Fiber type populations and
Ca2+-activation properties of
single fibers in soleus muscles from SHR and WKY rats
Susan K.
Bortolotto1,
D. George
Stephenson2, and
Gabriela M. M.
Stephenson1
1 School of Life Sciences and
Technology, Victoria University of Technology, Melbourne, Victoria
8001; and 2 School of Zoology, La
Trobe University, Melbourne, Victoria 3083, Australia
 |
ABSTRACT |
Electrophoretic analyses of muscle proteins in whole muscle
homogenates and single muscle fiber segments were used to examine myosin heavy chain (MHC) and myosin light chain 2 (MLC2) isoform composition and fiber type populations in soleus muscles from spontaneously hypertensive rats (SHRs) and their age-matched
normotensive controls [Wistar-Kyoto (WKY) rats], at three
stages in the development of high blood pressure (4 wk, 16 wk, and 24 wk of age). Demembranated (chemically skinned with 2% Triton X-100),
single fiber preparations were used to determine the maximum
Ca2+-activated force per
cross-sectional area, calcium sensitivity, and degree of cooperativity
of the contractile apparatus and
Ca2+-regulatory system with
respect to Ca2+. The results show
that, at all ages examined, 1) SHR
soleus contained a lower proportion of MHCI and MLC2 slow (MLC2s) and a
higher proportion of MHCIIa, MHCIId/x, and MLC2 fast (MLC2f )
isoforms than the age-matched controls;
2) random dissection of single fibers from SHR and WKY soleus produced four populations of fibers: type I (expressing MHCI), type IIA (expressing MHCIIa), hybrid type
I+IIA (coexpressing MHCI and MHCIIa), and hybrid type IIA+IID (coexpressing MHCIIa and MHCIId/x); and
3) single fiber dissection from SHR
soleus yielded a lower proportion of type I fibers, a higher proportion
of fast-twitch fibers (types IIA and IIA+IID), and a higher proportion
of hybrid fibers (types I+IIA and IIA+IID) than the homologous muscles
from the age-matched WKY rats. Because the presence of hybrid fibers is
viewed as a marker of muscle transformation, these data suggest that
SHR soleus undergoes transformation well into adulthood. Our data show
also that, for a given fiber type, there are no significant differences
between SHR and WKY soleus muscles with respect to any of the
Ca2+-activation properties
examined. This finding indicates that the lower specific tensions
reported in the literature for SHR soleus muscles are not due to
strain- or hypertension-related differences in the function of the
contractile apparatus or regulatory system.
single muscle fiber; myosin heavy chain; myosin light chain; muscle
proteins; hypertension; spontaneously hypertensive rat; Wistar-Kyoto
rat
 |
INTRODUCTION |
THE MOST WIDELY USED ANIMAL model for exploring the
pathophysiology of genetic hypertension in humans is the spontaneously hypertensive rat (SHR), which was originally established by Okamoto and
Aoki (20) from a colony of normotensive Wistar rats [Wistar-Kyoto (WKY)]. According to Pickar et al. (23), in SHR the development of hypertension occurs in three stages: the rising of blood pressure (4-8 wk), the arrival of blood pressure to peak value (14-18
wk), and the stabilization of blood pressure and the appearance of adaptive changes in various organs (
24 wk).
Although there is general agreement that skeletal muscles of
hypertensive humans contain a higher percentage of fast-twitch fibers
than the normotensive controls (10, 15), the fiber type composition of
skeletal muscles from SHRs is the subject of an ongoing controversy.
Thus Gray and colleagues (2, 11) have reported that soleus muscles from
6- to 28-wk-old SHRs contain more slow-twitch (type I) and fewer
fast-twitch (type II) fibers than the homologous muscles of age-matched
WKY rats. In contrast, Benbachir-Lamrini et al. (3, 4) and Lewis et al.
(19) have reported that soleus muscles from SHRs aged 4-17 wk
contain fewer type I fibers and more type II fibers than the soleus of age-matched WKY rats.
It is important to note that all three groups cited above determined
the fiber type composition of soleus muscles from SHR and WKY rats
using myosin ATPase-based (2-4, 11, 19) and succinate
dehydrogenase-based (2, 11) histochemistry. However, as it has been
stated previously, both enzyme-based histochemistry (14) and
immunohistochemistry (5) are strategies of limited suitability for
studying fiber type composition in transforming muscles, because they
cannot convincingly identify fibers coexpressing two or three myosin
heavy chain (MHC) isoforms (hybrid fibers). More recently, analysis of
muscle proteins from whole muscles and from single muscle fiber
segments using SDS-PAGE has become the preferred strategy in research
concerned with the effect of various conditions on myosin isoform
expression/fiber type composition in skeletal muscle. Indeed, the major
advantage of this latter strategy is that it can rapidly detect and
quantitate MHC isoform expression in whole muscles as well as in hybrid
fibers (21, 22). Furthermore, this strategy allows detection, at the
whole muscle or single fiber level, of changes in myosin light chain (MLC) isoforms expression (21, 22, 25) that can also occur in
transforming muscles.
