Am J Physiol Cell Physiol 302: C228–C239, 2012.
First published October 5, 2011; doi:10.1152/ajpcell.00266.2011.
Absolute amounts and diffusibility of HSP72, HSP25, and ␣B-crystallin in
fast- and slow-twitch skeletal muscle fibers of rat
Noni T. Larkins, Robyn M. Murphy, and Graham D. Lamb
Department of Zoology, La Trobe University, Melbourne, Victoria, Australia
Submitted 29 July 2011; accepted in final form 29 September 2011
heat shock protein; inducible heat shock proteins; stress response;
skinned fiber; chaperones
HEAT SHOCK PROTEINS (HSPs) enable skeletal muscle to cope
with physiological stresses, such as glycogen depletion (6),
Ca2⫹ increases (42), heat (39), and exercise (21). HSPs play
essential roles in maintaining cellular homeostasis by acting as
molecular chaperones and as stress sensors and by conferring
direct cytoprotection. They show a high degree of homology
across species but are a diverse family of molecules, including
both constitutively expressed and stress-inducible members.
Three HSPs known to have significant roles in cellular protection and adaptation in skeletal muscle are HSP72 (the inducible
HSP70 isoform), HSP25 (murine isoform, homologous to
human HSP27), and ␣B-crystallin. These HSPs bind to and
stabilize damaged proteins and thus protect against protein
degradation (14). Specifically, HSP72 has been shown to
stabilize both the structure and function of the sarcoplasmic
reticulum Ca2⫹ pump (SERCA1a and SERCA2a) in skeletal
Address for reprint requests and other correspondence: G. D. Lamb, Dept. of
Zoology, La Trobe Univ., Melbourne, Victoria 3086, Australia (e-mail: g.
lamb@latrobe.edu.au).
C228
and cardiac muscle subjected to heat stress (8, 43). HSP25 and
␣B-crystallin on the other hand are thought to bind predominantly to cytoskeletal/myofibrillar proteins, including desmin,
titin, ␣-actinin, actin, and myosin, protecting them against
denaturation following potentially stressful insults (1, 11, 16,
19, 21, 22, 31, 36).
Data from various studies suggest that HSP72, HSP25, and
␣B-crystallin are each expressed at relatively higher levels in
type I muscle fibers than in type II muscle fibers (1, 11, 12, 18,
29). However, the actual amounts of each HSP present in
skeletal muscle are unknown; even the relative abundance of
the three HSPs is not known. Skeletal muscle fibers likely
contain numerous potential binding sites for the various HSPs,
and it is currently unclear whether the expression levels of any
of the HSPs in unstressed muscle are sufficient to protect all or
only a fraction of the relevant target molecules. It is well
known that the levels of expression of the HSPs increase to
various degrees with stress (5, 21, 29, 31, 33), but to quantitatively interpret the relevance of such increases and their
implications for cellular function it is important to know how
much of the given HSP is initially present and how much the
increase actually represents.
Most previous studies have suggested that, in unstressed
muscle fibers, the majority of each of the three HSPs is located in the cytosol (2, 11, 16, 21, 32). This has been concluded
largely from centrifugation of muscle tissue into “soluble” and
“insoluble” fractions, with the cytosolic constituents thought to
partition predominantly into the soluble fraction. However,
there is some uncertainty as to whether the cytosolic and
noncytosolic constituents are completely separated in this way,
particularly since cytosolic components might become bound,
and noncytosolic components unbound, during the homogenization and centrifugation procedures. Additionally, to permit
truly quantitative interpretation of the data, no fraction at all
should be discarded during the procedures, and the proportion
of HSP present in a given fraction should be quantified by
directly comparing the HSP content across all of the fractions
in absolute terms, rather than by normalizing the HSP content
of a fraction by its protein content, a measure that differs for
each individual fraction.
In this study, we performed quantitative Western blotting on
entire constituents of muscle samples to determine the absolute
amounts of HSP25, HSP72, and ␣B-crystallin present in muscles composed primarily of either type I or type II fibers, and
also that present in individual muscle fibers of known type. By
peeling off the sarcolemma from a muscle fiber, it was also
possible to determine the proportion of HSP readily diffusible
in the cytoplasm and whether any of the HSP associated with
the sarcolemma. It was hypothesized that 1) the absolute
amounts of the various HSPs differ considerably in unstressed
muscle, possibly in inverse relation to their relative increases
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Larkins NT, Murphy RM, Lamb GD. Absolute amounts and
diffusibility of HSP72, HSP25, and ␣B-crystallin in fast- and
slow-twitch skeletal muscle fibers of rat. Am J Physiol Cell Physiol
302: C228 –C239, 2012. First published October 5, 2011;
doi:10.1152/ajpcell.00266.2011.—Heat shock proteins (HSPs) are
essential for normal cellular stress responses. Absolute amounts of
HSP72, HSP25, and ␣B-crystallin in rat extensor digitorum longus
(EDL) and soleus (SOL) muscle were ascertained by quantitative
Western blotting to better understand their respective capabilities and
limitations. HSP72 content of EDL and SOL muscle was only ⬃1.1
and 4.6 ␮mol/kg wet wt, respectively, and HSP25 content approximately twofold greater (⬃3.4 and ⬃8.9 ␮mol/kg, respectively). ␣Bcrystallin content of EDL muscle was ⬃4.9 ␮mol/kg but in SOL
muscle was ⬃30-fold higher (⬃140 ␮mol/kg). To examine fiber
heterogeneity, HSP content was also assessed in individual fiber
segments; every EDL type II fiber had less of each HSP than any SOL
type I fiber, whereas the two SOL type II fibers examined were
indistinguishable from the EDL type II fibers. Sarcolemma removal
(fiber skinning) demonstrated that 10 –20% of HSP25 and ␣Bcrystallin was sarcolemma-associated in SOL fibers. HSP diffusibility
was assessed from the extent and rate of diffusion out of skinned fiber
segments. In unstressed SOL fibers, 70 –90% of each HSP was readily
diffusible, whereas ⬃95% remained tightly bound in fibers from SOL
muscles heated to 45°C. Membrane disruption with Triton X-100
allowed dispersion of HSP72 and sarco(endo)plasmic reticulum
Ca2⫹-ATPase pumps but did not alter binding of HSP25 or ␣Bcrystallin. The amount of HSP72 in unstressed EDL muscle is much
less than the number of its putative binding sites, whereas SOL type
I fibers contain large amounts of ␣B-crystallin, suggesting its importance in normal cellular function without upregulation.
HEAT SHOCK PROTEINS IN UNSTRESSED SKELETAL MUSCLE FIBERS
following stress, and also differ between fiber types, 2) each
HSP is present at much lower density than its potential binding
sites, 3) the majority of each HSP is readily diffusible within
unstressed fibers and hence able to reach and bind to numerous
possible targets as needed, and 4) heat stress causes tight
binding of all three HSP types, but differentially so to membranous and cytoskeletal targets.
