From the
Death of dystrophin-deficient muscle purportedly results from
increases in [Ca]
Mutations in the gene for dystrophin that lead to the
protein's absence underlie the muscular degeneration that occurs
in Duchenne muscular dystrophy (DMD)
Muscle
from DMD humans and mdx mice contain abnormally high concentrations of
intracellular calcium during necrosis, which is believed to contribute
to muscle cell death through the activation of proteases. Evidence for
the pathological increase in intracellular [Ca] has been
obtained in several studies using a variety of technical approaches
(5, 6, 7, 8, 9, 10, 11, 12, 13, 14) .
One hypothesis to explain the elevated intracellular calcium
concentrations states that dystrophin's normal function is to
stabilize a calcium channel, and its absence results in the
deregulation of that channel
(14) . Alternatively, the increase
in [Ca] may result from mechanical damage to the cell
membrane that permits unregulated entry of calcium into the cell
(15) .
The relationship between the increase in
[Ca]
Although there are no published data to show whether
calpains are activated in dystrophic muscle, several studies provided
indirect evidence consistent with a role for calpain activation in the
pathology of dystrophin-deficient muscle
(23, 24, 25, 26, 27) . First,
data show that when [Ca]
In
vitro studies have demonstrated that autolysis of amino-terminal
peptides from each calpain subunit occurs upon activation and results
in increased calcium sensitivity
(40, 41) . Thus,
proteolytic modification of the enzyme can be used as a direct
indication of calpain activation. Although autolysis of calpains has
not been demonstrated previously in vivo, calpain
autoproteolysis in platelets coincides with activation of platelets
in situ. Calpain autolysis in situ has been shown to
coincide with relocalization of the enzyme to the membrane and cleavage
of the membrane skeletal proteins, talin, spectrin, and filamin
(42, 43, 44, 45) .
In the present
investigation, we measured changes in the concentration and
autoproteolytic modification of m-calpain and µ-calpain over the
course of mdx dystrophy. By performing these analyses on muscles from
4-week (peak necrosis) and 14-week (regenerated) mice, the
autoproteolytic modification of calpain can be correlated with the
stage of the disease process. The mRNA levels and protein
concentrations of the specific inhibitor of calpains, calpastatin, were
also measured to determine whether changes in calpain's
autoproteolytic modification could be secondary to changes in the
regulation of calpastatin.
For preparation
of whole muscle extracts to be analyzed by SDS-PAGE, mouse hind limb
muscles previously frozen in liquid nitrogen were homogenized in 30
µl of reducing sample buffer (80 mM Tris-HCl, pH 6.8, 0.1
M dithiothreitol, 70 mM SDS, glycerol) per mg wet
weight using a Dounce homogenizer. The samples were boiled for 1 min,
centrifuged at 4 °C for 3 min at 12,000
Samples were loaded onto the Mono Q column in
buffer A and washed to baseline in the same buffer. Samples were eluted
with a linear gradient of buffers A and B (20 mM Tris, pH 7.6,
3 mM
The analyses of m- and µ-calpain were performed
slightly differently. Because the autoproteolyzed, p78 (active) and
non-autoproteolyzed, p84 (inactive) forms of µ-calpain are resolved
by SDS-PAGE, the extent of autoproteolysis of µ-calpain can be
assessed directly by densitometric analysis of immunoblots of
µ-calpain ion exchange fractions
(54) . For m-calpain, the
autolyzed 80-kDa subunit is not resolved from the unmodified subunit,
and autolysis was assessed with anti-NTm calpain which recognizes the
peptide lost from the 80-kDa subunit through autolysis. Therefore,
anti-NTm calpain recognizes only inactive m-calpain. Thus, the extent
of autoproteolysis was measured by comparing m-calpain reactivity with
anti-CCD (total m-calpain) (Fig. 5 a), and the reactivity
of the same samples with anti-NTm calpain (preautolysis only)
(Fig. 5 b). The activation index (extent of
autoproteolysis) was derived using the following formula: [mdx
total calpain/C57 total calpain]
Previous investigations have relied upon indirect evidence
and speculation to conclude that calpains play a role in the wasting of
dystrophic muscle. Recent progress in understanding the biochemistry of
the calpain family of proteases has provided the tools to finally
address this issue more definitively. The present investigation
provides the first direct evidence that calpains are active components
in the pathology of dystrophin-deficient muscle in that their
concentrations and/or levels of autoproteolytic modification vary over
the course of the disease.