In the present study we used the microelectrophoretic methodology to
compare the MHC isoform, MLC2 isoform, and fiber type composition of
soleus muscles from SHRs and WKY rats, at the three stages of
development of hypertension defined by Pickar et al. (23). A secondary
aim of the study was to assess whether the reduced specific tetanic
force and contractile kinetics parameters reported by Gray and
colleagues (2, 7, 12, 13) and Lewis et al. (19) for SHR soleus are due
in part to functional alterations of the myofibrillar compartment. For
this purpose we used the skinned muscle preparation, which allows
direct activation of the myofibrillar compartment in solutions of
carefully controlled free calcium concentrations (26, 28, 29).
 |
METHODS |
Animals. The male normotensive WKY
rats and SHRs used in this study were bred at the Baker Medical
Research Institute (Melbourne, Victoria, Australia) from stock obtained
in 1985 from one of the original breeders of SHR (Y. Yamori) at the
Shimane Institute of Health Science. The rats were housed in a
temperature-controlled environment (22°C) with a 12:12-h light-dark
cycle and had access to food and water ad libitum. Before and on the
day of the experiment, systolic blood pressure was measured in
conscious rats by tail-cuff plethysmography (IITC Life Science
Instruments) while the animals were in a restriction chamber at
27°C (9, 16). The animals used for this study belonged to three age
groups, representing the three stages in the development of
hypertension (23): 1) the rising
phase of blood pressure, before the onset of hypertension (4 wk),
2) the arrival of blood pressure to
its peak value at the onset of hypertension (16-18 wk), and
3) the maintenance of blood pressure
at elevated levels (24 wk). On the day of the experiment, rats were
killed by deep halothane inhalation, and the soleus muscles were
quickly removed, blotted dry on filter paper, weighed, and placed under
paraffin oil saturated with water. One muscle was used for single fiber
analyses and the contralateral muscle was used for preparing whole
muscle homogenate.
Preparation of skinned, single muscle fiber
segments. Single fibers were isolated randomly under
paraffin oil using a dissecting microscope, fine jewelers forceps, and
iris scissors, as described in detail elsewhere (26). The paraffin oil
plays three important roles: 1) it
facilitates the visualization of the single fiber by having a different
refractive index, 2) it precludes
fiber swelling/fiber-water loss when fiber segments are prepared, and 3) it confers the fiber a
quasicircular cross-sectional area through the surface tension exerted
on the fiber at the fiber/oil interface. A video camera-monitor system
(Olympus) was used to measure the length and the width of the fiber
segment in at least three places along its length, while still under
paraffin oil. The volume of a fiber segment was calculated assuming it
to be a cylinder with a diameter equal to the mean value of the fiber
width. Some fiber segments were examined for calcium-activation
characteristics as well as MHC composition, whereas others were
examined for MHC composition only.
Calcium-activation characteristics of chemically
skinned, single muscle fibers. Single fiber segments
were mounted, at slack length, under oil, between a force transducer
(Sensonor, Horten, Norway) and a pair of fine Barcroft forceps or were
incubated directly in SDS solubilizing buffer. In this study, the slack length of a fiber segment was obtained by releasing the slightly stretched fiber segment until no measurable force could be detected (<2.5 µN) and the preparation was just "taut." After
mounting, the fiber segment was incubated for 10 min in a relaxing
solution (solution
A; see composition below) containing
2% Triton, a strategy known to disrupt cellular membranes without
affecting the contractile system (27). While in this solution, the
average sarcomere length was measured from the diffraction maxima of a
HeNe laser beam illuminating the mounted preparation as described
previously (28). At slack length, the average sarcomere length of the
preparation was 2.65 ± 0.02 µm (n = 45) for fibers from the 16- and 24-wk-old rats. This corresponds to
the "optimum" sarcomere length for maximum Ca2+ force development in skinned
rat muscle fibers from adult rats (28). The "slack" sarcomere
length of fibers segments from the 4-wk-old rats was 3.14 ± 0.01 µm
(n = 12). Preliminary experiments showed that at this sarcomere length, the maximum
Ca2+-activated force developed in
two fiber preparations from 4-wk-old rats was greater than that
produced when the fibers were allowed to shorten to a sarcomere length
of 2.7 µm.
The calcium-activation characteristics of single muscle fiber segments
were determined from force responses developed at 21 ± 2°C, in a
series of strongly buffered Ca2+
solutions prepared as described in detail elsewhere (26, 29). All
solutions contained (in mM) 117 K+, 36 Na+, 1 Mg2+ (free), 90 HEPES, 8 total
ATP, 10 creatine phosphate, and 1 sodium azide, pH 7.10 ± 0.01 at
22°C. The relaxing solution
(solution A) contained 50 mM
EGTA2
(pCa =
log
[Ca2+]
9), the
maximally Ca2+-activating solution
(solution
B) contained 49.8 mM
CaEGTA2
and 0.2 mM excess EGTA2
(pCa = 4.3), and the preactivating solution
(solution
C) contained 49.75 mM
hexamethylenediamine tetraacetate (HDTA2
) and 0.25 mM
EGTA2
(pCa
8). A series of solutions with a range of
pCa values of 6.5-4.3 was obtained by mixing
solutions
A and
B in different proportions. The
osmolality of all solutions was 295 ± 2 mosmol/kg. Before exposure
to Ca2+-activating solutions, the
fiber segment was incubated in
solution C. Each fiber preparation was first
maximally activated at pCa 4.3 (solution
B), then relaxed in
solution
A (pCa
9), equilibrated in the
preactivating solution, and then exposed to a set of 10 activating
solutions in decreasing order of pCa values (6.5 to 4.3). In this
study, the second maximum
Ca2+-activated force
response was on average 8.19% (±5.74 SD; range 0.4-21%)
less than the first response. For calculating the submaximal level of
activation in a solution of a particular pCa value, the steady level of
force recorded in that solution was divided by the interpolated value
of the maximum Ca2+-activated
force response as described in detail elsewhere (24). This strategy
corrects for the small degree of deterioration in force production
associated with the repeated activation of the skinned fiber preparation.
