Research Article
2413
Myoferlin regulation by NFAT in muscle injury,
regeneration and repair
Alexis R. Demonbreun1,2, Karen A. Lapidos2,3, Konstantina Heretis2, Samantha Levin2, Rodney Dale1,
Peter Pytel4, Eric C. Svensson1,3 and Elizabeth M. McNally1,2,3,*
1
Committee on Developmental Biology, 2Department of Medicine, 3Department of Molecular Genetics and Cell Biology, and
Department of Pathology, The University of Chicago, 5841 South Maryland Avenue, MC 6088, Chicago, IL 60637, USA
4
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 9 April 2010
Journal of Cell Science 123, 2413-2422
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jcs.065375
Summary
Ferlin proteins mediate membrane-fusion events in response to Ca2+. Myoferlin, a member of the ferlin family, is required for normal
muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model
of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased
in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of
the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin
expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy
myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin
promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle
disease. Myoferlin modulates the response to muscle injury through its activity in both myoblasts and mature myofibers.
Key words: NFAT, Myoferlin, Muscle regeneration, Myoblast, Myofiber
Introduction
Skeletal muscle is composed of multinucleated myofibers. Muscle
growth and repair occur as a result of mononucleated myoblasts
fusing with one another as well as myoblasts fusing to existing
myotubes (Ontell et al., 1988). In addition, damaged muscle
membranes also require fusion to reseal membrane disruption.
Recently, two members of the ferlin family, dysferlin and
myoferlin, have been implicated in muscle-membrane fusion.
The gene encoding dysferlin is mutated in patients with Limb
Girdle Muscular Dystrophy type 2B, a disorder of progressive
muscle weakness (Bashir et al., 1998; Liu et al., 1998). Dysferlin
encodes a 220-kDa membrane-associated protein with six C2
domains. The first C2 domain of dysferlin, C2A, binds negatively
charged phospholipids, only in the presence of Ca2+ (Davis et al.,
2002). In the absence of dysferlin, there is a marked delay of
resealing of muscle-membrane tears (Bansal and Campbell, 2004;
Lennon et al., 2003). This delay is associated with vesicle
accumulation under the membrane and reflects defective vesiclemediated resealing.
Myoferlin is highly related to dysferlin and, like dysferlin,
contains six C2 domains. As with dysferlin, the C2A domain of
myoferlin binds phospholipids in the presence of Ca2+ (Doherty et
al., 2005). Myoferlin is broadly expressed and is highly expressed
during muscle development, specifically in myoblasts undergoing
fusion (Davis et al., 2002). In normal, mature skeletal muscle there
is relatively low expression of myoferlin (Davis et al., 2000).
However, in the adult mdx mouse – a mouse model of Duchenne
muscular dystrophy, in which there is ongoing degeneration and
regeneration – myoferlin mRNA is upregulated (Davis et al., 2000;
Doherty et al., 2005). Myoferlin mRNA is also increased in human
muscle affected by Duchenne muscular dystrophy (Haslett et al.,
2003). The increase of myoferlin in muscular dystrophy supports
a role for myoferlin not only in muscle regeneration, but also in
muscle repair.
The defects in muscle growth and repair seen in myoferlin-null
muscle are reminiscent of what was described in muscle lacking
nuclear factor of activated T cells (NFAT)c2 (Horsley et al., 2001).
NFATc2-null mice have defective myoblast fusion, resulting in
fibers with a decreased cross-sectional area and delayed muscle
repair (Horsley et al., 2001). Of the five NFAT isoforms, all are
expressed in skeletal muscle and are required for proper muscle
development and repair (Abbott et al., 1998; Calabria et al., 2009;
Cho et al., 2007; Friday et al., 2000; Horsley et al., 2001; Kegley
et al., 2001; O’Connor et al., 2007). NFATs 1-4 are responsive to
calcineurin activation. Calcineurin, a serine/threonine protein
phosphatase, responds to intracellular Ca2+ levels and regulates
NFAT-dependent transcription. The activity of calcineurin A is
elevated during myoblast differentiation and during muscle
regeneration in mdx mice, a model of muscular dystrophy (Friday
et al., 2000; Stupka et al., 2006b). Furthermore, regeneration of
skeletal muscle fibers is enhanced in transgenic mice
overexpressing calcineurin A (Stupka et al., 2007). Inhibition of
calcineurin with the immunosuppressive drug cyclosporine A (CsA)
causes inhibition of muscle repair in normal mice (Abbott et al.,
1998; Sakuma et al., 2003) and mdx mice (Stupka et al., 2004).
We now used laser-guided microdissection to collect damaged
and intact myofibers from a mouse model of muscular dystrophy.
We examined differential gene-expression profiles in these two
populations and found that myoferlin mRNA was upregulated
sevenfold in damaged myofibers compared with neighboring intact
myofibers. We characterized the sequences responsible for
myoferlin gene expression and identified a 1543-bp promoter that
drives expression 80-fold over baseline. We investigated the role
of NFAT factors in driving myoferlin expression by identifying
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functional NFAT-binding consensus sequences in the 1543-bp
myoferlin promoter. This myoferlin promoter sequence was
sufficient to respond to calcineurin and NFAT activation, CsA
inhibition, and to mediate the response to cardiotoxin-induced
damage in multinucleate myotubes. Transgenic mice expressing
GFP under the control of the myoferlin promoter express GFP in
response to cardiotoxin exposure and in muscular dystrophy,
providing a ‘damage sensor’.
Results
Journal of Cell Science
Myoferlin is upregulated in dye-positive damaged
myofibers and surrounding mononuclear cells
Evans Blue dye (EBD) is a reliable marker for identifying myofibers
with disrupted sarcolemma (Matsuda et al., 1995; Straub et al.,
1997). g-sarcoglycan (Sgcg)-null mice, similar to other models of
muscular dystrophy, develop focal regions of damage in muscle so
that there is both degeneration and regeneration occurring within
the same muscle (Hack et al., 1998). We isolated muscle from
EBD-injected Sgcg-null mice and then rapidly stained it with an
anti-spectrin antibody to identify those fibers that had taken up dye
but retained an intact cytoskeleton. The same number of dyepositive and -negative myofibers was isolated by laser-capture
microdissection (Fig. 1A), and total RNA was extracted and
subjected to one round of linear amplification. Each sample of
amplified RNA was hybridized to an Affymetrix Mouse 430A
chip.