In this study, three isoforms of calpains
were quantified. Immunoblots of whole muscle extracts document that
each calpain isoform appears to be regulated independently throughout
the progression of necrosis and regeneration. Thus, each isoform may
make distinct contributions to the process of muscle degeneration in
muscular dystrophy. Results from immunoblotting also demonstrated that
the total concentration of calpains is increased in necrotic muscle
from mdx mice relative to controls. The increase is largely
attributable to an increase in the m-calpain isoform and correlated
with the onset of necrosis. The increased m-calpain protein did not
result from an accompanying increase in its mRNA suggesting that
regulation was not at the level of transcription. The total
concentration of the other major calpain isoform, µ-calpain, was
not changed throughout the progress of muscle necrosis and
regeneration.
This is the first study to document the presence of
the skeletal muscle-specific calpain, p94 protein, in muscle tissues
and to show that its concentration decreases at peak muscle necrosis.
In a previous study, p94 was not detected in fractionated rat muscle
extracts, which the authors interpreted to suggest that p94 was very
short-lived
(22) . The discrepancy between the two reports may
lie in differences in sample preparation, antibody titer, and/or
antibody specificity. The difference is apparently not due to species
differences as the anti-CCD also detected p94 in rat muscle extracts
(not shown). The decrease in p94 concentration accompanying necrosis
may reflect a decreased stability and increased turnover.
The
protein concentration of calpains, however, may not be directly related
to enzyme activity in vivo. It is well established that µ-
and m-calpain undergo autoproteolysis which precedes, or accompanies,
substrate hydrolysis (16-18, 40, 50, 54, 56-58). Evidence
also clearly demonstrates that calpains become more sensitive to
calcium after autoproteolysis and that removal of an amino-terminal
peptide of the µ-calpain catalytic subunit is prerequisite for
cleavage of other proteins
(59) . It is still controversial
whether the autoproteolysis of m-calpain is a prerequisite for
substrate hydrolysis
(50, 57, 58, 60) although it is agreed that if m-calpain has been
proteolytically modified it is, or has been, activated. Thus, the loss
of the amino-terminal peptide provides a minimal estimate of activation
of m-calpain in vivo. We therefore examined covalent changes
to the calpain catalytic subunits to assess their activation status
within dystrophic muscles in vivo. Despite no significant
change in µ-calpain concentration at peak necrosis, the fraction of
autoproteolyzed, active µ-calpain increased substantially.
Importantly, the activation status returned to control values in
regenerated muscle. These changes in µ-calpain activation do not
result from any regulatory influence of calpastatin, because we found
no change in the concentration of calpastatin or its mRNA at the ages
sampled. Our findings also show that m-calpain concentration increases
during necrosis, although the extent to which it is autoproteolyzed
does not change. However, these findings indicate that the total mass
of autoproteolyzed m-calpain increases during necrosis because
autolyzed m-calpain is a constant proportion of an increased mass of
total m-calpain.
The results of the present study provide strong
evidence that calpains are active components in the pathology of
dystrophin-deficient muscle necrosis in that their concentrations
and/or levels of activation of each isoform vary independently with the
progress of the disease state. Although the data show here that calpain
activation increases only 2-3-fold above controls, these values
were averaged over the whole muscle. Even at 4 weeks of age, the
proportion of the fibers that undergo necrosis at any one time is
small, so that a 2-3-fold increase in the entire muscle
represents a much higher level of calpain activation in the
subpopulation that is experiencing necrosis. For example, Carnwath and
Shotten
(61) have shown that at the peak of mdx muscle
necrosis, only 7% of the muscle cross-section is occupied by
degenerating fibers. If the majority of the increased calpain
activation occurs in these fibers, the actual increase in calpain
activation may be as high as 20-30-fold in fibers undergoing
degeneration. These studies show directly for the first time in any
whole animal system that calpain activation varies during a biological
process and is pertinent to events occurring in vivo. It
remains for future studies to establish whether calpain plays an
initiating role in proteolysis encountered in mdx dystrophy.