MATERIALS AND METHODS
min). In a subset of experiments, fiber segments were immersed in
buffer A with 1% Triton X-100 for 10 min. Most fibers were vortexed
briefly (⬃2 s) at least one time during the exposure time, in particular
just before removing the fiber from the bathing solution. It was
subsequently found that there was no detectable difference in results
between fibers that were or were not vortexed. After the required time,
the fiber was removed and placed in another microcentrifuge tube
containing the same volume of buffer A, and 2.5 ␮l of 3⫻ SDS
loading buffer was then added to both tubes, thus obtaining a matched
set with the fiber segment and corresponding wash solution in separate
tubes [as previously described (24, 28)].
Western blotting for protein diffusibility and absolute quantification. As
described previously (20), protein samples were loaded and separated
on 12% SDS polyacrylamide gels, or in some cases on Criterion
Stainfree gels (26). Proteins were then transferred onto nitrocellulose
membrane and blocked with blocking buffer (5% skim milk in
Tris-buffered saline with Tween 20) for 2 h. Following blocking,
primary antibodies were applied for 2 h [room temperature (RT)] and
overnight (4°C) on a rocker. Membranes were cut into two sections at
a molecular mass position of ⬃40 kDa, and the sections were probed
as required for ␣B-crystallin, HSP25, HSP72, MHCI, MHCII,
SERCA1, SERCA2, and actin, diluted in 1% BSA PBS with 0.025%
Tween (23). Following washes, secondary antibodies were applied for
1 h (RT), either goat anti-mouse horseradish peroxidase (HRP)conjugated (Pierce) or goat anti-rabbit HRP-conjugated (Pierce) (both
diluted 1: 20,000 in blocking buffer). Following transfer, BioSafe
Coomassie Stain (Bio-Rad) was used to stain the SDS-polyacrylamide
gel for the detection of MHC, which was used as an indicator for
the presence of myofibrils and also of the relative amounts of sample
loaded. Images of the membrane were collected following exposure to
chemiluminscence substrate (Thermo Scientific SuperSignal West
Femto; Pierce) using a charge-coupled device camera attached to
ChemiDoc XRS (Bio-Rad), and Quantity One software was used for
detection as well as densitometry. The relative positions of molecular
mass markers were captured under white light prior to chemiluminescent imaging without moving the membrane. For the examination of
protein diffusibility, the individual fiber segments and their corresponding wash solution were run side by side and analyzed for the
relevant proteins by Western blotting (24, 28).
For analyses of the absolute amount of specific proteins, whole
muscle homogenates (2.5–50 ␮g total muscle mass) were loaded onto
a gel together with known amounts of purified recombinant proteins
(mouse HSP25, rat HSP72, and bovine ␣B-crystallin), the latter
allowing a calibration curve to be generated (see Fig. 1). When
quantifying absolute amounts of ␣B-crystallin, HSP25, or HSP72, the
density of the relevant band was converted to an equivalent protein
amount, according to the calibration curve derived from the pure
protein samples run on the same Western blot. This amount was
expressed relative to the mass of muscle loaded in that lane, and the
average was calculated for all repeated samples run on the same gel.
The validity of this Western blot quantification procedure was verified
as described in RESULTS (see Fig. 2).
Statistics. Data are expressed as means ⫾ SE, with the number of
samples studied denoted as n, being muscles or individual fibers as
indicated. Statistical significance was examined using Student’s t-tests
(paired or unpaired as appropriate), or, in cases where data were not
normally distributed (e.g., data in Figs. 3 and 9), significance was
tested using the nonparametric Mann Whitney unpaired rank test; a
probability value (P) ⬍0.05 was deemed as significant. All statistical analyses and data fits were performed using GraphPad Prism version 4.
RESULTS
Measurement of absolute amounts of HSPs in rat muscle.
Absolute amounts of ␣B-crystallin, HSP25, and HSP72 present
in rat EDL and SOL muscle were each assessed using the
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Materials and antibodies. All chemicals were obtained from Sigma
(St. Louis, MO) unless otherwise stated. Antibodies used were against
HSP25 (1:2,000 rabbit polyclonal, SPA-801; Stressgen), HSP72 (1:
500 mouse monoclonal, SMC100A; Stressmarq), ␣B-crystallin (1:
1,000 mouse monoclonal, SPA-222; Stressgen), actin (1:300 rabbit
polyclonal, A2066; Sigma), myosin heavy chain (MHC) I (1:200
mouse monoclonal, A4.840), MHCII (1:200 mouse monoclonal,
A4.74), SERCA1 (1:1,000 mouse monoclonal, Ca F2–5D2; from
Developmental Studies Hybridoma Bank), and SERCA2a (1:5,000,
gift from Dr. F Wuytack, Leuven, Belgium). Purified proteins used
were HSP25 (mouse recombinant, ADI-SPP-510; Stressgen), HSP72
(rat recombinant, ADI-SPP-758; Stressgen), and ␣B-crystallin (bovine native, ADI-SPP-226; Stressgen).
Tissue preparation. With approval of the La Trobe University
Animal Ethics Committee, male Long-Evans hooded rats (⬃6 –10 mo
old) or C57/BL10 mice (⬃5–7 mo) were killed by overdose with
inspired isoflurane (4% vol/vol). The extensor digitorum longus
(EDL) and soleus (SOL) muscles were quickly excised. Bovine
muscle, supplied through a butcher, was obtained from the upper and
lower hind limb of an animal ⬃24 h postdeath and carcass storage at
4°C. To verify that the level of ␣B-crystallin was not greatly altered
by the postmortem storage, additional experiments were carried out
comparing SOL muscle excised from a rat carcass that had been
stored at 4°C for 24 h after death; there was no significant difference
in the amount of ␣B-crystallin found in muscle excised immediately
after death and muscle excised 24 h after death and 4°C storage (ratio:
1.1 ⫾ 0.2, n ⫽ 3).
Muscle homogenates were prepared (1:10 wt/vol) in a physiological-like solution with free Ca2⫹ buffered at very low levels with
EGTA: buffer A (in mM): 126 K⫹, 36 Na⫹, 1 free Mg2⫹ (10.3 total
Mg), 90 HEPES, 8 ATP, 8 creatine phosphate, and 50 EGTA (pH
7.1), with protease inhibitor cocktail (COMplete, Roche Diagnostics,
Sydney, NSW, Australia). Muscle homogenates were diluted further
1:20 (vol/vol) using the same solution and then 2:1 (vol/vol) with SDS
loading buffer (0.125 M Tris·HCl, 10% glycerol, 4% SDS, 4 M urea,
10% mercaptoethanol, and 0.001% bromphenol blue, pH 6.8). Samples were stored in ⫺20°C until analyzed by Western blotting.
Single fiber experiments. For single fiber experiments, rat muscles
were pinned immediately after excision at resting length under paraffin oil and then either slowly cooled and maintained at ⬃10°C on an
ice pack (unstressed, control muscle) or first heated for 30 min to 45°C
and then slowly cooled and maintained at ⬃10°C (heated muscle).