The following Ca2+-activation
characteristics were determined for each individual fiber segment from
the isometric force-pCa curves: 1)
maximum Ca2+-activated force per
cross-sectional area (CaFmax/CSA;
kN/m2), determined from the
amplitude of the first force response of a fiber in the maximally
Ca2+-activating solution and from
its cross-sectional area; 2)
pCa50 (representing the pCa where
50% maximal force was produced); and 3) the Hill coefficient
nCa, which
relates to the steepness of the force-pCa curve, described by the
theoretical relation
|
(1)
|
where
Pt is the fraction of maximal
force developed by the fiber segment at a given
Ca2+ concentration. For each
force-pCa curve, the values of
pCa50 and
nCa were
determined using the nonlinear regression analysis option of the
GraphPad Prism software.
SDS-PAGE analysis of MHC and MLC
isoforms. MHC isoform analyses were performed on whole
muscle homogenates and single muscle fiber segments, whereas MLC
isoform analyses were performed on whole muscle homogenates only. Whole
muscles were homogenized in six volumes of
solution
A, and protein concentration was
determined using the Bradford protein assay (6). Each homogenate was
diluted with SDS-PAGE solubilizing buffer (62.5 mM Tris, 2.3% SDS, 5%
-mercaptoethanol, 12.5% glycerol, 13.6% sucrose, 0.01%
Bromophenol blue, 0.1 mM phenylmethylsulfonyl fluoride, 2 µM
leupeptin, and 1 µM pepstatin) and boiled for 5 min. The single
fibers to be analyzed by SDS-PAGE were placed in 12 µl SDS-PAGE
solubilizing buffer either immediately after dissection under paraffin
oil or after being first examined for contractile characteristics as
described above. In both cases, the single muscle fiber segments were
incubated in the solubilizing buffer for 24 h at room temperature and
then were boiled for 5 min. MHC isoforms in whole muscle homogenates and single muscle fibers were analyzed on 0.75-mm-thick slab gels using
the Hoefer Mighty Small gel apparatus and the Laemmli procedure (18)
with minor modifications. Briefly, the separating gel contained 6.7 or
7.9% acrylamide, 0.15 or 0.096% bis-acrylamide, 375 or 425 mM Tris pH
8.8, 40% (vol/vol) glycerol, and 0.1 or 0.3% (wt/vol) SDS and was
polymerized with ammonium persulfate and
N,N,N',N'-tetramethylethylenediamine. The stacking gel contained 4% acrylamide and the running buffer contained 32.5 mM Tris, 288 mM glycine, and 0.1% SDS, and the gels
were run for 24 h at 16°C and at constant voltage (100-120 V).
A 6-µl sample aliquot containing 0.05 mg/ml muscle protein or
0.025-0.05 nl fiber/µl was applied per electrophoretic well. The
gels were stained using Bio-Rad Silver Stain Plus. The MHC isoforms
were identified based on data reported in the literature and on
SDS-PAGE analysis of MHC isoforms in rat muscles containing one
dominant isoform: soleus (MHCI), diaphragm (MHCIID), and levator ani
(MHCIIB) (14). The nomenclature used for fiber types (e.g., IIA, IID/X)
and for MHC isoforms (e.g., IIa, IId/x) follows that of Pette and
Staron (21, 22).
Low-molecular-weight myofibrillar proteins were separated on SDS-PAGE
using a separating gel containing 18% acrylamide, 0.09% bis-acrylamide, 0.75 M Tris pH 9.3, 10% glycerol, 0.1% SDS, a stacking gel containing 4% acrylamide, and a running buffer containing 50 mM Tris, 380 mM glycine, and 0.1% SDS. For each homogenate, a
10-µl sample containing 0.15 mg protein/ml was loaded per
electrophoretic well. Gels were run at a constant current (10 mA/gel)
until the lowest molecular weight marker reached the bottom of the gel
(4-5 h) and then were stained with Pharmacia Biotech silver stain. The MLC2 band was identified on the basis of data reported in literature, of the electrophoretic mobility of purified rabbit MLC2
(gift from Dr. T. Walsh, QUT, Australia), and of SDS-PAGE analysis of
low-molecular-weight myofibrillar proteins in segments of single muscle
fibers containing only one fast or one slow MHC isoform.
Estimation of MHC isoform and MLC2 isoform composition in whole
muscle homogenates.
The gels for both high- and low-molecular-weight proteins were analyzed
using a Molecular Dynamics Personal Densitometer and the volumetric
quantitation method, with background correction provided by the
ImageQuaNT software version 4.1 (Molecular Dynamics).
Figure
1A
illustrates the relationship between the computed difference between
the optical density of the protein band corresponding to MHCi (where i = I or IIa or IId/x) and that of the background (
OD1MHCi) and the total
protein concentration in the homogenate sample (over the range
0.01-0.06 mg/ml). As seen in Fig.
