKgsg line 2 mice (Fig. 4, E and
G). These data suggest that increased cytoplasmic
-sarcoglycan may be toxic to myofibers and sufficient to cause
muscular dystrophy. Mislocalization of the remaining sarcoglycans is
not required for the development of muscular dystrophy, but abnormal
assembly of the sarcoglycans may contribute to the development of
muscle degeneration.
View larger version (75K):
[in a new window]
Fig. 4.
Expression of DGC proteins in
-sarcoglycan-overexpressing mice. Quadriceps
muscle from MCKgsg line 4 (A, C, E,
and G) and normal controls (B, D,
F, and H) were stained for components of the DGC.
Shown in A and B is staining for
-sarcoglycan.
C and D represent
-sarcoglycan staining.
-Sarcoglycan (E and F) and dystrophin
(G and H) are also shown. I, Western blot
analysis of
-,
-, and
-sarcoglycans
(
-sg,
-sg, and
-sg), dystrophin (dyst), and myosin
in MCKgsg high copy lines (lines 2 and 4), a low copy line (line1), and
a wild type control (wt). Coomassie blue (CB)
staining is shown as a loading control. J, Northern blot
analysis of
- and
-sarcoglycan in MCKgsg high copy line (line 4)
and wild type control. Ribosomal RNA is shown as a loading
control.
- and
-sarcoglycan.
- and
-Sarcoglycans were significantly
up-regulated in muscle from the MCKgsg high copy lines (lines 2 and 4)
compared with MCKgsg line 1, a low copy number line and wild type
littermate muscle (Fig. 4I).
-Sarcoglycan and dystrophin
levels were not significantly changed by overexpression of
-sarcoglycan at any level. Surprisingly, myosin expression appeared
reduced in the high copy MCKgsg transgenic muscles suggesting that
cytoplasmic
-sarcoglycan expression may disrupt sarcomeric function.
At the light microscopic level, sarcomeres did not appear disrupted in MCKgsg line 2 (data not shown).
- and
-sarcoglycans is
accompanied by an increase in
- or
-sarcoglycan mRNA, we compared the mRNA expression in muscle from MCKgsg high copy
transgenic and wild type mice.
-Sarcoglycan mRNA in MCKgsg line
4 was similar to that in wild type (Fig. 4J, top
panel), suggesting that up-regulation of
-sarcoglycan is
related to an inability of myocytes to regulate stoichiometric ratios
at the sarcolemma.
-Sarcoglycan mRNA exists as three isoforms
(4.4, 3.0, and 1.4 kb) that are thought to encode the same protein. We
found that the 3.0- and 1.4-kb fragments were increased, whereas the
4.4-kb fragment was decreased (Fig. 4J, middle
panel). It is possible that these alternative splicing forms may
regulate the overall mRNA and consequently
-sarcoglycan protein
expression. Thus, multiple factors might be involved in the
mechanism underlying the up-regulation of
-sarcoglycan protein.
-sarcoglycan overexpression under the control of the MCK promoter.
We examined limb muscles from MCKgsg high copy mice and compared soleus
(predominantly slow) to gastrocnemius (predominantly fast) muscle for
ATPase activity. Fig. 5 shows both soleus
(lower right portion of each muscle section) and
gastrocnemius (upper left portion). Compared with normal
muscles (Fig. 5A), muscles from MCKgsg line 2 (Fig. 5B) showed pervasive degeneration in areas, such as the
gastrocnemius, where fast fibers are concentrated. Only the fast fibers
in the soleus of MCKgsg line 2 showed overt degeneration. In MCKgsg
line 2, slow fibers, seen abundantly in the soleus, showed less damage. Soleus also showed an increased percentage of slow fibers, consistent with their decreased susceptibility to muscular dystrophy and decreased
level of the MCK promoter-directed
-sarcoglycan overexpression. It
was previously shown that the MCK promoter directed dystrophin protein
expression at a level 3-4 times higher in fast fibers than in slow
fibers (22). It is possible that reduced expression of MCKgsg may
occur, accounting for some of the protection of slow skeletal
fibers.
View larger version (87K):
[in a new window]
Fig. 5.
Fast fibers are preferentially affected by
marked overexpression of -sarcoglycan.