that cause the activation of
calpains. We have tested whether calpains play a role in this process
by assaying for changes in calpain concentration and activation in peak
necrotic mdx mice (4 weeks of age) and in completely regenerated mdx
mice (14 weeks of age). Biochemical fractionation and immunoblotting
with epitope-specific antisera allowed measurement of the
concentrations of m- and µ-calpains and the extent of
autoproteolytic modification. Our findings show that total calpain
concentration is elevated in both 4-week and 14-week mdx mice. This
increase in concentration was shown to result primarily from a
significant increase in m-calpain concentration at 4 weeks. Northern
analysis demonstrated that neither m- nor µ-calpain mRNA
concentrations differed between mdx and controls suggesting that the
increased calpain concentration results from post-translational
regulation. Immunoblotting with antibodies directed against
amino-terminal peptides revealed an increase in autoproteolysis of
µ-calpain, indicative of increased activation. The extent of
autoproteolysis of µ-calpain returns to control levels during
regeneration. This is not a consequence of increased calpastatin mRNA
or protein. The findings reported here support a role for calpains in
both the degenerative and regenerative aspects of mdx dystrophy.
(
)
and mdx
mouse dystrophy. However, the mechanisms by which dystrophin's
absence leads to muscle cell death are unknown. Both mdx and DMD
pathologies are characterized by myofibrillar protein loss, Z-disc
disorganization, plasma membrane defects, and dilation of the
sarcoplasmic reticulum
(1, 2) . In DMD, these
pathological changes first appear at about age two, when muscle
necrosis begins and continues until death. mdx mice display these
pathological changes at approximately 28 days when their muscles
undergo one bout of necrosis, but then spontaneously regenerate
(3) . mdx muscles do not undergo cycles of necrosis regeneration
subsequent to this first regenerative episode
(4) .
and the pathology of dystrophin-deficient
muscle suggests that calpains, ubiquitous calcium-dependent cysteine
proteases, play a key role in degeneration of dystrophic muscle.
Calpains exist as two main isoforms designated µ-calpain (or
calpain I) and m-calpain (or calpain II)
(16) . The two forms
are classified based on their calcium requirements, with µ-calpain
requiring between 1 and 70 µM calcium and m-calpain
requiring 10-800 µM calcium for half-maximal
activity in vitro(17, 18, 19) .
Recently, two tissue-specific calpain isoforms have been described at
the cDNA level
(20, 21) . One form specific to skeletal
muscle is designated p94. It is reported to be rapidly turned over and
present at low concentrations relative to the m- and µ-isoforms
(22) .
is experimentally
elevated in normal, healthy muscle, cytosolic cysteine proteases are
activated that initiate widespread proteolysis
(28, 29, 30) . Thus, calpains, which are
calcium-sensitive cysteine proteases, may feasibly play a similar
proteolytic role in dystrophin-deficient muscle. Calpains are capable
of proteolyzing a broad range of structural proteins found in muscle,
which also supports their possible involvement in muscular dystrophy.
Z-disc proteins appear to be particularly susceptible to calpains.
Treatment of myofibrils with purified calpain removes Z-discs and
releases
-actinin
(31, 32, 33, 34, 35, 36) ;
similar Z-disc disruption or ``streaming'' is commonly cited
as an early event in DMD and mdx
(37) . In addition, many
myofibrillar and other cytoskeletal proteins are substrates for calpain
such as troponins I and C, tropomyosin, filamin, desmin, talin,
vinculin,
-actinin, and integrin
(38) . Finally, current
evidence has shown that the collective concentration of calpains is
increased in extracts of DMD muscle
(26, 39) , which
also supports the possibility that calpains play a role in degeneration
of dystrophin-deficient muscle. The previous studies, however, measured
the concentration and activation of calpain in tissue homogenates using
conditions under which all calpains present would be activated, so that
the actual in vivo activity could not be assessed.