1A,
OD1MHCi was linearly related to
the total protein concentration for each MHC isoform detected
(r2 for
MHCI = 0.98;
r2 for
MHCIIa = 0.96;
r2 for
MHCIId = 0.95).

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Fig. 1.
A: relationship between
OD1MHCi (computed difference between optical density of
the protein band corresponding to MHCi isoform and that of the
background) expressed in arbitrary units and the total protein
concentration in the homogenate sample.
B: relationship between
OD2MHCi (an optical parameter
derived from OD1MHCi as
explained in METHODS) expressed in
arbitrary units and the total protein concentration in the homogenate
sample. , MHCI; , MHCIIa; x, MHCIId/x.
|
|
The linear functions represented in Fig.
1A are described by the equation
|
(2)
|
where
C is the total protein concentration in the homogenate sample,
ai is the slope
of the line for MHCi, and
bi is the intercept of the line for MHCi with the
y-axis. Direct proportionality between
total protein concentration in the homogenate sample and a second
optical parameter (
OD2MHCi),
derived from
OD1MHCi, can be
obtained by transferring
bi from the right
to the left side of Eq.
2
|
(3)
|
The
result of this mathematical manipulation is shown in Fig.
1B. The concentration of each MHC
isoform in the muscle homogenate sample ([MHCi]) is
proportional to the total protein concentration
|
(4)
|
where
i is the fraction of total
protein representing the MHCi component. Substituting C in
Eq. 3 with
[MHCi]/
i from
Eq. 4 results in a direct
proportionality between
OD2MHCi
and [MHCi]
|
(5)
|
If the silver stain reacts in a similar manner with all MHC isoforms,
then the ratio
ai/
i
should have a constant value that is independent of the MHC isoform.
This simple mathematical manipulation then allows the direct estimation
of the relative proportion of each MHC isoform in a given homogenate
sample, from the data generated by the densitometer software, using the
expression
|
(6)
|
Because
OD1MLC2s-i was found to be
linearly related to the total protein concentration in the sample over
the range of sample protein concentrations used (data not shown), the
strategy described above was also used to estimate the relative
proportion of MLC2 isoforms in a given muscle homogenate.
Statistics. Unless stated otherwise,
all data are expressed as means ± SE and statistical comparisons were
performed on groups with at least three data points using a two-way
analysis of variance followed by the Bonferroni test. Statistical
significance was accepted at P < 0.05.
 |
RESULTS |
Physical characteristics. In Table
1 are shown the average systolic blood
pressure (BP) readings for the WKY rats and SHRs examined in this
study. The 4-wk-old SHRs had a mean BP value of 105.6 mmHg, which was
25% higher than that of the age-matched WKY (84.8 mmHg), but was 28%
lower than the mean BP value for the 24-wk-old normotensive rats (146.0 mmHg). In contrast, the more mature SHRs (16 and 24 wk old) had BP
values that were 43-56% higher than the BP values for the
age-matched WKY and markedly higher than the upper limit for
normotensive animals. The highest average BP value for normotensive WKY
rats was 146 mmHg for the 24-wk-old animals, which is in full agreement
with the highest average BP value of 150 mmHg reported for WKY rats
aged 4-52 wk (4). Because the highest BP value recorded in WKY
rats in this study was 165 mmHg, all rats that had a BP reading
165
mmHg were regarded as normotensive.
The body weight of SHRs at 4 and 24 wk of age was not significantly
different from that of age-matched WKY rats, whereas the 16-wk-old SHRs
were slightly (8%) but significantly heavier
(P < 0.05) than the age-matched
normotensive rats (Table 1). The mean soleus muscle wet weight per body
weight values did not change with age in either of the two strains
examined. The mean muscle wet weight per body weight values were,
however, significantly higher in SHR than in WKY rats at all three ages examined.
MHC isoform composition of whole
muscles. Electrophoretic analyses of whole tissue
homogenates prepared from WKY rat and SHR soleus muscles revealed three
high-molecular-weight protein bands of decreasing electrophoretic
mobility, corresponding to slow MHCI, fast MHCIId/x, and fast MHCIIa
(Fig. 2). It is important to note for the
same amount of protein applied, the SHR soleus sample displayed MHCI,
MHCIIa, and MHCIId/x bands whereas the WKY soleus sample displayed only
MHCI and MHCIIa. The percentages of the MHC isoforms in muscle
homogenates of 4-, 16-, and 24-wk-old SHRs and WKY rats are shown in
Table 2.

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Fig. 2.
Representative electrophoretic profile of MHC isoforms detected in
soleus muscles from 24-wk-old Wistar-Kyoto (WKY) rats
(lane
2) and spontaneously hypertensive
rats (SHRs) (lane
3). Whole tissue homogenates were
prepared from WKY rat and SHR soleus muscles, and proteins were
analyzed by SDS-PAGE as described in
METHODS.
Lane
1: laboratory MHC isoform marker
containing the 4 MHC isoforms known to be expressed by adult rat
hindlimb skeletal muscles: MHCI, fast MHCIIb, fast MHCIId/x, and fast
MHCIIa. Amount of protein applied per electrophoretic well: 0.3 µg.
|
|
At 4 wk of age, the soleus muscle from SHRs contained a lower
proportion of MHCI (
14.2%) and a higher proportion of MHCIId/x (+12.5%) than the soleus muscle from the age-matched WKY rats. The
proportion of MHCIIa isoform was, however, similar in SHRs and WKY rats.