ATPase patterns indicate slow (light) and fast
(dark) fibers. The lower right of each panel
represents soleus (slow) muscle, and the upper left is
gastrocnemius (fast) muscle. A, normal. B, MCKgsg
line 2. Note preservation of slow fibers.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sarcoglycan to
study replacement approaches for the sarcoglycan gene products.
-sarcoglycan. These mice typically
died within 2-3 months of age, were markedly reduced in size, and
displayed limited mobility. In the high level
-sarcoglycan-overexpressing mice, we noted that fast fibers were
preferentially affected. This may be consistent with fiber type
specificity of the MCK promoter (22) or may be related to observations
made in DMD muscle where fast fibers are more sensitive to damage (21). Lower levels of
-sarcoglycan expression from the same MCK promoter were not associated with muscle damage. The lower levels of expression seen in the hearts of these transgenic animals resulted in near normal
-sarcoglycan expression and no evidence of toxicity or damage (data
not shown) and suggests that lethality associated with
-sarcoglycan
overexpression is related to skeletal muscle toxicity. Because
cardiomyopathy typically develops later in mice with sarcoglycan gene
mutations (6, 7), the premature death in MCKgsg high copy transgenic
mice may have limited our ability to detect cardiomyopathy.
-sarcoglycan expression resulting from the MCKgsg
transgenes produced several molecular consequences. Overexpression of
-sarcoglycan resulted in reduced
-sarcoglycan at the plasma membrane of myofibers. Loss of
-sarcoglycan expression at the membrane is found in DMD and sarcoglycan-mediated muscular dystrophies. Indeed, a human muscular dystrophy patient carrying the common
521-T
founder mutation was described who maintained expression of
-,
-,
and
-sarcoglycan and preferentially displayed reduced
-sarcoglycan (24). Thus, the loss of
-sarcoglycan at the plasma membrane of myofibers is likely a contributor to the pathogenesis seen
in this overexpression model. A second consequence of
-sarcoglycan overexpression was the accumulation of
-sarcoglycan immunoreactive aggregates in the cytoplasm of myofibers. Because the sarcoglycan complex is associated with both mechanical and signaling functions (25), mislocalization of
-sarcoglycan in the cytoplasm could lead to
abnormal signaling or alteration of cytoskeletal elements that interact
with the
-sarcoglycan. In its cytoplasmic domain,
-sarcoglycan
interacts with a muscle-specific form of filamin (
-filamin or
filamin 2), and loss of
-sarcoglycan produces an increased plasma
membrane
-filamin level (26). Once antibodies that recognize murine
-filamin are available, it will be interesting to determine whether
filamin localization is altered in
-sarcoglycan-overexpressing mice.
A third consequence of marked
-sarcoglycan expression is the
increased expression of
- and
-sarcoglycan. Up-regulation of
-sarcoglycan is possibly due to an inability to regulate protein assembly at the sarcolemma. However, regulation at both transcription level as well as protein level may be responsible for the up-regulation of
-sarcoglycan. The increase in these proteins likely produces abnormal sarcoglycan complexes that, in turn, may produce muscular dystrophy. The aberrant ratio of sarcoglycans may cause abnormal mechanical and signaling functions that may be pathologic to the muscle.
-sarcoglycan
overexpression-mediated muscular dystrophy and muscular dystrophy
caused by dystrophin- or
-sarcoglycan-null mutation, disparities
exist. In muscle degeneration where sarcoglycan is absent, instability of the muscle membrane is demonstrated by increased permeability. Normal muscle is impermeable to Evans blue dye, a small molecular weight vital tracer. Mutations in dystrophin or sarcoglycan lead to
abnormal membrane permeability, and Evans blue dye uptake is a
significant feature of the membrane defect in mdx and
-sarcoglycan mutants (7, 27, 28). We were able to assess Evans blue dye uptake in MCKgsg line 5 (copy number 29) and found no evidence for
Evans blue dye uptake (data not shown). The expression and membrane
localization of the remaining sarcoglycan subunits in
-sarcoglycan-overexpressing muscle likely has a protective effect on
membrane permeability that is absent in
-sarcoglycan-null muscle
(7). Thus, although overexpression of
-sarcoglycan leads to muscular
dystrophy, the mechanism by which this muscular dystrophy develops
likely differs from
-sarcoglycan deficiency.