Fibers from each heated muscle were collected over the same period
as fibers from the unheated contralateral muscle (control) from the
same rat. Single fibers were separated by dissection, and segments ⬃3
mm in length were collected for Western blotting or first “skinned” by
microdissection to remove the sarcolemma (i.e., surface membrane).
The latter was achieved by pulling a small number of myofibrils away
from the rest of the fiber, causing the sarcolemma to roll back along
the fiber (27). Only one segment was examined from any given fiber
to avoid sampling bias. A small piece of suture thread was tied around
each fiber segment to allow ready transfer between solutions as
required. To examine the diffusibility of ␣B-crystallin, HSP25, and
HSP72, single mechanically skinned fiber segments (each ⬃10 –15 nl
in volume) were immersed in 5 ␮l of buffer A in a microcentrifuge
tube for a specified time (30 s, 2 min, 10 min, 30 min, 60 min, or 120
C229
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HEAT SHOCK PROTEINS IN UNSTRESSED SKELETAL MUSCLE FIBERS
methodology shown in Fig. 1. Very small amounts of unfractionated muscle homogenate were run on a given gel together
with a range of amounts of the relevant purified HSP. A
calibration curve was generated for each gel by plotting the
density of the Western blot band for each purified HSP sample
vs. the amount of HSP loaded (e.g., Fig. 1B), and this was used
to assess the amount of HSP present in homogenate samples
run on the same gel. The entire muscle homogenate was run
without any spinning or removal of cell debris to ensure that
there was no omission of any muscle proteins. The relative
amount loaded was also verified for each muscle type by
reprobing the membrane for actin and also by the MHC signal
after Coomassie staining of the SDS gel posttransfer. Such
analysis also suggested that rat SOL muscle likely has ⬃10%
less MHC and actin per unit muscle wet mass than does rat
EDL muscle (mean: 90 ⫾ 6%, n ⫽ 8; 90 ⫾ 12%, n ⫽ 3,
respectively), as expected given the greater mitochondrial
content per unit volume in SOL muscle.
This HSP quantification method is only valid if the HSP
present in the muscle homogenate is detected with the same
efficacy as the pure HSP run alone as the standard. This was
tested by comparing band intensities in lanes containing just
purified HSP, or just muscle homogenate, or purified HSP
mixed together with another sample of the same homogenate
Fig. 2. Verification of HSP72 quantification. A: Western blot of lanes containing purified HSP72 alone (6.6 ng, lane 2; 11 ng, lane 5; and 20 ng, lane 6),
SOL muscle homogenate alone (lanes 1 and 4), or the same amounts of
homogenate (15 or 33 ␮g) mixed with 6.6 or 11 ng of HSP72 (lanes 3 and 7,
respectively). Coomassie blue-stained gel showing MHC and membrane reprobe for actin both verified that similar amounts of muscle homogenate were
present in corresponding lanes (lanes 1 and 3, 4, and 7). B: density of band for
purified HSP72 plotted against the amount of protein. Band densities indicate
that HSP72 in the mixture of muscle homogenate and purified HSP72 was
transferred, detected, and quantified with similar efficacy as purified HSP72
run by itself (see RESULTS).
(Fig. 2). Such data were obtained on three to five independent
gels for each HSP. For all three HSPs, the amount of HSP
gauged as present in the muscle homogenate with added HSP
was virtually identical to the sum of that found when running
the homogenate and pure HSP samples separately (ratio: 1.0 ⫾
0.1 for HSP72, n ⫽ 3; 1.0 ⫾ 0.1 for HSP25, n ⫽ 5; and 1.1 ⫾
0.1 for ␣B-crystallin, n ⫽ 3), verifying the reliability of the
quantification method.
Table 1 lists the absolute amounts of the three HSPs present
in rat EDL and SOL muscles in the unstressed state, deemed as
such because the animals were healthy and had not been
subjected to any heat or exercise stress. Of note, in EDL
Table 1. Absolute amount of HSPs in rat EDL and SOL
muscle
Rat EDL
n
Rat SOL
n
HSP72
HSP25
␣B-Crystallin
1.1 ⫾ 0.2
5
4.6 ⫾ 1.0
7
3.4 ⫾ 0.5
5
8.9 ⫾ 0.3
5
4.9 ⫾ 1.4
5
139 ⫾ 11
7
Values are mean ⫾ SE amount of heat shock proteins (HSPs) (in ␮mol/kg
wet mass) determined from Western blotting of entire constituents of muscle
homogenates, as in Figs. 1 and 2; n indicates the no. of independent muscles
examined, each obtained from a different rat. EDL, extensor digitorum longus;
SOL, soleus.
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Fig. 1. Quantification of heat shock protein (HSP) 25 in rat extensor digitorum
longus (EDL) and soleus (SOL) muscle. A: protein from rat EDL and SOL total
muscle homogenates (17 ␮g muscle wet wt) and 1– 8 ng purified HSP25,
detected on the same Western blot. Relative loading amounts were verified by
membrane reprobe with actin and also by staining gel posttransfer with
Coomassie blue to show myosin heavy chain (MHC). B: calibration curve
derived by plotting band density for each purified HSP25 sample (see A)
against the amount of protein. HSP25 content of EDL and SOL homogenate
samples (⬃1.9 and 4.2 ng in 17 ␮g total muscle wet wt, respectively) derived
using the calibration curve to convert the average band intensity for the
duplicate homogenate samples into the amount of HSP25 present. AU, arbitrary units.
HEAT SHOCK PROTEINS IN UNSTRESSED SKELETAL MUSCLE FIBERS
and SOL type I fibers. Rat SOL muscle of this rat strain
typically is composed of ⬃80% type I fibers and ⬃20% type II
fibers (40), and, of the 22 SOL fibers examined here, 20 were
assessed as being type I and the other 2 as type II. In both of
these SOL type II fibers, each of the three HSPs was present at
lower levels than in the SOL type I fibers, levels indistinguishable from that present in the type II fibers from EDL muscle
(Fig. 3B).
Proportion of HSPs associated with sarcolemma. Immunostaining of muscle fibers has indicated that some HSPs appear
to be localized at relatively high levels at or just beneath the
sarcolemma (surface membrane) (11). To determine whether
particular HSPs are actually directly associated with the sarcolemma and related structures, segments of individual SOL
and EDL muscle fibers were skinned by microdissection to
completely remove the sarcolemma, and the amount of the
given HSP present in the skinned segments was compared with
that in “intact” segments where the sarcolemma was still
associated. In both EDL and SOL fibers, the amount of ␣Bcrystallin found in the skinned segments was significantly
lower than in the intact segments (see Table 2), indicating that
some ␣B-crystallin had been closely associated in some way
with the sarcolemma and removed by the skinning. This
amount was only ⬃10% of the total in SOL fibers, but
proportionately much more (⬃60%) in EDL fibers, consistent
with a substantial amount of ␣B-crystallin being associated
with the sarcolemma in both types of muscle fiber and the total
present being larger in SOL fibers (Table 1 and Fig. 3B).