An increase in age from 4 to 24 wk was accompanied by clear changes in
the relative proportion of MHC isoforms in the soleus muscles of both
SHRs and WKY rats. The direction of these changes was MHC isoform
dependent, with the percentage of MHCI isoform increasing and the
percentage of MHCII isoforms decreasing with age in both strains. For
all MHC isoforms, the major age-related changes occurred during the rat
development from 4 to 16 wk. Thus the proportion of MHCI isoform
increased between 4 and 16 wk, by 20.1% in WKY and 15.0% in SHR, but
it did not change significantly between 16 and 24 wk of age in either
strain. The proportion of MHCIIa decreased significantly (by 18.6%) in
WKY between 4 and 16 wk, but did not change in SHR. Between 16 and 24 wk no significant change was detected in the proportion of MHCIIa in
either strain.
Finally, the proportion of MHCIId/x in WKY rats decreased from low
(1.5% at 4 wk) to nondetectable levels at 16 and 24 wk, whereas in the
SHR it decreased from 14.0% at 4 wk to ~2.4% at 16 wk and this
level remained unchanged in 24-wk-old rats. In summary, at all ages
examined, the soleus muscle from SHRs contained a statistically
significant higher proportion of fast MHC isoforms (comprising both
MHCIIa and MHCIId/x) and a lower proportion of slow MHC isoform than
soleus muscle from age-matched WKY rats.
MLC2 isoform composition of whole
muscles. Each myosin molecule contains two regulatory
light chains (MLC2) that exist in two isoforms: MLC2f (fast isoform)
and MLC2s (slow isoform). In SDS gels, MLC2f has a higher
electrophoretic mobility than MLC2s (Fig.
3A). In
agreement with the MHC isoform data, soleus muscles from SHRs were
found to contain a statistically significant lower proportion of MLC2s
and a higher proportion of MLC2f than their WKY counterparts at all
ages examined. This can be seen also in Fig. 3. In Fig.
3A are shown side-by-side two
representative electrophoretograms of low-molecular-weight proteins
produced by SDS-PAGE analyses of SHR and WKY soleus muscle homogenates
from 24-wk-old rats, and the bar graph in Fig.
3B illustrates the differences between the calculated values for the ratio
MLC2s/MLC2t (where
MLC2t = MLC2s + MLC2f) in soleus
muscles of 4-, 16-, and 24-wk-old SHRs and WKY rats.

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Fig. 3.
A: representative electrophoretogram
of low-molecular-mass (<45 kDa) muscle proteins from 24-wk-old WKY
rat (lane
1) and SHR
(lane
2) soleus muscles. Arrows indicate
the bands corresponding to actin, slow MLC2 isoform (MLC2s), and fast
MLC2 isoform (MLC2f). B: bar chart for
ratio MLC2s/MLC2t (where
MLC2t = MLC2s + MLC2f) in soleus
muscles of 4-, 16-, and 24-wk-old SHRs (filled bars) and WKY (open
bars) rats. # Statistically significant difference between
4-wk-old and adult (16 wk and 24 wk) rats of same strain.
* Statistically significant difference between SHR soleus and
age-matched WKY rat soleus. Amount of protein applied per
electrophoretic well: 3 µg.
|
|
Fiber type populations yielded by random
dissection. Electrophoretic analyses of MHC isoform
composition in muscle homogenates prepared from soleus muscles of SHRs
and WKY rats provide very useful information on strain-related
differences in MHC expression at the whole muscle level, but give no
indication on the types of fibers that make up the muscles compared. To
obtain this information, microelectrophoretic analyses of MHC isoform
expression were performed in segments of single fibers, randomly
dissected from soleus muscles of SHRs and WKY rats. These analyses
showed that soleus muscles from 4-, 16-, and 24-wk-old WKY rats and
age-matched SHRs contain four fiber types: I (pure slow; containing
MHCI), I+IIA (hybrid slow/fast, containing different proportions of
MHCI and MHCIIa), IIA (pure fast, containing MHCIIa), and IIA+IID
(hybrid fast/fast containing different proportions of MHCIIa
and MHCIId/x) (Fig. 4). In agreement with
the MHC isoform composition data shown in Table 2, no fibers containing
the MHCIIb isoform were detected in any of the muscles examined.

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Fig. 4.
Representative electrophoretograms of MHC isoform composition of fiber
types yielded, on random dissection, by soleus muscles of SHRs and WKY
rats. Lane
1: laboratory marker containing the 4 MHC isoforms known to be expressed by adult rat hindlimb skeletal
muscles: MHCI, fast MHCIIb, fast MHCIId/x, and fast MHCIIa.
Lane
2: slow fiber (type I).
Lane
3: hybrid slow/fast fiber (type
I+IIA). Lane
4: fast fiber (type IIA).
Lane
5: hybrid fast/fast fiber (type
IIA+IID). Sample size: 0.3 nl fiber per electrophoretic well. Details
regarding the preparation of single fibers, their solubilization in
SDS-PAGE solubilizing buffer, and their microelectrophoretic analysis
by SDS-PAGE are described in
METHODS.
|
|
In Table 3 are shown, for each strain- and
age-fiber group examined, the proportions of each fiber type, expressed
as percent of the number of fibers examined per rat (%fR),
the actual number of fibers, and percent of the total number of fibers
examined per age group (%fT).