We identified 43 differentially regulated genes (29 upregulated
and 14 downregulated) in dye-positive compared with dye-negative
myofibers (supplementary material Table S1). The genes include
those expressed not only in myofibers, but also in invading
inflammatory cells as well as neighboring fibroblasts. The genes
with the greatest fold increases are predominately involved in
modifying the cytoskeleton, in cell adhesion and in migration. The
third highest differentially expressed gene in our data set, Fer13L,
encoding myoferlin, was upregulated 6.8-fold in dye-positive
damaged myofibers compared with dye-negative fibers. The
upregulation of myoferlin mRNA in damaged myotubes is
consistent with prior reports that surveyed gene expression in
whole muscle from human or mouse models of muscular dystrophy
(Davis et al., 2000; Haslett et al., 2002).
Immunolocalization of focally upregulated myoferlin in
dystrophic muscle
Immunofluorescence microscopy with an anti-myoferlin antibody
was performed on dye-injected muscle from wild-type mice and
two different models of muscular dystrophy, Sgcg-null and mdx. In
wild-type control skeletal muscle, myoferlin protein was found at
low levels at the sarcolemma (Fig. 1B, top left). Myoferlin was
Fig. 1. Myoferlin is focally upregulated in regions of degeneration and regeneration. (A)g-sarcoglycan-null (Sgcg null) mice are a model of muscular
dystrophy and develop focal areas of muscle damage (Hack et al., 1998). Sgcg-null mice were injected with EBD to mark compromised skeletal myofibers. The
muscle was counterstained with an anti-spectrin antibody to mark myofibers that were both intact and positive for EBD, consistent with early-phase degeneration.
Examples of dye-positive myofibers are shown before (left) and after (right) microdissection. (B)Myoferlin is focally upregulated in regions of degeneration and
regeneration. Wild-type, Sgcg-null or mdx mice were injected with EBD, and muscles were studied. Increased myoferlin (green) is concentrated near or within
EBD-positive fibers (red; co-staining appears yellow). DAPI staining of nuclei is shown in blue. (Bottom right) Immunoblot analysis demonstrates myoferlin
protein is increased in Sgcg-null and mdx muscle compared with normal (WT) muscle. Myosin heavy chain is shown as a loading control. Scale bars: 50mm.
(C)Endogenous myoferlin expression is shown in a mixed cell culture containing myoblasts (arrowheads) and multinucleate myotubes (arrows; note linear
arrangement of nuclei consistent with myotubes). In the absence of injury, myoferlin expression is readily detected in the mononuclear cells within the culture (left,
–CTX) but is nearly absent from the neighboring multinucleate myotubes. With cardiotoxin exposure (+CTX), myoferlin expression was present in the elongated
myotubes and appeared unchanged in the neighboring singly nucleated myoblasts. (D)Endogenous myoferlin (red) is also expressed in Mac-1-positive
lymphocytes (green; coexpression appears yellow) that surround myoferlin-positive myofibers in mdx mouse quadriceps muscle. The right three panels are a
magnified view of the white boxed area in the left panel.
Journal of Cell Science
Myoferlin regulation by NFAT
upregulated similarly in both Sgcg-null and mdx dye-positive
regenerating myofibers (Fig. 1B, top right and bottom left,
respectively). Myoferlin staining was also increased in mononuclear
cells surrounding dye-positive myotubes (Fig. 1B). Immunoblots
of skeletal muscle lysates from Sgcg-null and mdx musculardystrophy models both showed an increase of myoferlin protein
compared with wild-type muscle (Fig. 1B, bottom right). The
C2C12 line is a myoblast-like cell line that can be induced to form
multinucleated myotubes. C2C12 myotubes were treated with
cardiotoxin to induce damage and to determine whether myoferlin
was similarly upregulated in this cell-culture-based model.
Cardiotoxin is a 60- to 65-amino-acid protein that induces
microperforations along the plasma membrane leading to an
increase in intracellular Ca2+ and cell injury in multiple cell types,
including skeletal muscle. Endogenous myoferlin is expressed at
low levels in healthy multinucleated myotubes (arrows in Fig. 1C,
left) and is more abundantly detected in mononuclear myoblasts
(arrowheads in Fig. 1C). After 1 mM cardiotoxin treatment,
endogenous myoferlin was upregulated in multinucleated myotubes
and myoblasts (Fig. 1C, right). These data demonstrate that
myoferlin expression is increased in response to injury in both
myoblasts and myotubes.
In addition to myoblast activation, muscle injury is associated
with an infiltration of inflammatory cells. Given the increase in
myoferlin seen in damaged muscle and the known broad expression
pattern of myoferlin, we queried whether the mononuclear cells
seen surrounding the damaged fibers were at least partly
immunogenic in nature. Quadriceps muscle from the mdx mouse
model of muscular dystrophy was analyzed by immunofluorescence
microscopy. Myoferlin-positive fibers were surrounded by
macrophage-1 antigen (Mac-1; also known as integrin M2 and
Cd11b)-positive mononuclear cells that also express myoferlin (Fig.
1D). Mac-1 marks macrophages, natural killer cells, monocytes and
granulocytes (Ault and Springer, 1981). We conclude that myoferlin
is expressed in myoblasts, injured myotubes and the surrounding
inflammatory cells seen in injured muscle.
The myoferlin promoter contains conserved, active
NFAT-binding sites
To characterize the molecular signature of muscle injury and repair,
we dissected the myoferlin promoter. Sequences directly upstream
of the myoferlin translational start site were compared between the
mouse and human genomes up to 20 kb (Fig. 2A). The first 1543
bp of the myoferlin promoter showed high conservation across
species and was inserted into the pGL2-basic vector to test the
ability of the promoter to drive expression in C2C12 cells. In
C2C12 myoblasts, the 1543-bp–pGL2 construct induced luciferase
activity 80-fold above background. Smaller constructs induced
high-level promoter activity ranging from 50- to 60-fold above
background, but each of these smaller constructs was consistently
less effective than the full-length 1543-bp–pGL2 construct (Fig.
2B).
On the basis of the known role of NFAT proteins in muscle
development, the 1543-bp sequence upstream of the myoferlin
translational start site was analyzed for NFAT-binding consensus
sequences (WGGAAANH) (Rao et al., 1997) (Fig. 3A). Multiple
additional conserved transcription-factor-binding sequences were
also identified, including for Myc, MEF2, CEBP, Sp1 and AP1
(data not shown). Chromatin immunoprecipitation (ChIP) assays
were performed using cross-linked chromatin from confluent
C2C12 cells in growth media to confirm that these sequences
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Fig. 2. The 1543-bp myoferlin promoter drives high-level expression.
(A)Sequence conservation of the first 1500 bp of the myoferlin promoter from
humans and mice. ‘0’ refers to the transcriptional start site and ‘275’ marks the
initiator methionine. Regions as high as 75% similarity were identified. Below
is a schematic of four different promoter constructs generated and tested.