Skinned segments from SOL fibers also contained ⬃20% less
HSP25 than comparable intact segments (Table 2). In EDL
fibers, however, the difference in HSP25 amount (⬃12%) was
not statistically significant. It was nevertheless apparent that
some HSP25 is associated with the sarcolemma in at least some
EDL fibers, because in a few cases the sarcolemma excised
from a segment of fiber was analyzed by Western blotting
alongside the skinned segment, and, in one of the EDL fiber
cases shown in Fig. 4, a substantial amount of HSP25 was
found to be present with the sarcolemma. In the case of HSP72,
the mean amount present in skinned fiber segments was not
significantly different from that in intact segments for either
EDL or SOL fibers (Table 2); no attempt was made to directly
measure whether HSP72 was associated with the sarcolemma
owing to the low absolute amounts involved and detection
limitations of the Western blotting.
Diffusibility of HSPs in unstressed muscle fibers. To gauge
how much of a given HSP is freely diffusible in the cytoplasm
in unstressed muscle fibers, segments of individual fibers from
rat SOL and EDL muscles were skinned by microdissection
under paraffin oil and then transferred for a set period into a
comparatively large volume of an aqueous solution that
broadly mimicked the normal cytosol (see MATERIALS AND
METHODS). Figure 5 displays representative Western blots of the
proteins remaining within the fiber and those that diffused out
into the wash solution; note that there was no detectable loss of
either actin or myosin to the wash solution. Each of the fiber
and wash samples was run in its entirety without any spinning
or fractionation. For each HSP, the total of that found in the
wash solution and fiber lanes together was not noticeably
different from that seen when running either unskinned fiber
segments or skinned segments taken straight from paraffin oil
without any washing. (Note all such comparisons of HSP
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muscle, there was only ⬃1.1 ␮mol HSP72/kg wet muscle
mass, whereas SOL muscle contained approximately fourfold
more HSP72 (⬃4.6 ␮mol/kg). In both muscle types, there was
approximately two to three times more HSP25 than HSP72
(⬃3.4 and 8.9 ␮mol/kg in EDL and SOL, respectively). Perhaps the most surprising finding was the very large absolute
amount of ␣B-crystallin present in rat SOL muscle, ⬃139
␮mol/kg, with EDL muscle containing only ⬃4.9 ␮mol/kg.
Thus, in SOL muscle, the amount of ␣B-crystallin is ⬃16
times greater than the amount of HSP25 and ⬃30 times greater
than the amount HSP72.
HSP measurements in murine and bovine muscle. The pure
proteins used to quantify HSP25 and ␣B-crystallin were murine and bovine proteins, respectively. To validate their use as
standards for quantifying the HSP content of rat muscle, and
additionally to enable comparison between different species,
these same pure proteins were also used to assess the HSP
content of murine and bovine muscle. The absolute amounts of
HSP25 in mouse EDL and SOL muscles were determined,
respectively, to be 3.5 ⫾ 0.7 and 11.1 ⫾ 8 ␮mol/kg muscle
mass (n ⫽ 4 mice), which were similar to the amounts
determined in the corresponding rat muscles (Table 1). The
amount of ␣B-crystallin in bovine muscle was 132 ⫾ 17
␮mol/kg in the three muscle samples examined from the distal
hind limb of one animal and 69 ⫾ 17 ␮mol/kg (n ⫽ 3) for
samples from the proximal hind limb. The density of MHCI
was approximately two times greater in the distal hind limb
than in the proximal hind limb (2.3 ⫾ 0.2-fold, n ⫽ 3),
indicative of a higher proportion of type I fibers in the distal
muscles. These data suggest that the ␣B-crystallin content of
bovine type I fibers is quantitatively similar to that in the
predominantly type I SOL muscle of the rat.
Relative amounts of HSPs in individual fibers. The above
HSP measurements were made on muscle homogenates that
consisted of a range of different fiber types. It was thus
important to also consider whether individual SOL and EDL
fibers showed the same disparity as the homogenate data
because the total HSP in a given muscle might be determined
predominantly by higher HSP levels in some subset of fibers in
that muscle. To investigate this, single segments of individual
muscle fibers were obtained by microdissection and analyzed
in their entirety by the same Western blotting procedure used
for the muscle homogenate measurements. Because it was not
possible to accurately measure the weight of the individual
fiber segments (each ⬃3 mm long and likely ⬃10 to 15 ␮g wet
wt), the Coomassie MHC band density was taken as a measure
of the relative sample mass and used to normalize the corresponding HSP band density for each fiber sample run on a
given gel. This normalized HSP amount for each fiber was then
expressed relative to the mean of that for all SOL type I fibers
run on the same gel (mostly 4 to 6); in effect, this defined the
HSP amount in an “average” SOL type I fiber as unity and
indicated how the HSP amount in a given fiber compared with
that level (see Fig. 3). SOL fiber segments were denoted as
being either type I (slow-twitch) or type II (fast-twitch) based
on the MHC type present (as assessed by Western blotting of
the same membranes). As seen in Fig. 3B, the EDL fibers,
which were all type II fibers, formed a relatively discrete
population with much less of each HSP than present in the SOL
type I fibers (P ⬍ 0.05 in all cases); in fact, there was no
overlap in the amount of HSP present in the EDL type II fibers
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HEAT SHOCK PROTEINS IN UNSTRESSED SKELETAL MUSCLE FIBERS
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Fig. 3. Relative amounts of HSPs in individual SOL and
EDL fibers. A: Western blots of multiple proteins detected
in same fiber segment; examples of type II EDL fibers and
of type I and type II SOL fibers (all SOL fiber segments run
on same gel). B: comparison of relative amount of HSPs in
individual fiber segments. For each fiber segment, relevant
HSP band intensity was first normalized by the density of
Coomassie stain of MHC for that sample, and the resulting
value was expressed relative to the mean for all SOL type
I fibers run on the same gel. Compilation of data from 5
independent gels. Filled symbols denote two SOL type II
fibers, distinguished both by the presence of MHCII and a
relatively high density of sarco(endo)plasmic reticulum
Ca2⫹-ATPase (SERCA) 1 pumps; these same two fiber
segments were examined for all three HSPs. Horizontal bars
indicate mean values for SOL type I and EDL fibers.
amounts between different fiber samples took into account the
relative mass of the fiber sample, based on the associated MHC
signal.) Figure 6 plots the extent and rate of diffusional loss of
each of the HSPs in SOL and EDL fibers. Most of the HSP72
diffused out into the wash solution within 10 min, but ⬃5–15%
still remained within the fiber even after 2 h (Fig. 6A). With
HSP25, most washed out of the fiber within several minutes,
but there was a nondiffusing component of ⬃24% in SOL
fibers and ⬃50% in EDL fibers (Fig. 6B). Similar to HSP25,
most ␣B-crystallin was readily diffusible in SOL fibers, with a
maximum washout of ⬃89% attained within ⬃2 min (Fig. 6C),
whereas, in EDL fibers, the washout was only ⬃30%. Interestingly, comparison of the rates of diffusional loss of the three
HSP in SOL fibers (where values are probably more accurately
determined than in EDL fibers because of the larger absolute
amounts of HSP involved) showed that HSP72 diffused much
more slowly out of fibers (time constant ⬃2.2 min) than did the
HSP25 and ␣B-crystallin (time constants ⬃0.25 and 0.34 min,
respectively) (see Fig. 6). This difference was not explained
simply by the relative mass of the HSP proteins (see DISCUSSION).