The data presented in Table 3 were generated by analyses of 472 randomly dissected fibers (minimum 10 fibers/rat and 73 fibers/age
group). It is important to note that a relatively large number of
hybrid fibers were detected among the fiber segments dissected from the
soleus muscle of SHRs of all ages and from soleus muscles of 4-wk-old
WKY rats.
The proportions of type I and type I+IIA fibers isolated from the
soleus muscles of 4-wk-old SHR were, on average, 1.4 and 1.9 times
lower than those for 4-wk-old WKY rats. However, the proportion of type
IIA fibers isolated from 4-wk-old SHR soleus muscle was 4.9 times
greater than that for soleus muscle of age-matched WKY. Moreover,
~13% of the fibers dissected from the 4-wk-old SHR soleus muscles
were type IIA+IID fibers, whereas no such fibers were found among the
fibers dissected from the age-matched WKY soleus.
An increase in age was found to be accompanied, in the two strains, by
an increase in the proportion of slow (type I) fibers and a general
decrease in the proportion of fast (type IIA, I+IIA, and IIA+IID)
fibers. It is important to note, however, that the proportion of IIA
fibers found in the soleus muscle of 24-wk-old SHRs was significantly
greater than that in the age-matched WKY rats. Moreover, the 24-wk-old
SHR soleus produced a sizable proportion of hybrid fibers (8.1% type
IIA+IID and 4.9% I+IIA), whereas no fibers of these two types were
detected among the fibers sampled from the soleus muscle of 24-wk-old
WKY rats. As seen in Table 3, the most striking difference with respect
to the proportion of fiber types produced by soleus muscles from SHRs
and WKY rats relates to the significantly larger proportion of fast
fibers (pure type IIA and hybrid fast/fast type IIA+IID) and hybrid
fibers (types I+IIA and IIA+IID) detected among the fibers sampled from soleus muscles of SHR of all ages examined.
Correlation between MHC isoform composition, MLC2
composition, and fiber type populations. As shown in
Fig. 5A,
the MHCI composition (%MHCI) of all muscles examined in this
study was directly proportional to their relative content of
MLC2s (MLC2s/MLC2t). Furthermore, for all muscles
analyzed, there was a tight correlation between the relative
content of each MHC isoform detected
(%MHCi) in the muscle and the proportion of
corresponding fiber type derived from the analysis of single fiber
segments obtained by random microdissection of the contralateral soleus
muscle (Fig. 5B). When calculating
the proportion of fibers corresponding to each MHC isoform (i.e., MHCI,
MHCIIa, and MHCIId/x) represented in the muscles examined, the hybrid
fibers that expressed two MHC isoforms (i.e., fiber types I+IIA and
IIA+IID) were apportioned with equal weights to each of the two
respective MHCi pools. The good correlation found in this study between
the MHC isoform composition of the whole soleus muscle and the
proportion of fiber types produced by single fiber dissection indicates
that, when the sample size is sufficiently large, the proportion of
fiber types in the sample of dissected fibers is a reasonable
approximation of the fiber type composition of the whole muscle.

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Fig. 5.
A: relationship between the proportion
of MLC2s isoform (MLC2s/MLC2t) and
MHCI isoform (%MHCI) in soleus muscles from SHRs and WKY rats. Data
points, representing mean values for 6-8 rats, were obtained from
Table 2 and Fig. 3B.
B: correlation between relative
content of each MHC isoform detected
(%MHCi) in soleus muscle and proportion of
corresponding fiber type derived from analysis of single fiber segments
obtained by random microdissection of the contralateral soleus muscle.
In calculating proportion of fibers corresponding to each MHC
isoform (i.e., MHCI, MHCIIa, and MHCIId) represented in the muscles
examined, hybrid fibers that expressed 2 MHC isoforms (i.e., fiber
types I+IIA and IIA+IID) were apportioned with equal weights to each of
the 2 respective MHCi pools.
|
|
Calcium-activation characteristics of single
fibers. Fiber diameter,
CaFmax/CSA, sensitivity to
Ca2+ of the isometric force
response (pCa50), and the degree
of cooperativity within the myofibrillar compartment with respect to
calcium regulation of the contractile response
(nCa) were
determined as described in the methods for individual fiber segments
isolated from soleus muscles of 4-, 16-, and 24-wk-old WKY and
age-matched SHR before electrophoretic analyses. The results are
summarized in Tables 4-6.
As seen in Table 4, for each strain and
each fiber type group that allowed a meaningful statistical analysis
(at least 3 data points per sample), an increase in rat age was
accompanied by a statistically significant increase in fiber diameter.
Also, the diameter of type I and type IIA fibers from 16-wk-old SHRs appeared to be slightly but significantly larger than that of age-matched controls. The lack of statistically significant differences between diameter values of other groups of fibers may be due to the low
number of fibers in the respective groups.
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Table 4.