-sarcoglycan overexpression. This
suggests that the skeletal myocyte has different mechanisms for the
maintenance of stoichiometric ratios of the different components of the
DGC.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Donna Fackenthal, Josephine M. Y. Lam, and Kelly Smith for technical assistance, Mike Allikian and Ahlke Heydemann for proofreading the manuscript, and members of the McNally laboratory for helpful discussions. The monoclonal anti-myosin antibody developed by H. M. Blau was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Research Service Award HL10288.
** Supported by Muscular Dystrophy Association, National Institutes of Health Grant HL61322; a Charles Culpeper Medical Scholar. To whom correspondence should be addressed: Section of Cardiology, The University of Chicago, 5841 S. Maryland, MC 6088, Chicago, IL 60637. Tel.: 773-702-2672; Fax: 773-702-2681; E-mail: emcnally@medicine.bsd.uchicago.edu.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M101877200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
DGC, dystrophin-glycoprotein complex;
DMD, Duchenne muscular dystrophy;
MCK, muscle creatine kinase;
kb, kilobase pair(s);
gsg, -sarcoglycan.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Stedman, H., Wilson, J. M., Finke, R., Kleckner, A. L., and Mendell, J. (2000) Hum. Gene Ther. 11, 777-790[CrossRef][Medline] [Order article via Infotrieve] |
2. | Zatz, M., Vainzof, M., and Passos-Bueno, M. R. (2000) Curr. Opin. Neurol. 13, 511-517[CrossRef][Medline] [Order article via Infotrieve] |
3. | Lim, L. E., Duclos, F., Broux, O., Bourg, N., Sunada, Y., Allamand, V., Meyer, J., Richard, I., Moomaw, C., Slaughter, C., Tomé, F. M. S., Fardeau, M., Jackson, C. E., Beckmann, J. S., and Campbell, K. P. (1995) Nat. Genet. 11, 257-265[Medline] [Order article via Infotrieve] |
4. | Noguchi, S., McNally, E. M., Ben Othmane, K., Hagiwara, Y., Mizuno, Y., Yoshida, M., Yamamoto, H., Bonnemann, C. G., Gussoni, E., Denton, P. H., Kyriakides, T., Middleton, L., Hentati, F., Ben Hamida, M., Nonaka, I., Vance, J. M., Kunkel, L. M., and Ozawa, E. (1995) Science 270, 819-822[Abstract] |
5. | Bonnemann, C. G., Modi, R., Noguchi, S., Mizuno, Y., Yoshida, M., Gussoni, E., McNally, E. M., Duggan, D. J., Angelini, C., and Hoffman, E. P. (1995) Nat. Genet. 11, 266-273[Medline] [Order article via Infotrieve] |
6. |
Hack, A. A.,
Lam, M. Y.,
Cordier, L.,
Shoturma, D. I.,
Ly, C. T.,
Hadhazy, M. A.,
Hadhazy, M. R.,
Sweeney, H. L.,
and McNally, E. M.
(2000)
J. Cell Sci.
113,
2535-2544 |
7. |
Hack, A. A.,
Ly, C. T.,
Jiang, F.,
Clendenin, C. J.,
Sigrist, K. S.,
Wollmann, R. L.,
and McNally, E. M.
(1998)