The above diffusional data were obtained from fibers that
had been skinned at various different times after removing the
muscle from the rat (up to ⬃150 min). To ascertain whether
this time period in vitro had any influence on HSP diffusibility,
the percentage HSP washout value found in each fiber washed
for 10 min or more was plotted against the time delay before
skinning the fiber (data not shown). Linear regression analysis
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HEAT SHOCK PROTEINS IN UNSTRESSED SKELETAL MUSCLE FIBERS
Table 2. Relative amounts of HSPs in skinned vs. intact fiber segments
␣B-Crystallin
HSP25
Rat EDL
n
Rat SOL
n
HSP72
Intact
Skinned
Intact
Skinned
Intact
Skinned
100 ⫾ 7
6
100 ⫾ 4
6
88 ⫾ 8
6
81 ⫾ 6*
6
100 ⫾ 7
3
100 ⫾ 7
3
39 ⫾ 9*
3
91 ⫾ 3*
3
100 ⫾ 13
3
100 ⫾ 16
6
95 ⫾ 18
3
89 ⫾ 9
6
Shown is the amount of indicated HSP (normalized to MHC) in skinned fiber segments, expressed as a percentage of that in intact fiber segments quantified
on the same gel (minimum of 3). *Significantly less than intact fibers.
Fig. 4. HSP25 associated with sarcolemma in some EDL fibers. Three
examples in which an ⬃2- to 3-mm section of an EDL fiber was isolated by
microdissection and the sarcolemma rolled back and removed. The resulting
skinned fiber segment (F) and excised sarcolemma (SL) were run in adjacent
lanes on SDS-PAGE, and Stainfree images were obtained (bottom) before
Western blotting for HSP25 and laminin (top). Fiber 1 was run on Criterion
10% SDS gel; fibers 2 and 3 were run on Criterion 4 to 12% SDS gradient gel.
Note that SL samples have no MHC and little or no actin. In fiber 1, ⬃30% of
the total HSP25 was found with the sarcolemma, whereas, in fibers 2 and 3,
there was little or none associated. The presence of laminin with the SL of
fibers 1 and 2 indicates that, in those cases, some basal lamina had remained
with the fiber when it was dissected free from its neighbors, and it was then
peeled back and removed with the sarcolemma.
isoform could be detected not only in SOL type II fibers but
even in a proportion of type I SOL fibers, although at comparatively low levels. (In some experiments, membranes were
probed only for SERCA1, but, in later experiments, they were
probed for both SERCA1 and SERCA2.) As seen in the
example in Fig. 7, left, after 10 min exposure to the standard
wash solution without Triton X-100, all of the SERCA1 still
remained within the fiber. Neither SERCA1 nor SERCA2 was
ever seen to partition into the wash solution in any fiber in the
absence of detergent. In contrast, when Triton X-100 was
present, 80 ⫾ 5% of SERCA1 was lost to the wash solution
after 10 min in the three SOL fibers examined here with
detectable levels of SERCA1, consistent with the expected
effect of the detergent on membrane-bound proteins. This
Triton X-100 treatment did not, however, detectably increase
the extent of diffusional loss of any of the HSPs in those fibers
nor in the three other SOL fibers examined; the percentage
remaining after 10 min was 9 ⫾ 3% (n ⫽ 6) for HSP72, 16 ⫾
4% (n ⫽ 6) for HSP25, and 15 ⫾ 4% (n ⫽ 6) for ␣B-crystallin,
values that do not differ significantly from the respective
percentage remaining after the same wash time without detergent (HSP72, P ⫽ 0.6; HSP25, P ⫽ 0.8; and ␣B-crystallin,
Fig. 5. Majority of each HSP is freely diffusible in unstressed SOL muscle
fibers. Representative Western blots of HSP72, HSP25, and ␣B-crystallin
remaining within the fiber (F) or lost to the surrounding “wash solution” (W)
after bathing individual mechanically skinned SOL fiber segments in physiological intracellular solution for the indicated time. Each fiber segment was run
with its corresponding wash solution in adjacent lane. Coomassie-stained
MHC and actin reprobe indicate the relative amount of tissue constituting the
given muscle fiber segment, and the absence of such MHC and actin signals in
the W lanes shows there was little, if any, myofibril contamination in the wash
solutions.
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showed no significant relationship in any case except for
␣B-crystallin washout in SOL fibers, which displayed somewhat reduced washout if the time before skinning the fiber
exceeded 80 min. Consequently, for the latter case, the HSP
washout data presented here were restricted to fibers examined
⬍80 min after muscle removal.
To further investigate the binding properties of the HSPs in
unstressed fibers, we examined the effects of supplementing
the wash solution with 1% (vol/vol) of the detergent, Triton
X-100. Membrane-bound proteins become diffusible in the
presence of the detergent, whereas those bound to sarcomeric
structural proteins evidently remain within the fiber (24). The
membrane-bound protein examined was the sarcoplasmic reticulum (SR) calcium pump protein, SERCA, which is a known
binding site for HSP72 and situated in the SR membranes
around each myofibril throughout a muscle fiber. The SERCA1
C234
HEAT SHOCK PROTEINS IN UNSTRESSED SKELETAL MUSCLE FIBERS
Fig. 6. Time course of diffusional loss of HSPs in unstressed SOL or EDL
skinned fibers. Mean percentage (⫾ SE) of HSP72 (A), HSP25 (B), and
␣B-crystallin (C) diffusing out of a skinned unstressed muscle fiber in
indicated time. Data obtained as in Fig. 5, expressing the amount of given HSP
found in wash solution (W) as a percentage of the total present in the given
fiber (F) and wash (W) pair. No. of fibers examined is shown with each data
point. Each data set was fit with a one-phase exponential function, y ⫽
M/100(1-e⫺t/␶). Most HSP72 was able to diffuse out in both EDL fibers
(⬃84%) and SOL fibers (⬃95%). HSP25 rapidly diffused out of SOL fibers,
although the maximum washout was only ⬃76% and, in EDL fibers, was only
⬃44%. ␣B-crystallin showed maximum washout of ⬃89% in SOL fibers but
only of ⬃30% in EDL fibers.
P ⫽ 0.4). Thus the great majority of all three HSPs are readily
diffusible in the cytoplasm of SOL fibers from unstressed
muscles, and the small proportion of each remaining within the
fibers is not detectably altered by disrupting and dispersing at
least some of the internal membranes.
Diffusibility of HSPs in fibers from heat-stressed muscle.
Finally, HSP amounts and diffusion were examined in fibers
from heat-stressed muscles. Rat SOL and EDL muscles were
heated in vitro to 45°C for 30 min, and the fibers were
compared with those from the nonheated contralateral muscles.