Reagents
The following materials were used in
this investigation: cesium trifluoroacetate, oligolabeling kit, and
restriction enzymes (Pharmacia Biotech Inc.),
[-
P]dCTP (ICN), purified m-calpain (Sigma),
recombinant calpastatin (Takara Biochemicals). Calpain cDNA probes to
m- and µ-calpain were generously donated by Dr. John S. Elce
(46) . Calpastatin cDNA probe was kindly donated by Dr.
Masatoshi Maki (Kyoto University)
(47) . The
-actin probe
was generously donated by Dr. Adrian Casillas (Dept. of Medicine, UCLA)
who transcribed it from the original plasmid. Other chemicals were the
highest grade obtainable from Fisher Scientific.
Animals
C57 and mdx mice were obtained from a
colony generated at UCLA using breeding pairs originating from The
Jackson Laboratories. Animals were housed at the UCLA vivarium and were
2, 4, or 14 weeks old at the time of sacrifice.
Antibody Production and Antisera
Purification
Antisera were raised against oligopeptides of two
different calpain sequences. One antigen corresponding to chicken
calpain sequence (residues 282-301)
(48) represents a
highly conserved catalytic domain sequence present in m-, µ-, and
the muscle-specific p94 calpains. The antisera were raised and
characterized as described earlier
(49) ; however, the antibody
used for these experiments was of a much higher titer than in
previously performed studies
(49) , enabling sensitive detection
of all known calpain isoforms. In this report it is referred to as
anti-CCD (Conserved Catalytic Domain sequence). Immunoreactivity with
this serum is independent of the autolytic modification of the calpain
isoform. The other peptide antigen corresponded to the amino-terminal
sequence for mammalian m-calpain (residues 4-23 from human
sequences). The antiserum raised against this peptide was generated and
characterized as described earlier
(50) , which demonstrated its
isoform specificity and selective reactivity with calpain prior to
autoproteolysis, thereby recognizing only the un-autoproteolyzed
m-calpain catalytic subunit. In this report, we refer to this serum as
anti-NTm calpain (amiNo-Terminal-m). Anti-calpastatin was produced by
immunizing a rabbit with recombinant human calpastatin domain I. The
recombinant protein was initially purified on a Mono Q ion exchange
column (Pharmacia) with a linear NaCl gradient at pH 7.6. The antisera
were affinity-purified with antigen coupled to cyanogen bromide
(CNBr)-activated Sepharose beads, prepared according to the
manufacturer's instructions (Pharmacia Biotech Inc.). Following
purification, the anti-calpastatin recognized one band of crude whole
muscle extracts on immunoblots at 107 kDa.
Immunoblots
Proteins fractionated by ion exchange
chromatography and an m-calpain standard were separated using 8% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
(51) . Samples were then electrophoretically transferred to
nitrocellulose for antibody overlays
(52) . Nitrocellulose
strips were incubated with the primary antibody, and immunoblots were
performed as described earlier
(49) except the secondary
antibody (goat anti-rabbit) was conjugated to alkaline phosphatase.
Color development was performed using nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate as substrate.
g, and the
supernatant was collected. The protein concentration was determined,
and 50-75 µg were loaded onto 8% polyacrylamide gels. For
each immunoblot, three C57 and three mdx samples were electrophoresed,
and at least three experiments were performed for each age group.
Ion Exchange Chromatography
mdx and C57 mice were
sacrificed in pairs, by cervical dislocation, and the hind limb muscles
were dissected and homogenized with a Dounce homogenizer for 2 min in 5
volumes of buffer A (20 mM Tris, pH 7.6, 3 mM
2-mercaptoethanol, 5 mM EGTA, 10 mM NaCl) and
centrifuged at 10,000 g at 4 °C for 20 min. The
supernatant was then filtered through a 0.22 µM filter,
and the concentration of the samples was adjusted with buffer A so that
they were equal.
-mercaptoethanol, 5 mM EGTA, 1.0 M
NaCl) increasing from 0% B to 70% B in 21 ml. One-ml fractions were
collected, and 65 µl of each fraction were loaded onto SDS-PAGE
gels and blotted onto nitrocellulose paper. Duplicate sets of column
fractions were analyzed; one for anti-CCD analysis (total calpain
concentration) and one for anti-NTm calpain analysis (unproteolyzed
m-calpain). The relative concentrations of calpain isoforms separated
by ion exchange chromatography were measured by densitometry of
immunoblots, and mdx values were expressed relative to control values.