(B)Activity of the myoferlin promoter fragments shown in A in C2C12
myoblasts. The 1543-bp–pGL2 reporter activates luciferase expression 82-fold
above background.
bound endogenous NFATc1 and NFATc3. IgG and NFATc2 were
used as controls because NFATc2 translocates to the nucleus in
nascent myotubes and not myoblasts (Abbott et al., 1998). As
expected, interactions with endogenous NFATc2 or IgG were not
detected. NFATc4 was not analyzed because the majority of NFATc4
translocates to the nucleus 48 hours after differentiation, not in
growth media conditions (Cho et al., 2007). NFATc5 was not
studied because NFATc5 is constitutively nuclear in a variety of
cell types and is calcineurin insensitive (Lopez-Rodriguez et al.,
1999). We found that NFATc3 bound to all four promoter fragments,
whereas only fragments 1 and 2 were found to interact with
NFATc1 (Fig. 3B).
To examine further the role of NFATs in modulating myoferlin
expression, we mutated five NFAT-binding consensus sites that
were identified by the TESS and VISTA algorithms (Fig. 3C).
Mutation of NFAT site 1 or NFAT site 3 significantly, but
incompletely, reduced luciferase induction to 25-fold or 12-fold
above background, respectively (P<0.004). An electrophoretic
mobility shift assay (EMSA) was performed to verify further that
mutating NFAT sites 1 and 3 causes a reduction in binding to the
1543-bp myoferlin promoter (supplementary material Fig. S1).
Mutating NFAT sites 2, 4 or 5 in the 1543-bp–pGL2 vector had no
effect on luciferase induction as compared to the 1543-bp–pGL2
construct (Fig. 3C). Together these results demonstrate that NFATc3
and NFATc1 interact with the myoferlin promoter and that NFAT
sites 1 and 3 of the myoferlin promoter are crucial for full promoter
function.
The myoferlin promoter responds to activated calcineurin
A and is inhibited by CsA
We next investigated the calcineurin responsiveness of the
myoferlin promoter. In C2C12 cells, transfection with 1543-bp–
pGL2 resulted in a 12-fold induction of luciferase expression above
background (Fig. 3D). Because of the need for co-transfection,
these experiments used fivefold less of the 1543-bp–pGL2 plasmid
than previous experiments and therefore have a lower level of
induction (Fig. 2B). Co-transfection of 1543-bp–pGL2 along with
constitutively active calcineurin plus NFATc1 and NFATc3 further
promoted luciferase expression 25-fold above background.
Similarly, co-transfection with 1543-bp–pGL2 and either NFATc1
alone or NFATc3 alone resulted in expression that was less than
the combined transfection (not shown). The induction caused by
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and 20-fold above background in the 1 mM or 5 mM CsA treated
cultures, respectively (Fig. 3E; P0.05). These results provide
additional support that the calcineurin-NFAT pathway modulates
the activity of the 1543-bp myoferlin promoter. The inability of
CsA to fully suppress myoferlin-reporter expression suggests that
other factors regulate basal myoferlin expression.
Journal of Cell Science
The myoferlin promoter recapitulates endogenous
myoferlin expression during development and repair in
C2C12 cells
Fig. 3. NFAT binds the myoferlin promoter and regulates its activity.
(A)The position of the five predicted NFAT-binding sites are designated by
arrows. The four different regions amplified in the ChIP assay are indicated
below the line. (B)ChIP assays using anti-NFAT antibodies for endogenous
NFAT proteins from confluent C2C12 cells. NFATc1, NFATc2 and NFATc3
were tested, and NFATc3 was detected occupying all NFAT sites tested. Lowlevel occupancy of NFATc1 was also detected. NFATc2 was not detected at the
myoferlin promoter. (C)pGL2 constructs were generated containing mutations
in five predicted NFAT-binding sites in the 1543-bp myoferlin promoter
labeled NFAT site 1 through 5. Constructs were transfected into C2C12
myoblasts and luciferase expression analyzed. Mutagenesis of NFAT sites 1
and 3 reduced luciferase expression by approximately 30% and 50%,
respectively, compared with the 1543-bp–pGL2 construct or mutants 2, 4 or 5
(*P<0.004). (D)The 1543-bp–pGL2 reporter construct or pGL2-basic control
vectors were transfected into C2C12 myoblasts in combination with activated
calcineurin A (CnA), NFATc1 and/or NFATc3 expression plasmids. Luciferase
expression was increased by twofold with the addition of CnA, NFATc1 and
NFATc3 compared with the 1543-bp–pGL2 vector alone (P<0.0001). (E)The
1543-bp–pGL2 construct was transfected into C2C12 myoblasts and, 18 hours
post-transfection, 1mM or 5mM CsA was added to the cultures. After 24
hours, luciferase expression was analyzed. The addition of 5mM CsA reduced
the 1543-bp myoferlin-promoter activity by 30% compared with 1543-bp–
pGL2 (*P<0.05).
the addition of constitutively active calcineurin, NFATc1 and
NFATc3 was statistically significant compared with 1543-bp–pGL2
(P<0.0001) and 1543-bp–pGL2 plus constitutively active
calcineurin (P0.038).
CsA blocks the Ca2+-calcineurin-dependent activation of NFAT
(Schreiber and Crabtree, 1992). C2C12 cells were transfected with
1543-bp–pGL2 in the presence of 1 mM or 5 mM CsA. The addition
of CsA caused a dose-dependent decrease in promoter activity as
seen by the reduction of luciferase expression from 28-fold above
background in the 1543-bp–pGL2-transfected cultures to 23-fold
To determine whether expression from the myoferlin promoter
fragment mirrored endogenous myoferlin expression, the 1543-bp
region was ligated to a promoterless GFP expression plasmid. The
1543-bp–GFP plasmid was transfected into C2C12 cells and GFP
expression analyzed. At 24 hours after transfection, strong GFP
expression was seen in the singly-nucleated myoblasts transfected
with 1543-bp–GFP (Fig. 4B). The level of induction produced
from the myoferlin promoter was qualitatively equivalent to that
seen from the strong CMV viral promoter (Fig. 4A). Transfected
C2C12 cells were allowed to differentiate into multinucleated
myotubes for 3 days, then the cultures were analyzed for GFP
expression. Myotubes transfected with 1543-bp–GFP displayed
reduced levels of GFP expression compared with myoblasts,
whereas singly-nucleated cells within these same cultures continued
to express high levels of GFP (Fig. 4D). By contrast, CMV-GFPtransfected myotubes retained a high level of GFP expression in
both multinucleate and singly nucleated cells (Fig. 4C). These
results were corroborated by immunoblotting for GFP
(supplementary material Fig. S2). This pattern of GFP expression,
in which high-level expression is seen in myoblasts followed by
reduction after fusion to multinucleated myotubes, parallels what
is seen for myoferlin protein expression in development (Doherty
et al., 2005).