This heat treatment caused no acute change in the amounts of
the HSPs; the amounts measured in homogenates from three
Fig. 7. Nondiffusible HSP pools in unstressed fibers are unaffected by treatment with Triton X-100. Western blots showing HSP72, HSP25, and ␣Bcrystallin remaining in fiber (F) or lost to surrounding wash solution (W) when
individual skinned SOL fiber segments were bathed for 10 min in physiological solution with or without 1% Triton X-100. SOL fibers shown had
detectable levels of SERCA1, albeit much less than in EDL fibers. When
Triton X-100 was present, most of the SERCA1 was lost to the wash solution,
but the proportion of nondiffusing HSPs was not noticeably altered (see text),
and no MHC or actin could be found in the wash solution.
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heated SOL muscles expressed relative to that in the corresponding three contralateral muscles were 1.0 ⫾ 0.1 for
HSP25, 0.9 ⫾ 0.1 for ␣B-crystallin, and 0.9 ⫾ 0.1 for HSP72
(no significant change in any case).
Individual fibers from heated and nonheated muscles were
skinned and bathed for 10 min in physiological intracellular
solution as described earlier, and the fiber and wash solution
samples were run in adjacent lanes, as seen in Fig. 8A. The
HSPs in the fibers from the unheated control muscles were
largely lost to the wash solution, consistent with the results in
the previous section, whereas, in the fibers from the heated
muscles, all three types of HSPs were found to remain almost
entirely within the fiber (e.g., Fig. 8A, wash solution and fiber
data on right), even though aldolase, a 40-kDa cytoplasmic
protein, still readily diffused out of the fiber in every case
examined. The mean data for the three HSPs are shown in
Fig. 9. The HSPs all still remained in the fibers even if the wash
time was increased to 30 min (data not shown). Very similar
tight binding was also seen in all the fibers examined from
heated EDL muscles (data not shown, only HSP25 and HSP72
were examined). Consistent with the homogenate data reported
above, there was no significant difference in any case between
the HSP amounts in the fibers obtained from the control and the
heated SOL muscles; the relative amounts for the fibers from
the control and the heated muscles were 1.0 ⫾ 0.1 (n ⫽ 16) and
1.0 ⫾ 0.1 (n ⫽ 12), respectively, for HSP25, 1.0 ⫾ 0.1 (n ⫽
16) and 1.0 ⫾ 0.1 (n ⫽ 14) for ␣B-crystallin, and 1.0 ⫾ 0.1
(n ⫽ 10) and 1.1 ⫾ 0.1 (n ⫽ 15) for HSP72. (These values
were calculated from the sum of the HSP in each individual
HEAT SHOCK PROTEINS IN UNSTRESSED SKELETAL MUSCLE FIBERS
C235
fiber and wash set, each normalized by respective MHC
amount, expressing all values relative to the mean for the
control fibers run on the same gel.)
Importantly, when 1% Triton X-100 was present in the wash
solution used to bathe SOL fibers from the heated muscles
(e.g., Fig. 8B), 52 ⫾ 12% of the HSP72 and 70 ⫾ 7% of the
SERCA were lost to the wash solution within 10 min in the six
fibers examined, whereas virtually all of both the HSP25 and
the ␣B-crystallin still remained in the fibers (mean data shown
in Fig. 9).
DISCUSSION
The absolute amounts of the three major HSPs found here in
unstressed skeletal muscle provide novel insights into their
relative capabilities and limitations. The absolute amounts
(Table 1) were in the order HSP72 ⬍ HSP27 ⬍ ␣B-crystallin
in both EDL and SOL muscle, which are composed primarily
of type II (fast-twitch) and type I (slow-twitch) fibers, respectively. In EDL muscle, the amount of ␣B-crystallin (⬃4.9
␮mol/kg muscle mass) was approximately fourfold greater
than the amount of HSP72 but only slightly more than the
HSP25. The most striking finding was that SOL muscle contained a very large amount of ␣B-crystallin (⬃140 ␮mol/kg
muscle mass), almost 30-fold higher than in EDL muscle,
whereas the amount of HSP25 was only 2.5-fold higher in SOL
fibers compared with EDL fibers (⬃8.9 vs. 3.4 ␮mol/kg). The
large amount of ␣B-crystallin found in the SOL muscle was
not a peculiarity of rat, because bovine hind limb muscles were
also found to contain very high amounts (⬃130 and 70
␮mol/kg in distal and proximal muscles). Like rat SOL muscle,
these bovine muscles would be expected to have relatively high
densities of type I fibers, and the relative amounts of ␣Bcrystallin found in the distal and proximal muscles approximately matched the relative density of MHCI in those muscles.
The relative amounts of the HSPs found here in rat SOL
muscle compared with EDL muscle (Table 1) are in good
accord with previous studies in rat and mouse, where it was
found that SOL muscle had ⬃3-fold more HSP25 (35), ⬃6fold more HSP72 (12), and ⬃15- to 40-fold more ␣B-crystallin
(1, 11, 13, 35) than did EDL or tibialis anterior muscles (which
both consist predominantly of type II fibers). The absolute
amounts of the HSPs present in the muscles, however, are not
apparent from those earlier studies because HSP content was
typically examined only in enriched fractions rather than in
total homogenates, and the amounts of the HSPs were reported
relative to protein contents, which could not be related back to
muscle amounts. One early report (9), however, did determine
the ␣B-crystallin content of rat total heart homogenate as being
⬃0.6% of total cellular protein, and, if the latter is assumed to
be ⬃230 g/kg muscle mass, this indicates that the ␣B-crystallin
content of cardiac muscle is ⬃70 ␮mol/kg. Furthermore, the
same group later found (11) that the ␣B-crystallin content in
total homogenates of rat SOL muscle was very similar to that
of rat cardiac muscle, consistent with the very high absolute
levels of ␣B-crystallin found here.
Using our recently described methodology for quantitative
Western blotting of single fiber segments (20, 25, 28), the
present study also provided the first quantitative assessment of
the HSP levels present in individual muscle fibers. This single
fiber analysis demonstrated that the type I fibers in rat SOL
muscle are a broadly homogenous group, in all cases having
greater levels of each of the HSPs than found in any of the EDL
fibers (which were all type II fibers) (Fig. 3). Interestingly, both
of the two type II fibers from SOL muscle examined in this
study had lower levels of all three HSPs than was present in
any SOL type I fiber, with the amounts being indistinguishable
from those present in the EDL type II fibers (Fig. 3). The
MHCII antibody used in this study detected all MHCII isoforms, and the fibers were not classified into type II subclasses.