RNA Blot Analysis
Total RNA was prepared from hind
limb muscles using cesium trifluoroacetate, prepared according to the
manufacturer's instructions. Total RNA (25 µg) was
fractionated on 1.2% agarose gels containing 2.2 M
formaldehyde
(53) . Samples were transferred to uncharged nylon
membranes electrophoretically and cross-linked to the membrane in a UV
cross-linker (Bio-Rad) with 125 mJ. DNA probes were labeled with
P using the random priming method. Membranes were
hybridized with 2
10
cpm/ml of probe in the
pre-hybridization fluid for 15-18 h. Washing was done with 0.2
SSC, 0.1% SDS at 42 °C for 2 h with at least 6 changes of
wash buffer. Following washes, blots were exposed to Kodak
autoradiographic film for 2-6 days.
Statistical Analysis
All quantitative data were
analyzed using statistical analysis. For all experiments normalized to
controls, Mann-Whitney analysis was utilized. Results were given as
p to indicate the
level.
Assays for Calpain Concentration in Prenecrotic,
Necrotic, and Regenerative mdx Muscle
Calpain concentration was
measured in mdx mouse muscle to determine if its elevation corresponds
to a particular stage of the disease process. The antibody utilized was
generated using a synthetic polypeptide from a highly conserved domain
of calpain
(49) that is approximately 70% conserved between
m-calpain, µ-calpain, and p94; thus, this antibody recognizes all
three isoforms of the catalytic subunit of calpain. Immunoblots of
whole muscle extracts with anti-CCD revealed three bands migrating at
molecular sizes of 78/80 kDa, 84 kDa, and 94 kDa (Fig. 1). The
immunoreactive proteins at 78/80 kDa represent a mixture of the
catalytic subunits of m-calpain, both pre- and post-autoproteolysis of
the amino-terminal peptide of m-calpain, and autoproteolyzed
µ-calpain. These are not resolved under the electrophoretic
conditions used
(54) . The 84-kDa band represents the
unproteolyzed µ-calpain catalytic subunit. The 94-kDa band is
believed to represent the skeletal muscle-specific p94 since the
immunogen utilized is highly conserved between the 3 known isoforms of
calpain.
Figure 1:
Immunoblot showing
specificity of anti-conserved catalytic domain peptide (anti-CCD).
SDS-PAGE separation of high molecular mass protein standard stained
with Coomassie Blue ( lane a), whole muscle extract of C57
muscle stained with Coomassie Blue ( lane b), immunoblot with
anti-CCD on 500 ng of purified m-calpain ( lane c), and
immunoblot, with anti-CCD, of the whole muscle extract shown in
lane b ( lane d). Double arrows point to
p78/80 ( lower band) and p84 kDa ( upper band) doublet.
Arrowhead points to p94. Numbers indicate molecular
mass in kilodaltons.
Muscle extracts were prepared from mdx mice at three stages
of dystrophy: 2-week prenecrotic, 4-week peak necrotic, and 14-week
regenerated and their age-matched controls. Samples were analyzed by
immunoblotting with the anti-CCD serum. Calpain concentration was
expressed relative to myosin heavy chain concentration so that changes
in the concentration of fibrous components would not influence
measurements of calpain concentration. Previous investigators
(55) have shown that there is more connective tissue in mdx
muscle than in age-matched controls. There are also significant
increases in connective tissue concentration of mdx mice as they
mature. If data were not expressed relative to a muscle protein, the
change in concentration of muscle cells in the tissue during the
necrotic or aging processes would cause an apparent change in calpain
concentration in the tissue that would not necessarily reflect a change
in calpain concentration in the muscle cells. Therefore, data were
normalized to myosin heavy chain. Quantitation of total immunoreactive
protein at 78/80 and 84 kDa by this method revealed a 1.81-fold
increase (* p < 0.05) in the concentration of calpain in
4-week mdx muscles and a 1.59-fold increase (* p < 0.05) in
calpain in adult mdx mouse muscles relative to age-matched controls
(Fig. 2). This increase was attributable primarily to a
significant increase in the amount of p78/80 which corresponds to a
mixture of calpain isoforms. In contrast, p94 showed a significant
decrease at the 4-week time point in mdx (63% of controls, * p < 0.05) but returned to control values in the adult mdx animals
(91% of controls, * p < 0.3) (Fig. 2). Prenecrotic
(2-week) mdx muscles were also assayed to determine whether calpain was
constitutively expressed at higher levels in mdx muscle than in
controls. This analysis showed no difference in calpain concentration
between 2-week mdx or C57 muscles (Fig. 2). The increased
concentration of total calpain in 4-week and 14-week mdx mice but not
in 2-week prenecrotic mice indicates that the increase in calpain
coincides with the onset of mdx muscle pathology.