We treated transfected myotubes with cardiotoxin to induce
damage. After cardiotoxin exposure, the 1543-bp–GFP-transfected
myotubes demonstrated high-level GFP expression (Fig. 4F) as
compared with the untreated 1543-bp–GFP-transfected myotubes
(Fig. 4D). Notably, CsA blocked the cardiotoxin-induced activity
of the myoferlin promoter (Fig. 4H). As a control, cardiotoxin or
cardiotoxin plus CsA did not affect the level of GFP expression in
the CMV-GFP-containing myotubes, because GFP levels remained
high in all conditions (Fig. 4C,E,G). GFP expression levels were
verified by immunoblot using an anti-GFP antibody (data not
shown). Thus, these results suggest that the 1543-bp promoter
contains the region responsible for upregulating myoferlin in
response to cardiotoxin-induced damage.
The myoferlin promoter is responsive to
cardiotoxin-induced damage in vivo
Multiple independent lines of transgenic mice were generated
carrying the 1543-bp myoferlin promoter driving GFP (Fig. 5A).
Four lines were tested for transgene expression before and after
muscle damage (lines 17, 36, 53 and 68). Consistent with the
known pattern of endogenous myoferlin expression, relatively low
GFP expression was present in uninjured adult muscle (Fig. 5B).
However, after intramuscular cardiotoxin injection to induce injury,
each of these lines demonstrated an increase in GFP protein (Fig.
5B). To verify that the GFP response mirrored myoferlin expression,
equal amounts of protein from uninjured and cardiotoxin-injured
skeletal muscle from a transgene-positive mouse were
immunoblotted for both GFP and myoferlin. As expected, the
Myoferlin regulation by NFAT
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Journal of Cell Science
Fig. 5. The 1543-bp myoferlin promoter drives GFP expression in
damaged muscle in vivo. (A)Schematic of the 1543-bp–GFP transgene
construct used to generate multiple lines of transgenic mice. (B)Immunoblot
analysis of four transgenic lines showing induction of GFP in response to
muscle damage induced by cardiotoxin (CTX) injection into the muscle.
(C)Immunoblot analysis of transgene-positive muscle lysates with and
without cardiotoxin-induced muscle damage, confirming that GFP expression
mirrors endogenous myoferlin expression in response to muscle injury.
Fig. 4. The 1543-bp myoferlin promoter drives GFP expression in C2C12
myotubes in response to cardiotoxin-induced damage. (A-H)The CMVGFP or 1543-bp–GFP constructs were transfected into C2C12 cells and cells
were imaged for GFP expression by live-cell microscopy. (A,B)Mononuclear
myoblasts transfected with CMV-GFP or 1543-bp–GFP expressed high levels
of GFP, similar to endogenous myoferlin expression. (C,D)After
differentiation and fusion into myotubes, the CMV promoter, but not the
myoferlin 1543-bp promoter, drives expression of GFP in multinucleate
myotubes. (E,F)Myotubes were treated with CTX in the absence of CsA (E,F)
or in the presence of CsA (G,H). The 1543-bp myoferlin promoter expresses
GFP in cardiotoxin-treated myotubes (F), whereas GFP expression is blocked
by the presence of CsA (H). Cardiotoxin or cardiotoxin and CsA did not alter
CMV-GFP expression (C,E,G). Scale bars: 10mm.
levels of both GFP and myoferlin increased in response to
cardiotoxin-induced muscle damage (Fig. 5C). Thus, the 1543-bp
myoferlin promoter contains the region responsible for upregulating
myoferlin in response to cardiotoxin-induced damage in vivo.
The 1543-bp myoferlin promoter drives GFP expression in
myoblasts and damaged myofibers in vivo
To identify the cell type expressing GFP in the transgenic 1543bp–GFP mice, we enzymatically digested muscle from newborn
uninjured mice and isolated only the mononuclear cells for FACS
analysis. FACS analysis detected GFP fluorescence in the 1543bp–GFP suspension, indicating that GFP positivity derives from
mononuclear muscle-derived cells (Fig. 6A). This culture protocol
from newborn uninjured muscle typically yields cultures that are
more than 90% myoblasts and less than 10% fibroblasts (Rando
and Blau, 1994; Doherty et al., 2008). Myoblasts in culture are
distinguished from fibroblasts on the basis of their refractive
properties and elongated shape (Klamut et al., 1986). GFP
expression in transgene-positive mononuclear muscle-derived
myoblasts was confirmed by immunoblotting with an anti-GFP
antibody. GFP expression was not detected in transgene-negative
wild-type littermates or a wild-type control line, confirming
myoferlin-promoter activity in myoblasts (Fig. 6B). We injected
skeletal muscle of transgenic and littermate control mice with
cardiotoxin to induce local muscle damage and with EBD to mark
damaged muscle fibers. At 3 days post-injection, tissue was
harvested. Schematics of isolated muscle are included in Fig. 6C
identifying healthy fibers (white) and damaged fibers (gray).
Dissected fibers were fixed on glass slides and stained with an
anti-myoferlin antibody. Myoferlin was upregulated in dye-positive
damaged fibers (numbered 2, 3 and 6 in Fig. 6C, upper panel),
whereas little to no myoferlin expression was seen in healthy dyenegative fibers (numbered 1, 4, 5, 7 and 8 in Fig. 6C, upper panel).
Similarly, live imaging of myofibers revealed GFP expression in
dye-positive damaged myofibers (numbered 3, 5 and 6 in Fig. 6C,
bottom panel), whereas little or no expression of GFP was seen in
neighboring undamaged, EBD-negative myofibers (numbered 1, 2,
4, 7 and 8 in Fig. 6C, bottom panel). Therefore, GFP expression
mirrors endogenous myoferlin expression; both are upregulated in
dye-positive damaged muscle fibers.
To assess the expression of myoferlin in damaged myotubes in
a more physiological form of muscle damage, we bred mice
containing the 1543-bp–GFP transgene to the mdx mouse model of
Duchenne muscular dystrophy. mdx mice have continual muscle
degeneration that exceeds the capacity for muscle regeneration,
resulting in muscle wasting similar to what is observed in the
Sgcg-null mice. The 1543-bp–GFP:mdx muscle displays signs of
muscle degeneration and regeneration, namely centrally nucleated
fibers (Fig. 7). Furthermore, basophilic degenerating and/or
regenerating fibers are also evident (bluish fibers in Fig. 7, top
left). These basophilic fibers are the same fibers that stain positive
for EBD, indicating membrane damage (Fig. 7, top row). With
membrane disruption, there is an increase in intracellular Ca2+,
visualized by Alizarin Red. 1543-bp–GFP:mdx mice accumulated
EBD-positive, Alizarin-Red-positive fibers within the diaphragm
muscle, and GFP expression was present in the same myofibers
(Fig. 7, top row). No basophilic, EBD-positive, Alizarin-Redpositive or GFP-positive myofibers were seen in 1543-bp–GFP
mice in the absence of the mdx allele (Fig. 7, bottom row). These
data show that the 1543-bp promoter is activated in vivo in a
mouse model of muscular dystrophy in damaged, Ca2+-loaded
myofibers, which is consistent with the role of myoferlin in injury
response.