Nevertheless, it can be confidently assumed that the two type II
SOL fibers examined here were not IIB fibers because previous
studies have established that type II fibers in rat SOL muscle
are all either IIA or less often IIX/D fibers and never IIB fibers
(1, 4), in contrast to rat EDL muscle where the great majority
are either IIB or IIX fibers. The findings here are in general
accord with conclusions of immunohistochemical staining of
muscle cross sections from rat muscle, which, although not
truly quantitative, indicated that most if not all type I fibers
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Fig. 8. Heating SOL muscle to 45°C causes the
majority of all three HSPs to bind within the
fiber. Two representative Western blots showing
HSPs remaining in a skinned fiber (F) or diffusing into the surrounding wash solution (W) after
bathing individual skinned fiber segments for 10
min in physiological solution or in the same
solution with 1% Triton X-100. Fibers were isolated from untreated (control) SOL muscle or
from contralateral SOL muscle heated at 45°C in
paraffin oil for 30 min. A: in a skinned fiber from
control muscle, HSPs readily diffused into the
wash solution (W & F set on left), whereas, in a
skinned fiber from the 45°C heat-treated muscle,
all of the HSPs remained within the fiber (W & F
set on right). Note that aldolase remained diffusible even after heat treatment. B: in skinned fibers
from the heat-treated muscle that were examined
with Triton X-100 present in the wash solution,
the majority of both the SERCA2 and the HSP72
dispersed into the wash solution, whereas all
HSP25 and ␣B-crystallin still remained entirely
within the fiber.
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HEAT SHOCK PROTEINS IN UNSTRESSED SKELETAL MUSCLE FIBERS
contain much higher levels of ␣B-crystallin than type II fibers
(1, 11), and also higher levels of HSP72 (3), whereas the
differences between type I and type II are less marked with
respect to HSP25 (11).
Comparison of HSP72 and SERCA amounts. HSP72 binds
to both SERCA1 and SERCA2 (8, 43), as well as to many other
proteins, including the Na⫹-K⫹-ATPase (34) and K⫹ channels
(7). SERCA proteins are present in very significant amounts,
far more than the Na⫹-K⫹-ATPase, particularly in fast-twitch
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Fig. 9. Triton X-100 increases diffusional loss of HSP72, but not HSP25 or
␣B-crystallin, in fibers from 45°C-treated muscle. Mean (⫹SE) of percentage
of HSP72, HSP25, and ␣B-crystallin found in wash solution for skinned fibers
from control or heated muscles, obtained as in Fig. 8. A subset of the skinned
fibers from heated muscles was examined with 1% Triton X-100 present in the
wash solution, with alternate fibers examined without Triton. For each fiber
segment, the total amount of the given HSP was taken as the sum of that found
in corresponding F and W samples. n, No. of fibers/no. of muscles for each
case. #Significant temperature-related difference compared with control (unheated) SOL muscle. *Significant difference compared with 45°C heated SOL
muscle fibers examined without Triton X-100 (P ⬍ 0.05).
muscle fibers, and it appears that the SERCA must be an
important target in quantitative terms, at least in rodent muscle.
Specifically, in rat EDL muscle, there are ⬃110 ␮mol of
SERCA1 molecules/kg, with SERCA2 below detection limits,
and, in rat SOL muscle, there is ⬃20 and 15 ␮mol/kg of
SERCA1 and SERCA2, respectively (44). Similarly in mouse
muscle, the amounts equate to ⬃100 ␮mol/kg muscle for
SERCA1 and ⬃1 ␮mol/kg of SERCA2 in EDL, and ⬃20 and
1.5 ␮mol/kg, respectively, in SOL (30). Thus, the amount of
HSP72 present in unstressed EDL muscle (⬃1.1 ␮mol/kg,
Table 1) is ⬃100-fold lower than the density of the SERCA
(⬃100 to 110 ␮mol/kg), one of its putative targets. This
disparity between HSP72 and SERCA densities apparent in
EDL whole muscle homogenates also evidently pertains at the
single fiber level because individual EDL fibers were all found
to have both relatively high SERCA1 density (e.g., Fig. 3A and
Ref. 25) and low HSP72 density (Fig. 3B). Even in SOL
muscle fibers, where there is more HSP72 (Table 1) and fewer
SERCA molecules, the number of SERCA molecules is still on
average three- to fourfold greater than the number of HSP72
molecules present. Previous studies have shown that, following
heat shock, at least some of the HSP72 molecules present
become tightly bound to SERCA, which evidently helps protect both SERCA1 and SERCA2 molecules from thermal
inactivation (8, 42, 43). It was found here that, in fibers from
heat-treated EDL and SOL muscles, virtually all of the HSP72
remained tightly bound at sites within the fibers even with
extensive washing, but, when the internal membranes were
disrupted with the detergent Triton X-100, the HSP72 dispersed into the surrounding solution in tandem with the
SERCA (Figs. 8 and 9). If HSP72 does need to be bound to
SERCA to protect it from thermal inactivation, it is evident that
the amount of HSP72 present in unstressed EDL muscle would
be sufficient to bestow such protection only on at most ⬃1% of
the SERCA present, even without taking into account HSP72
binding to any other target molecules. This points to one reason
why greatly increasing HSP72 content, particularly in type II
fibers, might help enable them to cope in stressful situations
and reflect why HSP72 protein expression in muscles has been
observed in some cases to increase 10-fold or more following
heat stress (17) or acute exercise (15).
Binding sites for HSP25 and ␣B-crystallin. In contrast to
HSP72, ␣B-crystallin and HSP25 are reported to bind in
stressed conditions to structural and/or contractile proteins, and
this seems consistent with the findings here that disruption and
dispersion of intracellular membranes by Triton X-100 had no
apparent effect at all on the tight binding of ␣B-crystallin and
HSP25 in heat-stressed fibers (Figs. 8 and 9). It is unclear,
however, whether the target proteins differ for these two HSPs,
perhaps also differing with the type of stress involved. ␣Bcrystallin and HSP25 (HSP27 in humans) have been reported
to both accumulate at the Z-disk and with intermediate structures (thought to be desmin) in both mouse (16) and human
(31) muscle following damaging eccentric exercise. On the
other hand, with heat stress, ␣B-crystallin and HSP25 have
been both reported to bind and protect actin (22, 36), and
␣B-crystallin was found to bind at least transiently with myosin and help preserve its ATPase function (19). Other studies,
in contrast, found that, following ischemic stress, ␣B-crystallin
and HSP25 bound to titin and desmin and not at all to myosin,
actin, or ␣-actinin in cardiac muscle (10), and, in skeletal
HEAT SHOCK PROTEINS IN UNSTRESSED SKELETAL MUSCLE FIBERS
out the whole cross section (11). Furthermore, with HSP25,
there was less apparent difference in the staining between type
I and type II fibers, with an appreciable amount present around
the edges in both types (11), which is consistent both with the
much smaller disparity in HSP25 levels between type I and
type II fibers seen here (Table 1) and the comparatively large
amount (⬃20%) apparently associated with the sarcolemma
(Table 2), at least in some cases (Fig. 4). In contrast, no
evidence was found here that HSP72 associated with the
sarcolemma in unstressed fibers from rat (Table 2), and HSP70
immunohistochemistry of human fibers in control conditions
shows no obvious sarcolemmal staining (32, 41). Such findings
could result from HSP72 binding primarily at intracellular
sites, in particular on SERCA (8, 43), or alternatively from the
absolute levels of HSP72 in fibers being so low that the
sarcolemmal component fell below detection limits.