Fiber diameter and CaFmax/CSA of electrophoretically
typed, single soleus muscle fibers from WKY rats and age-matched
SHR
|
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The increase in age from 4 to 16 wk was accompanied by a statistically
significant increase in the
CaFmax/CSA developed by type I
fibers from the soleus muscle of WKY rats (+35%) and SHR (+57%) (see
Table 4). No further significant increase in average CaFmax/CSA was observed for either
strain when the rat age increased from 16 to 24 wk. A similar trend was
observed for all other fiber type groups.
For each fiber type group,
CaFmax/CSA developed by SHR soleus
muscle fibers were not significantly different from those recorded for
the age-matched WKY at all three ages examined. Assuming that the force
per cross bridge does not change, this suggests that there were no
differences in the number of cross bridges per one-half sarcomere
length per cross-sectional area.
Because SHR soleus contained a larger percentage of MHCIIa than MHCI
and yielded a larger proportion of type IIA fibers than the WKY rat
soleus (see Tables 2 and 3), it was of interest to examine whether type
IIA fibers developed a lower
CaFmax/CSA than the type I fibers,
which may explain in part the lower specific tetanic force reported in
the literature (2, 7, 12, 13, 19) for SHR soleus. The comparison of the
pooled data for type I and type IIA fibers from SHR and WKY rat soleus
showed that type IIA fibers developed a statistically significantly
lower CaFmax/CSA
(P < 0.02; Mann-Whitney test) than
type I fibers for 4-wk-old rats, but not for the other age groups.
Table 5 shows the summary of the
Ca2+-sensitivity data. Types IIA
and IIA+IID fibers showed a significantly lower sensitivity to
Ca2+ by a factor of 1.3 (lower
pCa50 values by ~0.1) than type
I fibers. Interestingly, hybrid (I+IIA) fibers exhibited either
pCa50 values characteristic of
type I fibers (4- and 16-wk-old WKY rats) or of type IIA (4-wk-old
SHRs) fibers. The hybrid fast fibers (type IIA+IID) displayed
Ca2+ sensitivities similar to
those of pure type IIA fibers. For all fiber type groups, no
significant differences were observed between the average
pCa50 values recorded for single
fibers of the same type from soleus muscles of WKY rats and age-matched
SHRs. Assuming that the length of the myosin and actin filaments does
not change with age, the consistently higher
pCa50 values by 0.1 log units noted for all fiber types from 4-wk-old animals of both strains may be
related to differences in sarcomere length or interfilament distance
(28).
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Table 5.
Sensitivity to calcium of the myofibrillar compartment in chemically
skinned, electrophoretically typed, single soleus muscle fibers from
WKY and age-matched SHR
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The degree of cooperativity within the myofibrillar compartment with
respect to contractile activation is indicated by the value of the Hill
coefficient (see METHODS). As shown
in Table 6, type I fibers have a lower Hill
coefficient than the fibers containing fast MHC isoforms (type IIA and
IIA+IID). The hybrid type (I+IIA) fibers displayed Hill coefficients
that were characteristic of either type I or type IIA fibers. No
significant age-related differences were detected between the
nCa values for
soleus fibers from either strain or between the
nCa values for
soleus fibers from SHRs and WKY rats.
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Table 6.
Degree of cooperativity within myofibrillar system with respect to
calcium regulation of contractile response in chemically skinned,
electrophoretically typed, single soleus muscle fibers from WKY
rats and age-matched SHR
|
|
 |
DISCUSSION |
This is the first study in which soleus muscles from SHRs and
normotensive (WKY) rats, at three stages in the development of high
blood pressure, were examined with respect to MHC isoform expression
(%MHCi), MLC2 isoform expression (MLC2s/MLC2t), and fiber type populations (%fT),
using electrophoretic analyses of muscle proteins from whole muscle
homogenates and segments of randomly dissected single muscle fibers.
For all muscles examined, the data produced by these three independent
electrophoretic methods were closely correlated (Fig. 5).
Calcium-activation properties of the contractile apparatus in
electrophoretically typed fibers from soleus muscles of the two strains
were also determined using permeabilized single fiber preparations.
There was good agreement between the MHC composition of soleus muscle
from WKY rats found in this study by SDS-PAGE and the MHC/fiber type
composition of soleus muscle from normotensive rats of similar age,
determined by others using histochemical techniques. For example, the
MHC composition of soleus muscles from 24-wk-old WKY rats found in this
study (MHCI: 89%; MHCIIA: 11%) (Table 2) is in close agreement with
the fiber type composition reported by Armstrong and Phelps (1) for 25- to 26-wk-old Sprague-Dawley rats (87% type I fibers, 13% type IIA
fibers), by Kovanen (17) for 25-wk-old Wistar rats (90% type I fibers,
10% type IIA fibers), and by Lewis et al. (19) for 12- to 17-wk-old
WKY rats (93% type I fibers, 7% type IIA fibers).