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Journal of Cell Science
Fig. 7. GFP expression from the myoferlin promoter drives expression in
the mdx mouse model of muscular dystrophy. Serial sections of 1543-bp–
GFP:mdx transgene (tg)-positive (top row) and wild-type transgene-positive
(bottom row) diaphragm were stained with hematoxylin and eosin (H&E) to
show gross muscle pathology. EBD-labeled fibers are shown in red. Alizarin
Red staining identifies increased Ca2+ within damaged myofibers and this
correlates with those fibers expressing GFP from the myoferlin promoter. No
positive fibers were ever seen in the wild-type transgene-positive tissue.
Arrows indicate the identical fiber.
Fig. 6. In response to in vivo muscle damage, the 1543-bp myoferlin
promoter drives GFP expression in myoblasts. (A)Muscle from newborn
mice was digested to release mononuclear cells and then subjected to FACS
analysis gated to detect GFP. Mononuclear cells from transgene (Tg)-positive
newborn muscle express GFP, reflecting expression in myoblasts.
(B)Immunoblot with an anti-GFP antibody, demonstrating GFP expression in
myoblasts cultured from transgene-positive neonatal mice. (C)Transgenenegative and transgene-positive mice were injected with cardiotoxin (CTX)
into skeletal muscle to induce focal damage. The animals were also injected
with EBD to mark damaged myofibers. A schematic of individual fibers
(white, healthy; gray, damaged) is shown, indicating that the myofibers run
vertical in the images. At 3 days post-injection, transgene-negative muscle was
harvested, and single live fibers were isolated and stained with an antimyoferlin antibody (upper panels). The image shows aligned fibers running
longitudinally from top to bottom. Myoferlin is upregulated in EBD-positive
fibers numbered 2, 3 and 6, and not in the adjacent, intact EBD-negative
myofibers numbered 1, 4, 5, 7 and 8 (upper panels). In transgene-positive
muscle, GFP expression parallels myoferlin expression. Arrows indicate
damaged fibers; arrowheads indicate healthy fibers.
Discussion
Muscle development is a multi-step process. During the first phase
of muscle development, termed myoinitiation, mononucleated
myoblasts fuse to each other to generate myotubes. Following this,
myoaugmentation occurs, in which myoblasts fuse with existing
myotubes, adding to the multinucleated syncytium and causing
muscle growth. After injury, muscle regeneration is also a multistep process. As in muscle development, activated myoblasts can
fuse with one another, generating new myofibers, or they can fuse
with existing myotubes, promoting muscle growth and repair
(Antonio and Gonyea, 1993; Kelley, 1996; Pena et al., 1995).
Cycles of degeneration and regeneration of skeletal myofibers are
a hallmark of the muscular dystrophy phenotype. In the muscular
dystrophies, as well as in other types of muscle damage,
degeneration and regeneration are tightly linked. Muscle repair
entails extensive cytoskeletal remodeling, cellular fusion, and
differentiation. We determined the molecular signature of damaged
versus intact myofibers in dystrophic muscle. In this setting,
differentially expressed genes are independent of the primary
genetic defect and are directly related to myofiber function and/or
repair. We detected a large number of upregulated genes that
mediate cytoskeletal remodeling and cell-cell signaling, suggesting
that these myofibers are attempting repair along with regeneration.
The identification of myoferlin as a focally upregulated gene
and protein product in damaged myofibers is consistent with muscle
regeneration because myoferlin is upregulated in pre-fusion
myoblasts and downregulated after fusion is complete (Davis et
al., 2002; Doherty et al., 2005). Immunolocalization studies
confirmed that myoferlin is expressed in the repairing multinucleate
myofibers and surrounding mononuclear cells. Nearly all of the
mononuclear cells in dye-positive regions, including Mac-1-positive
cells, showed upregulation of myoferlin, as did myofibers
themselves. These findings are important because dysferlin, the
gene responsible for limb girdle muscular dystrophy 2B, has been
shown to mediate resealing of damaged myofibers after laser injury.
Given the upregulation of myoferlin in response to muscle damage,
it is also possible that myoferlin contributes to membrane resealing
in the damaged fibers. Both myoferlin and dysferlin can bind
negatively charged phospholipids (Davis et al., 2002). Therefore,
it is likely that both of these proteins contribute to repair of the
multinucleate myofiber, although the molecular mechanisms might
not be completely overlapping. Recently, a role for myoferlin in
endocytic recycling was uncovered (Doherty et al., 2008); whether
dysferlin also participates in this process is unknown.
Owing to its role in repair and its complex expression profile,
modulation of myoferlin is tightly regulated. NFATc1 and NFATc3
regulate the myoferlin promoter. These data are consistent with the
expression of NFATc1 and NFATc3 in myoblasts, in which they
have the ability to translocate from the cytoplasm to the nucleus
(Delling et al., 2000). Consistent with this pathway, the myoferlin
promoter is sensitive to calcineurin activation. At the onset of
myogenic differentiation, calcineurin phosphatase activity is
increased (Delling et al., 2000). This increase in activation coincides
Journal of Cell Science
Myoferlin regulation by NFAT
temporally with the onset of myoferlin expression (Doherty et al.,
2005), as well as NFATc3 translocation to the nucleus (Delling et
al., 2000). The Ca2+-sensitive calcineurin-NFAT pathway is crucial
not only in muscle development but also in muscle repair. Both in
vivo and in vitro data have shown that blocking calcineurin and/or
NFAT function causes defects in muscle differentiation and repair
(Abbott et al., 1998; Friday et al., 2000; Horsley et al., 2001;
O’Connor et al., 2007). The addition of CsA, a calcineurin inhibitor,
to myoblast cultures causes a decrease in differentiation in a dosedependent fashion, with a maximum inhibition of 75-90% (Abbott
et al., 1998). This phenomenon is recapitulated in muscle of CsAtreated mice. Specifically, CsA-treated muscle displays a lack of
centrally located nuclei, a distinct marker of muscle regeneration
(Abbott et al., 1998). We found that the addition of CsA decreased
myoferlin promoter activity. We expect that at least some of the
negative effect of CsA on muscle regeneration can be explained by
the downregulation of myoferlin.