Figure 2:
Total calpain is increased in 4- week and
adult, but not 2-week mdx muscle extracts. Histogram of densitometric
measurements of calpain bands on immunoblots. Whole muscle extracts of
C57 and mdx mice were prepared by homogenization in SDS-PAGE sample
buffer. Samples were electrophoresed on 8% gels and immunoblotted with
anti-CCD. For each experiment, 3 control and 3 mdx samples were run on
the same gel and immunoblotted. Immunoreactive calpain areas were
normalized to myosin heavy chain within the same sample and expressed
relative to control values with control equal to 1. Values shown are
the mdx value expressed relative to controls and are the average of
three experiments. Error bars represent standard deviation
between experiments.
Characterization of Increased Calpain
Concentration
Whole muscle extracts from the gastrocnemius,
quadriceps, and hamstrings of 4-week- and 14-week-old mice were
separated by ion exchange chromatography and then by SDS-PAGE to
characterize further the increased 78/80-kDa and 84-kDa calpains.
Separation of the two isoforms, by ion exchange chromatography, allowed
for the quantitation of m- and µ-calpain concentrations separately.
In this procedure, µ-calpain elutes at approximately 180
mM NaCl and m-calpain at approximately 480 mM NaCl.
The fractions were then analyzed by immunoblot analysis (Fig. 3).
At the 4-week time point, only m-calpain concentration was shown to be
significantly elevated in mdx mice (1.81-fold, * p < 0.05).
At the 14-week time point, both m- and µ-calpains were elevated in
mdx (2.3-fold and 1.4-fold, respectively), although the increase in
calpain concentration relative to age-matched controls was not
significant (Fig. 3). The previously observed increase in total
calpain observed on immunoblots was therefore attributable to a
significant increase in m-calpain at 4 weeks and the combined increase
in both m- and µ-calpains at 14 weeks.
Figure 3:
The increase in total calpain is due to an
increase in m-calpain at the 4-week time point. Histogram showing
calpain concentrations obtained from immunoblots of ion
exchange-fractionated whole muscle extracts. mdx values are expressed
relative to control values with control equal to 1. Bars show
standard error between experiments.
Messenger RNA Analysis of C57 and mdx Muscles
We
then investigated whether the increase in calpain concentration
resulted from the induction of transcription of the genes for m- or
µ-calpain. mRNA concentrations were assessed using cDNA probes and
Northern blotting. The same blots were stripped and reprobed for
-actin mRNA to allow normalization of data. Analysis of total RNA
by Northern blots revealed that mRNA levels for these proteins remained
the same as control tissues at all time points in the disease process
(2-week, 4-week, and 14-week mice) (Fig. 4, 2-week not shown).
Therefore, it appears that an increased half-life of the calpain
proteins, rather than an increase in the rate of transcription, is
responsible for the increased concentration in mdx tissues.
Figure 4:
Northern analysis of mdx and C57 muscles;
calpain expression is not regulated at the transcriptional level in mdx
muscles. Twenty five micrograms of total RNA were electrophoresed on
formaldehyde gels and analyzed by cDNA probes. a, m-calpain,
3.5 kb; b, µ-calpain, 3.5 kb; c, -actin.
Lane 1, 4-week C57; lane 2, 4-week mdx; lane
3, 14-week C57; lane 4, 14-week
mdx.