Diffusibility of HSPs in unstressed muscle. In skinned muscle fibers, proteins that are freely diffusible in the cytoplasm
are readily lost to the bathing solution within ⬃1–2 min (28,
37). Such experiments here (Figs. 5 and 6) demonstrated that,
for all three HSPs, the majority of that present in rat SOL fibers
was readily diffusible within the fiber. Taking into account the
amounts associated and removed with the sarcolemma (Table
2), this diffusible component accounts for ⬃60 –70% of the
total HSP25 and ␣B-crystallin and ⬃90% of the total HSP72
present in SOL fibers. In EDL fibers, a similar proportion of the
total HSP72 is diffusible, but, in the cases of HSP25 and
␣B-crystallin, ⬍40% of the low total amount present is diffusible (Table 2 and Fig. 6). It is interesting to note that the HSP72
diffused out of the SOL fibers ⬃8 to 10 times more slowly
(time constant ⬃2.2 min) than did the HSP25 and ␣B-crystallin (time constants ⬃0.25 and 0.34 min, respectively). This
disparity is not seemingly explained by ⬃3-fold greater molecular mass of the HSP72, since, theoretically, this would be
expected to slow diffusion only ⬃1.5-fold (since diffusion rate
depends on the cubed root of molecular mass if spherical shape
is assumed). Moreover, we have previously shown that ␮-calpain, a protein with a slightly larger molecular mass than
HSP72 (80 kDa), washes out of rat skinned fibers with a time constant of ⬃0.4 min (28). There are a number of possible
explanations for the apparently slow diffusion rate of the
HSP72. One is that much of the HSP72 might actually be
loosely bound, coming off these sites with a time constant of
⬃1–2 min. An alternative possibility is that HSP72 is continually binding and unbinding rapidly on various target molecules it encounters, considerably slowing its rate of diffusional
loss. Presumably, the latter must happen to some degree in any
process in which HSPs must rapidly recognize and strongly
bind to damaged or unfolded proteins. Irrespective of this
relatively minor slowing in the apparent diffusional rate of
HSP72, a key finding here is that much of all three HSPs is
freely diffusible or in rapid equilibrium with the cytoplasm in
unstressed fibers, and hence could be expected to rapidly reach
any cytoplasmic-assessable sites as needed.
In conclusion, this study highlights how it is necessary to
know the absolute levels of each HSP present in skeletal
muscle to fully understand the possible roles and limitations on
each. Of particular note, it was found that the amount of HSP72
present in type II fibers in unstressed EDL muscle is much
lower than the density of its putative binding sites, indicating
why substantial upregulation would be advantageous in stress.
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muscle, ␣B-crystallin showed diffuse binding across the whole
I-band but none at the Z-disk (11). Hence, it is difficult to
quantitatively compare the densities of these HSPs and their
putative targets. Nevertheless, the following offers some important perspective. There is ⬃60 ␮mol of ␣-actinin/kg muscle
(38), and ⬃2–3 ␮mol/kg of titin (assuming a total of 8 titin
molecules tethering each myosin filament and 300 myosin
molecules/filament). Thus, the amount of ␣B-crystallin present
in EDL muscle (⬃5 ␮mol/kg) would be just sufficient to bind
at all the N-line sites (11) on the titin molecules but could bind
to only a small proportion of the ␣-actinin molecules present in
the muscle. The amount of ␣B-crystallin present in rat SOL
muscle (⬃140 ␮mol/kg muscle), however, would be sufficient
to bind in a 1:1 manner on all the titin and all alpha-actinin
molecules present, and even on many myosin molecules [⬃94
␮mol/kg (45)], although only on a fraction of the total actin
molecules [620 ␮mol/kg (45)]. The fact that virtually all of the
large amount of ␣B-crystallin present in rat SOL fibers (⬃140
␮mol/kg muscle) became bound in the heat-stressed fibers here
(Figs. 8 and 9) indicates that it indeed likely targets many of
the structural and sarcomeric proteins. In the case of HSP25,
however, the absolute amount present in unstressed muscle
fibers of either type (⬃3 to 9 ␮mol/kg) is insufficient to bind
and protect even a very limited range of the suggested target
molecules. Furthermore, if HSP25 and ␣B-crystallin compete
for the same binding site on particular target molecules, which
seems possible given their homology, the binding of ␣Bcrystallin would be expected to predominate, at least in type I
fibers.
In any case, the very high ␣B-crystallin content of type I
fibers stands out as an anomaly and is strongly suggestive that
the role of ␣B-crystallin in such fibers differs substantially in
some way from that of HSP25 and, furthermore, that it is
important for its role in the type I fibers that large amounts are
present even in the absence of overt stresses. The findings here
also make it clear that the relative importance of the various
HSPs should not be judged simply by the relative extent to
which each is upregulated with stress. In the case of ␣Bcrystallin in rat type I fibers, where there is ⬃140 ␮mol/kg
present in the unstressed state, even a 10% upregulation would
represent a very considerable increase in absolute terms, in fact
by an amount equal to the summed total of all HSP25 and
HSP72 present in the muscle.
HSPs and the sarcolemma. The results of the sarcolemma
removal experiments here (Table 2 and Fig. 4) also demonstrated that some of the HSP25 and ␣B-crystallin present in
unstressed muscle fibers is closely associated with the sarcolemma in some way, possibly bound or otherwise localized
there. In the case of ␣B-crystallin, the proportion associated
with the sarcolemma appeared to be much greater in the EDL
fibers (⬃60%) than in the SOL fibers (⬃10%) (all type II and
all type I fibers, respectively). This relative difference likely
simply reflects that some appreciable amount of ␣B-crystallin
is associated with the sarcolemma in both fiber types, but, in
the SOL fibers, this amount represents only a comparatively
small proportion of the total present, which is large in absolute
terms (Table 1). These observations and explanation appear to
fit well with the immunohistochemical staining patterns of rat
muscle fibers, in which ␣B-crystallin was seen in type II fibers
predominantly as a relatively bright ring around the fiber edge,
whereas in type I fibers there was quite bright staining through-
C237
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HEAT SHOCK PROTEINS IN UNSTRESSED SKELETAL MUSCLE FIBERS
On the other hand, type I SOL fibers contain very large
amounts of ␣B-crystallin, even in the unstressed state, suggesting its importance in normal cellular function in such fibers.
ACKNOWLEDGMENTS
We thank Maria Cellini and Heidy Latchman for technical assistance and
Dr. Frank Wuytack (Katholieke Universiteit, Leuven, Belgium) for the antiSERCA2a antibody.
The monoclonal antibodies directed against adult human MHC isoforms
(A4.84 and A4.74) were developed by Dr. Blau and those directed against
SERCA1 were developed by Dr. D. Fambrough. All were obtained from the
Development Studies Hybridoma Bank, under the auspices of the NICHD and
maintained by the University of lowa, Department of Biological Science, Iowa
City, IA 52242.
This study was supported by National Health and Medical Research
Council of Australia Grants 541938 and 602538.
DISCLOSURES
No conflicts of interest are declared by the authors.
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