The five NFAT isoforms are expressed in muscle; however,
each isoform is activated at different stages throughout muscle
growth and differentiation (Abbott et al., 1998; Cho et al., 2007;
O’Connor et al., 2007). The loss of individual NFAT isoforms
results in unique muscle defects, suggesting specific functions
for each NFAT isoform. Interestingly, the phenotype of the
myoferlin-null mouse is similar to the NFATc2-null mouse
(Doherty et al., 2005; Horsley et al., 2001). Both the myoferlinnull and NFATc2-null mice have decreased fiber cross-sectional
area, fusion defects and delayed muscle repair. Our data suggests
that NFATc3 binds to the myoferlin promoter in the early myoblast
growth stage, allowing for high expression of myoferlin, preparing
the myoblasts for the upcoming fusion events. Under our
experimental growth conditions, NFATc3 can translocate to the
nucleus, whereas NFATc2 and NFATc4 are not active until 48
hours after differentiation media is added (Abbott et al., 1998;
Cho et al., 2007). Later in differentiation, when large
multinucleate myotubes are present, NFATc2 and NFATc4 are no
longer active (Abbott et al., 1998; Cho et al., 2007). This decrease
in NFAT activity correlates with decreased myoferlin expression
in myotubes, suggesting that NFATs might differentially bind the
myoferlin promoter throughout muscle development.
Recently, myoferlin has been implicated in regulating growthfactor signaling that affects myoblast fusion and muscle growth
(Demonbreun et al., 2009). NFATc2 activates growth-factor
promoters such as that expressing IL-4, a protein that is secreted
by myotubes to recruit myoblasts and is crucial for
myoaugmentation (Horsley et al., 2003). We hypothesize that the
phenotypes of the NFATc2-null and myoferlin-null mice are similar
owing to the loss of these fusion signals. Both loss of NFATc2,
resulting in decreased production of growth factors, or the loss of
myoferlin, resulting in a decreased response to growth factors,
would result in similar muscle defects in growth and repair. The
role of NFATc1 has yet to be determined because NFATc1-null
mice die in utero from cardiac defects (de la Pompa et al., 1998;
Ranger et al., 1998). However, NFATc1 expression is upregulated
in the mdx mouse model of muscular dystrophy and after locally
induced injury in rat, suggesting a role for NFATc1 in muscle
repair similar to the endogenous myoferlin expression profile
(Sakuma et al., 2003; Stupka et al., 2006a). It has also been
reported that the NFATc3-null mouse has a decreased number of
myofibers (Kegley et al., 2001). Therefore, a combinatorial effect
of multiple NFATc factors probably regulates myoferlin expression
in vivo, consistent with our in-vitro-based studies.
2419
The expression pattern directed by the upstream region of the
myoferlin promoter recapitulates the expression of endogenous
myoferlin, including both its positive and negative regulation.
Myoferlin expression is highest in myoblasts that are elongated
and are preparing to fuse to myotubes (Davis et al., 2002).
Following fusion, myoferlin expression is reduced in mature,
undamaged muscle fibers. The 1543-bp myoferlin promoter
demonstrated this same pattern of expression when introduced into
cultured myoblasts and myotubes. When these myotubes were
damaged by brief exposure to cardiotoxin, the levels of myoferlin
expression in myotubes increased. This increase might represent a
contribution from myoblasts that have recently fused or from
myotube nuclei, or both. GFP expression was seen 48 hours after
cardiotoxin treatment of myotubes, and this time frame is sufficient
to support myoblast fusion in addition to gene expression from
myotube nuclei. Similarly, the pattern of myoferlin expression in
vivo was recapitulated using transgenesis. In vivo, we separated
the mononuclear component from the multinucleate myotubes.
Interestingly, the fibers that expressed the highest levels of GFP
were the same fibers that also displayed high levels of Ca2+. Thus,
we favor a model in which intracellular Ca2+ levels increase in
damaged myofibers, activating both calcineurin and NFAT
transcription factors. NFATs bind and activate the myoferlin
promoter, increasing the expression of myoferlin mRNA and protein
(Fig. 8). Furthermore, upon muscle damage, myoblasts are
activated, myoferlin is expressed, and myoblasts are recruited to
the site of damage for fusion to the injured fibers. Therefore, we
believe that myoferlin protein expression is increased from
myoferlin mRNA that derives from both myoblast as well as
myotube nuclei.
The 1543-bp–GFP transgenic mouse line is an ideal tool for
studying muscle repair in general, because this model serves as a
‘damage sensor’. To date, transgenic GFP-tagged mouse models
have been created for the key myogenic regulators, Pax7 and Pax3.
During muscle repair, these proteins are transiently expressed in
Fig. 8. Model for myoferlin expression in injured myofibers. Disruption of
myofiber membranes leads to increased intracellular Ca2+. This increase in
Ca2+ activates calcineurin, which results in NFAT dephosphorylation and
nuclear translocation. Within the nucleus, NFAT is now positioned to activate
myoferlin expression.
2420
Journal of Cell Science 123 (14)
activated satellite cells and are downregulated in daughter myoblasts
and myotubes (Buckingham et al., 2003; Seale et al., 2000). Thus,
although the Pax3-GFP and Pax7-GFP mouse models are well suited
for the study of satellite-cell activation, they do not allow the study
of damaged myotubes or myoblasts participating in the repair process.
Marking myotubes that are undergoing degeneration and/or
regeneration and myoblasts, which actively mediate muscle repair,
is crucial in furthering our understanding of the repair process both
in heritable and environmentally induced muscle damage.
Materials and Methods
Journal of Cell Science
Animals and tissue harvest for laser microdissection, verification and
muscle-injury assays
Mice lacking g-sarcoglycan (Sgcg null) were generated by targeting exon 2 (Hack et
al., 2000; Hack et al., 1998). mdx mice were obtained from Jackson Laboratories
(Bar Harbor, ME). Myoferlin (mko) mice were generated previously by deleting the
first exon (Doherty et al., 2005). For laser microdissection, two Sgcg-null mice, 13.5
weeks of age, were injected intraperitoneally with a 10 mg/ml solution of EBD at
1% of total body weight (Hack et al., 2000; Hamer et al., 2002). The mice were
sacrificed 24 hours post-injection. Quadriceps muscles were dissected and frozen in
cooled isopentane as described (Hack et al., 1998). For verification, EBD-injected
and non-injected wild-type, Sgcg null and mdx muscle was harvested as described
above. To induce muscle injury, cardiotoxin injections were done as 100 ml injections
of 10 mM cardiotoxin in PBS or PBS alone into the quadriceps or
gastrocnemius/soleus muscles from 14- to 20-week-old mice as described (Volonte
et al., 2005). After 3 or 5 days, muscle from injected and control legs were harvested
for immunoblot analysis and for immunofluorescence microscopy. For live-fiber
analysis, cardiotoxin-damaged fibers were isolated by manual dissection, placed in
PBS between a glass coverslip and slide, and immediately imaged using Ivision
software and a Zeiss Axiophot microscope.