Assay of Autoproteolytic Activation of µ- and
m-Calpain in mdx Dystrophy
The findings reported above show that
calpain concentration is modified during mdx dystrophy; however, the
data do not address changes in activation of the individual isoforms of
calpain in vivo. Therefore, direct analyses of the
amino-terminal propeptides of m- and µ-calpain were performed in
order to determine the extent of autoproteolysis of these enzymes. The
amino-terminal propeptide of calpain is cleaved upon calpain
activation; therefore, antibodies directed against the amino terminus
recognize only the inactive form of the enzyme. This difference between
inactive and active calpain was exploited in the following study and
utilized to determine the in vivo autoproteolytic modification
of the calpains.
[C57 pro-calpain/mdx
pro-calpain].
Figure 5:
Fractionation of calpain isoforms and
immunoblotting with anti-CCD and anti-NTmcalpain. Whole extracts of
adult mouse muscle were fractionated using ion exchange chromatography,
electrophoresed on 8% SDS-PAGE, and immunoblotted with anti-CCD or
anti-NTm calpain. Duplicate blots are shown where samples in a are overlaid with anti-CCD and samples in b with anti-NTm
calpain. Double arrowheads point to pro-µ-calpain (upper
band, 84 kDa) and autolyzed µ-calpain (lower band, 78 kDa).
Fraction numbers (11, 12 and 18, 19) off the ion exchange column are
indicated at the bottom of b. µ =
µ-calpain fractions, m = m-calpain fractions,
C57 = control fractions; MDX = mdx
fractions.
This analysis showed that the activation of
µ-calpain was the highest during peak necrosis (4-week mdx mice)
(2.9-fold increase, * p < 0.05) when compared to age-matched
controls and returned to levels near controls in regenerated muscle of
14-week mdx mice (Fig. 6). The extent of autoproteolysis of
m-calpain did not differ significantly between mdx and control muscle
at either time point.
Figure 6:
µ-Calpain has the highest
autoproteolytic activation at the stage of peak necrosis. Histogram
showing calpain activity obtained from propeptide analysis of pre- and
postautolysis isoforms of calpain. Six experiments were performed for
each time point. These data show that an increase in µ-calpain
activity occurs in the 4-week mice but returns to control levels in
adult, regenerated mice. mdx values are expressed relative to control
values with controls equal to 1. Bars show standard error
between experiments.
Calpastatin Concentration Is Not Elevated in Adult mdx
Mouse Muscle
All cells containing calpains also contain their
specific endogenous inhibitor known as calpastatin. Thus, changes
observed in the extent of autoproteolysis observed for µ-calpain
could result from changes in calpastatin concentration. To determine if
changes in calpain activation might be secondary to alterations in
calpastatin concentration, we used both Northern blotting and Western
blotting. Hind limb muscle from 4-week and adult mdx mice and
age-matched controls were subjected to immunoblot or Northern analysis
for calpastatin. No difference in calpastatin protein concentration was
observed between mdx and age-matched controls at either age
(Fig. 7). Furthermore, no difference in calpastatin mRNA
concentration was detectable in Northern blot analysis (Fig. 8).
Hence, up-regulation of calpastatin concentration is not the mechanism
by which mdx muscles regulate calpain during necrosis or regeneration.
Figure 7:
Calpastatin protein concentrations in mdx
muscles are similar to age-matched controls. Immunoblots of whole
muscle extracts from C57 and mdx hind limb muscles overlaid with
anti-calpastatin. a, immunoblot of 4-week whole muscle
extracts from mdx and C57 hind limb with anti-calpastatin. b,
immunoblot of adult whole muscle extracts from mdx and C57 hind limb
with anti-calpastatin. Anti-calpastatin recognizes a band in crude
muscle extracts at 107 kDa.
Figure 8:
Calpastatin mRNA concentrations in mdx
muscles are similar to age-matched controls. Autoradiograph of 25
µg of total RNA extracted from mdx and C57 hind limb and probed
with calpastatin cDNA probe (3.8 kb) ( a) or -actin cDNA
probe (1.7 kb) ( b). Lower stringency washes of the nylon
filter probed with calpastatin cDNA also revealed a faint band at 5.1
kb (not shown). Lane 1, 4-week C57 RNA; lane 2,
4-week mdx RNA; lane 3, 14-week C57 RNA; lane 4,
14-week mdx RNA.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.