J. Cell Biol.
142,
1279-1287 |
8. | Ohlendieck, K., Matsumura, K., Ionasescu, V. V., Towbin, J. A., Bosch, E. P., Weinstein, S. L., Sernett, S. W., and Campbell, K. P. (1993) Neurology 43, 795-800[Abstract] |
9. | Matsumura, K., Tome, F. M., Ionasescu, V., Ervasti, J. M., Anderson, R. D., Romero, N. B., Simon, D., Recan, D., Kaplan, J. C., Fardeau, M., and Campbell, K. P. (1993) J. Clin. Invest. 92, 866-871[Medline] [Order article via Infotrieve] |
10. | Matsumura, K., Ervasti, J. M., Ohlendieck, K., Kahl, S. D., and Campbell, K. P. (1992) Nature 360, 588-591[CrossRef][Medline] [Order article via Infotrieve] |
11. | Cox, G. A., Cole, N. M., Matsumura, K., Phelps, S. F., Hauschka, S. D., Campbell, K. P., Faulkner, J. A., and Chamberlain, J. S. (1993) Nature 364, 725-729[CrossRef][Medline] [Order article via Infotrieve] |
12. | Cordier, L., Hack, A. A., Scott, M. O., Barton-Davis, E. R., Gao, G., Wilson, J. M., McNally, E. M., and Sweeney, H. L. (2000) Mol. Ther. 1, 119-129[CrossRef][Medline] [Order article via Infotrieve] |
13. | Vincent, N., Ragot, T., Gilgenkrantz, H., Couton, D., Chafey, P., Gregoire, A., Briand, P., Kaplan, J. C., Kahn, A., and Perricaudet, M. (1993) Nat. Genet. 5, 130-134[CrossRef][Medline] [Order article via Infotrieve] |
14. | Ragot, T., Vincent, N., Chafey, P., Vigne, E., Gilgenkrantz, H., Couton, D., Cartaud, J., Briand, P., Kaplan, J. C., Perricaudet, M., and Kahn, A. (1993) Nature 361, 647-650[Medline] [Order article via Infotrieve] |
15. | Holt, K. H., Lim, L. E., Straub, V., Venzke, D. P., Duclos, F., Anderson, R. D., Davidson, B. L., and Campbell, K. P. (1998) Mol. Cell 1, 841-848[Medline] [Order article via Infotrieve] |
16. | Larochelle, N., Lochmuller, H., Zhao, J., Jani, A., Hallauer, P., Hastings, K. E., Massie, B., Prescott, S., Petrof, B. J., Karpati, G., and Nalbantoglu, J. (1997) Gene Ther. 4, 465-472[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Kim, S.,
Lin, H.,
Barr, E.,
Chu, L.,
Leiden, J. M.,
and Parmacek, M. S.
(1997)
J. Clin. Invest.
100,
1006-1014 |
18. | Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994) Manipulating the Mouse Embryo: A Laboratory Manual , 2nd Ed. , pp. 115-126, Cold Spring Harbor Laboratory Press, Plainview, NY |
19. | Zhu, X., Hadhazy, M., Wehling, M., Tidball, J. G., and McNally, E. M. (2000) FEBS Lett. 474, 71-75[CrossRef][Medline] [Order article via Infotrieve] |
20. | Punkt, K., Krug, H., and Bohme, R. (1987) Acta Histochem. 82, 109-113[Medline] [Order article via Infotrieve] |
21. | Webster, C., Silberstein, L., Hays, A. P., and Blau, H. M. (1988) Cell 52, 503-513[Medline] [Order article via Infotrieve] |
22. | Lee, C. C., Pons, F., Jones, P. G., Bies, R. D., Schlang, A. M., Leger, J. J., and Caskey, C. T. (1993) Hum. Gene Ther. 4, 273-281[Medline] [Order article via Infotrieve] |
23. | Hoffman, E. P., Brown, R. H., Jr., and Kunkel, L. M. (1987) Cell 51, 919-928[Medline] [Order article via Infotrieve] |
24. |
Crosbie, R. H.,
Lim, L. E.,
Moore, S. A.,
Hirano, M.,
Hays, A. P.,
Maybaum, S. W.,
Collin, H.,
Dovico, S. A.,
Stolle, C. A.,
Fardeau, M.,
Tome, F. M.,
and Campbell, K. P.
(2000)
Hum. Mol. Genet.
9,
2019-2027 |
25. |
Hack, A. A.,
Cordier, L.,
Shoturma, D. I.,
Lam, M. Y.,
Sweeney, H. L.,
and McNally, E. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
10723-10728 |
26. |
Thompson, T. G.,
Chan, Y. M.,
Hack, A. A.,
Brosius, M.,
Rajala, M.,
Lidov, H. G.,
McNally, E. M.,
Watkins, S.,
and Kunkel, L. M.
(2000)
J. Cell Biol.
148,
115-126 |
27. | Matsuda, R., Nishikawa, A., and Tanaka, H. (1995) J. Biochem. (Tokyo) 118, 959-964[Abstract] |
28. |
Straub, V.,
Rafael, J. A.,
Chamberlain, J. S.,
and Campbell, K. P.
(1997)
J. Cell Biol.
139,
375-385 |
29. | Tinsley, J., Deconinck, N., Fisher, R., Kahn, D., Phelps, S., Gillis, J. M., and Davies, K. (1998) Nat. Med. 4, 1441-1444[CrossRef][Medline] [Order article via Infotrieve] |