Rapid immunofluorescence microscopy and laser microdissection
Skeletal muscle was collected in 6 mm longitudinal sections. One slide was processed
for immunofluorescence microscopy with a rabbit polyclonal antibody to spectrin
(catalog number AB993; Chemicon, Temecula, CA) using RNase-free technique,
and staining was carried out as described previously (Bi et al., 2002). The antispectrin antibody was diluted (1:4) in PBS with 400 U/ml RNase inhibitor (Invitrogen,
Carlsbad, CA) and applied to the slides for 4 minutes followed by a 30-second PBS
wash. A goat anti-rabbit secondary antibody conjugated to Alexa Fluor 555 (Molecular
Probes, Eugene, OR) was diluted in PBS (1:50) and applied to the slide for 2
minutes. The slide was washed three times, for 30 seconds each, dipped in ethanol
for 3 seconds and then allowed to air dry for 2 minutes prior to laser microdissection.
A Leica AS LMD microscope was used to capture individual cells or cell clusters
and regions of interest were selected manually. Upon command, an ultraviolet laser
beam cut and released the cells into a PCR tube that contained lysis buffer from the
RNeasy micro kit (Invitrogen, Carlsbad, CA). A Rhodamine filter set was used to
detect both EBD and Cy3 fluorescence. For each group, a mean of 22.6⫻103±1.2⫻103
mm2 were collected (volume135.5⫻103±7.3⫻103 mm3) representing approximately
27⫻103±1.5⫻103 cells (each 6 mm in thickness) from each group.
RNA isolation, amplification hybridization and analysis
Reverse transcriptase (RT)-PCR was performed to test the integrity of the RNA
isolated using primers to dystrophin mRNA (data not shown). All samples within
each group (four groups total) were pooled prior to RNA extraction. The RNeasy
micro kit was used following the manufacturer’s instructions except that 20 ng
bacterial rRNA (Roche, Indianapolis, IA) was used as carrier RNA. The RiboAmp
OA Amplification kit (Arcturus, Mountain View, CA) was used to perform one
round of linear amplification prior to labeling, according to the manufacturer’s
instructions. A total of 200 ng Polyd(I-C) (Roche, Indianapolis, IA) was used as an
RNA carrier. According to Affymetrix protocols, antisense RNA (aRNA) was
streptavidin-labeled and each sample was hybridized to a Mouse 430A chip
(Affymetrix, Santa Clara, CA) at the Functional Genomics Core Facility. Raw
fluorescent data were imported into the Bioconductor platform (Gentleman et al.,
2004). The background fluorescence was corrected, data were normalized and
relative fluorescent values were determined by robust multichip analysis (RMA).
Fluorescent values were then imported into Microsoft Excel for Significance Analysis
of Microarray (SAM) to generate a differentially expressed gene list comparing the
EBD-positive and EBD-negative myofibers (Tusher et al., 2001). The fluorescent
data were analyzed by Student’s t-test in the Bioconductor platform and by Rank
products analysis by running a Windows script (rankproducts_FDR.exe) (Breitling
et al., 2004).
Comparative sequence analysis
1543 bp 5⬘ of the translational start site of both human and mouse myoferlin was
downloaded from http://genome.ucsc.edu and compared using the Vista program
(http://genome.lbl.gov/vista) with a window size of 100 bp and a minimum sequence
identity of 70%. Conserved NFAT-transcription-factor-binding sites were identified
using the rVISTA program. Predictions were made based on the TRANSFAC
Professional 9.2 library using the default core similarity value of 0.75 and the matrix
similarity value of 0.70. Conserved transcription binding sites were defined as
having greater than 80% similarity over a 24-bp window. Potential transcriptionfactor-binding sites were also determined using the Transcriptional Element Search
System (TESS) (http://www.cbil.upenn.edu) using the default settings (Frazer et al.,
2004; Loots et al., 2002; Mayor et al., 2000; Schug and Overton, 2007).
Plasmid construction
The 1543-bp fragment of the myoferlin promoter was amplified from genomic DNA
using primers from supplementary material Table S2. All products were sequenced
and verified. All promoter fragments were cloned into the pGL2-basic vector
(Promega, E1641) using KpnI and Sac1 to generate the reporter vectors 1543-bp–
pGL2, 918-bp–pGL2, 729-bp–pGL2 and 226-bp–pGL2. NFAT mutations were
introduced into the 1543-bp–pGL2 reporter construct sequences listed in
supplementary material Table S2. Using the restriction enzymes Sac1 and KpnI, the
1543-bp promoter was cloned into the pEGFP-N3 vector (Clontech, catalog number
6080-1). The CMV promoter was removed from the vector using AseI and BglII to
generate the 1543-bp–GFP plasmid. The transgene construct was generated from the
1543-bp–GFP construct using the restriction enzymes SspI and BglII.
Cell culture and isolation
C2C12 cells were obtained from ATCC (catalog number CRL-1772). For all
experiments, cells were grown to 95% confluency, the point at which C2C12 cells
are prefusion myoblasts, in DMEM supplemented with 10% fetal bovine serum and
1% penicillin/streptomycin at 37°C in 7% CO2. DMEM supplemented with 2%
horse serum and 1% penicillin/streptomycin was used as differentiation media.
Myoblasts were isolated as described previously (Doherty et al., 2008). Myoblasts
were lysed in 1 ml of lysis buffer [25 mM Tris (pH 7.4), 300 mM NaCl, 1 mM
CaCl2, 1% Triton-X 100 with Complete Mini Protease Inhibitor cocktail (Roche
Molecular Biochemicals)]. Cellular debris was removed, and the protein concentration
was determined using the Bio-Rad protein assay (Bio-Rad Protein Laboratories). A
total of 50 mg of protein was separated on a 12% acrylamide gel. Equivalently
loaded gels were stained with Gel Code Blue (Pierce) or transferred to PVDF
Immobilon-P membrane (Millipore).
Transfections and luciferase reporter assays
For transfections, 50,000 C2C12 cells were plated into each well of a 12-well tissueculture plate. After 24 hours, cells were transfected with 2.5 mg reporter plasmid, 10
ml Lipofectamine (Invitrogen), 10 ml Plus reagent (Invitrogen) and 50 ng of pRLSV40 Renilla luciferase plasmid (Promega, E2231) in Opti-MEM. The pGL2-basic
vector or the pGL2-SV40 control vector (Promega, E1631) replaced the reporter
vector as a negative or positive control, respectively. For co-transfection assays, 0.5
mg/well of 1543-bp–pGL2 reporter vector was transfected with 0.5 mg/well of
pcDL-Sra-DCnA, which was generously provided by Jeffery Molkentin (Oka et al.,
2005), pREP-NFATc1 (AddGene) and pREP-NFATc3 (AddGene), alone or in
combination. The total amount of DNA used per well was adjusted with the
promoterless pGL-2-basic vector. Cells were incubated with the transfection mix for
3 hours at 37°C and then the mix was replaced with growth media. Forty-eight hours
later, cells were lysed and analyzed for firefly and Renilla luciferase reporter gene
activity using the Dual Luciferase Reporter Assay system as specified by the
manufacturer (Promega, E1910). Data was normalized to Renilla luciferase activity.
All experiments were performed in triplicate and repeated at least three times. For
the CsA luciferase assay, cells were transfected with the 1543-bp–pGL2 construct as
described above. At 18 hours post-transfection, 1 mM or 5 mM CsA (Invitrogen,
PHZ1054) was added to the cells. Twenty-four hours later, cells were lysed and
analyzed for luciferase activity as described above. For transfections with cardiotoxin
exposure, 2.5 mg of the 1543-bp–GFP construct or CMV-GFP construct was
transfected into 50,000 C2C12 cells plated on glass coverslips in six-well plates.
Growth media was replaced with differentiation media after 24 hours. Cultures were
allowed to differentiate for 3 days. GFP expression was analyzed using live-cell
immunofluorescence microscopy. Cells were treated with 1 mM cardiotoxin
(Calbiochem, catalog number 217504) after having differentiated for 6 days or cells
were treated with 1 mM cardiotoxin and 1 mM CsA. GFP expression was analyzed
using live-cell immunofluorescence microscopy 48 hours after the addition of
cardiotoxin.
Chromatin immunoprecipitation
ChIP assays were performed in triplicate using the Upstate Chip Assay Kit according
to the manufacturer’s instructions (Upstate, catalog number 17-295) with Proteinase
K (Puregene, catalog number D50K5) and glycogen (Roche, catalog number 901393).
106 confluent C2C12 cells were sonicated to shear chromatin to 200-500 bp. For
immunoprecipitation, 2 mg of the antibodies anti-NFATc1, anti-NFATc2 or antiNFATc3 (Santa Cruz Biotechnology, catalog numbers sc-7294, sc-7296 and sc-8321,
respectively) were incubated with the cell lysates overnight at 4°C with rotation.
Immunoprecipitation with Immunopure IgG (Pierce, catalog number 31160) was
performed as a control. Myoferlin-promoter NFAT-binding sequences were amplified
using the primers in supplementary material Table S2.
Myoferlin regulation by NFAT
Electrophoretic mobility shift assay
EMSAs were performed using a double-stranded oligonucleotide containing a
hypothetical NFAT-binding site from the 1543-bp myoferlin promoter (see
supplementary material Table S1). Twenty-five mg of C2C12 nuclear extracts were
incubated with 30,000 cpm of 32P-labeled double-stranded oligonucleotide, 1 mg of
poly(dI-dC)-poly(dI-dC) and 1.5 ml of 10⫻ Ficoll binding buffer [20% Ficoll, 50
mM Tris (pH 7.5), 50 mM KCl, 5 mM EDTA, 5 mM dithiothreitol, 375 mM KCL]
on ice for 30 minutes in a 30 ml reaction. For super shift assays, 2 mg of anti-NFATc1
(Santa Cruz Biotechnology, catalog number sc-7294) or anti-NFATc3 (Santa Cruz
Biotechnology, catalog number sc-8321) was incubated with the reaction mixture.
Protein complexes were resolved from the free 32P-labeled probe using a 5%
acrylamide gel. Primers are shown in supplementary material Table S1.
Immunoblotting, immunofluorescence microscopy and antibodies
Journal of Cell Science
The anti-myoferlin antibody (MYOF3, 1:2000) (Doherty et al., 2005) and an antiGFP antibody (1:1000, Santa Cruz, sc-9996) were used. The secondary antibodies
goat anti-rabbit or goat anti-mouse conjugated to horseradish peroxidase (Jackson
ImmunoResearch) were used at a dilution of 1:5000. Blocking and antibody
incubations were done in Starting Block T20 (Invitrogen) for all antibodies. ECLPlus chemiluminescence (Amersham-Pharmacia) and Kodak Biomax MS film was
used for detection. For microscopy, MYOF3 was used at 1:100 or 1:500 dilution
(Doherty et al., 2005). Goat anti-rabbit conjugated to Alexa Fluor 594 was used at
1:2000 dilution or to Alexa Fluor 488 at 1:5000 dilution (Molecular Probes).
Phalloidin conjugated to Alexa Fluor 488 (Molecular Probes, A12379) was used at
1:2000. Mac-1 (BD Bioscience, 557395) was used at 1:100 and a strepavidin–AlexaFluor-488 secondary (Molecular Probes, S-11223) was used at 1:2500. Blocking and
antibody incubations were in 1⫻TBS and 5% fetal bovine serum. Coverslips were
mounted using Vectashield with DAPI. Images were captured using a Zeiss Axiophot
microscope and AxioVision software (Carl Zeiss).
FACS analysis
Mononuclear cells analyzed by flow cytometry were prepared as above. Cells were
washed and resuspended in PBS for analysis on a FACS Canto (BD Biosciences).
After gating to remove dead cells, aggregated cells and debris, a minimum of 10,000
events was scored per culture. Cells were derived from independent animals per
genotype. Fluorescent gates were set to include 0% wild-type cells. The percentage
of fluorescent cells was determined by the number of cells remaining in this gate.
FACS data was analyzed using FlowJo software (Tree Star).
Mdx 1543-bp–GFP transgenic mouse tissue analysis
The 1543-bp–GFP mouse line 68 was crossed to an mdx female mouse (Jackson
Laboratories). Diaphragm muscles from male transgene-positive mdx mice and male
transgene-negative littermates were analyzed using 7-mm serial sections. Unfixed
tissue sections were subjected to hematoxylin and eosin, 2% Alizarin Red (Chiu et
al., 2009) for 2 minutes then rinsed in acetone to visualize calcification, or 10 ng/ml
EBD for 30 minutes then three 5-minute PBS washes to identify damaged myofibers.
Tissues were fixed in ethanol for endogenous GFP expression. Coverslips were
mounted with permamount or Vectashield with DAPI. Images were captured using
a Zeiss Axiophot microscope and Ivision software.
This work was supported by NIH NS4776, HL61322 and T32 HL
7381, and the Muscular Dystrophy Association. We acknowledge the
Jain Foundation. Deposited in PMC for release after 12 months.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/123/14/2413/DC1
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