A major conclusion from this study is that at all stages in the
development of hypertension, soleus muscles of SHRs contain a
statistically significantly higher proportion of MHCII and MLC2f isoforms than soleus muscles from age-matched WKY rats. This conclusion was further supported by the data obtained from the fiber type population study, which showed that random sampling of single fiber
segments from the SHR soleus produced a higher proportion of fibers
expressing MHCII isoforms (type IIA; type I + IIA, and type IIA+IID)
than from soleus of age-matched WKY rats. As calculated from the data
in Table 3, the average percentages of type I and type I+IIA fibers
sampled from the soleus muscle of 4-wk-old SHRs were 1.4 times and 1.9 times lower than those from the age-matched WKY soleus. A similar
difference in the proportion of type I and type IIc/IntI (probably type
I+IIA) fibers was reported for soleus muscles from 4-wk-old SHRs and
WKY rats by Benbachir-Lamrini et al. (3, 4). By comparison, in the
present study it was found that soleus muscles from 4-wk-old SHRs
produced approximately eight times more fast fibers than the soleus
from the age-matched WKY rats. A major contributor to this difference
was the subpopulation of hybrid fast/fast (IIA+IID) fibers, which
amounted to 13.3% of the total number of fibers examined for this SHR
age group, but was not detected in the fiber population dissected from
WKY muscle. In the studies of Benbachir-Lamrini et al. (3, 4), the
4-wk-old SHR soleus contained only 1.7-2 times more type IIA fibers than the age-matched WKY soleus and displayed no IIA+IID fibers.
We attribute this difference between our and Benbachir-Lamrini's results to the higher sensitivity and resolving power of the
microelectrophoretic method particularly with respect to hybrid fibers
(5). An increase in age from 4 to 24 wk was accompanied by an increase
in the proportion of type I fibers and a decrease in the proportion of
type II fibers produced by soleus muscles from both rat strains. As a
result of the decrease in fast fibers, the 24-wk-old WKY soleus yielded only 2.5% fast fibers (type IIA), whereas the 24-wk-old SHR soleus yielded ~21% fast fibers (comprising both IIA and IIA+IID fiber types) (Table 3). The higher proportion of fast fibers sampled from SHR
soleus throughout maturation observed in this study is in agreement
with the data obtained by Benbachir-Lamrini et al. for 8- to 10-wk-old
(3) and 12-wk-old (4) animals and by Lewis et al. (19) for 12- to
17-wk-old rats, but strongly disagrees with the data of Atrakchi et al.
for 16- to 18-wk old rats (2) and Gray for 24- to 28-wk-old rats (11).
The microelectrophoretic analyses of single muscle fibers in this study
have provided valuable new information concerning the presence and the
percentage of hybrid fibers (I+IIA and IIA+IID) in soleus muscles from
SHRs and WKY rats. As indicated by Hämäläinen and
Pette (14) and Bottinelli et al. (5), identification of these fibers
can be achieved by microelectrophoretic methodology but not by
histochemical or immunohistochemical
techniques. In this study it was found
that at 4 wk of age, WKY rat soleus yielded almost twice as many hybrid
slow/fast fibers (I+IIA) as the SHR soleus; however, this population of
hybrid fibers decreased quite dramatically to nondetectable levels with
rat maturation. In contrast, maturation in SHRs was accompanied by only
a slight decrease in the percentage of type I+IIA fibers sampled from
soleus muscle. Furthermore, according to our data, soleus muscles from SHR of all ages produced a sizable proportion of hybrid fast/fast fibers (IIA+IID), ranging from ~13% at 4 wk to ~8% at 24 wk,
whereas only a very small proportion of such fibers (~1%) was
yielded by soleus of 16-wk-old WKY rats. The presence of hybrid fibers is regarded by Pette and Staron (22) as an indicator of muscle transformation and their MHC composition as an indicator of the direction of fiber type transition. Seen in this context, the size of
the hybrid fiber population and the combination of MHC isoforms
detected in soleus muscles from SHRs at the three ages examined suggest
that in SHR soleus, there is an active process of fiber type transition
in the direction IID
IIA
I. This process takes place not
only in the very young rats but also persists in mature animals. In
contrast, WKY rat soleus appears to undergo fiber transition (in the
direction IIA
I) only in young rats (from 4 to 16 wk of age).
Thus, with respect to fiber transformation, the adult SHR soleus
resembles more the soleus from 4-wk-old WKY rats than that of the adult
WKY rat, suggesting that many fibers of the adult SHR soleus muscle are
still subjected to a process of transition.
The higher percentage of fast fibers and hybrid fibers in SHR than in
WKY rat soleus muscle is in full agreement with previous reports that
skeletal muscles of hypertensive humans contain a higher percentage of
fast-twitch fibers than the normotensive controls (10, 15). One can
argue that this characteristic may not be related to high blood
pressure because it occurred in 4-wk-old SHRs before the onset of
hypertension. However, one cannot rule out a causal relationship
between hypertension and MHC expression in the soleus muscle because it
is clear from the present study that the BP of the 4-wk-old SHR was
significantly elevated compared with the age-matched controls (Table
1).
In this study we did not find any statistically significant difference
between single muscle fibers of the same type from SHR and WKY soleus
muscles with respect to the
CaFmax/CSA and the sensitivity to
Ca2+ of the contractile machinery.
Furthermore, the degree of cooperativity of the contractile apparatus
and Ca2+-regulatory system with
respect to Ca2+ regulation of the
contractile response, expressed by the Hill coefficient, was similar in
single fibers of the same type from soleus muscles of SHRs and WKY
rats. Nevertheless, the pooled data for type I and type IIA fibers from
SHR and WKY rat soleus showed that, in soleus muscles of 4-wk-old
animals, there was a clear tendency for the type IIA fibers to develop
lower CaFmax/CSA compared with the
type I fibers (69.2 ± 15.0 kN/m2,
n = 12, vs. 100.6 ± 7.6 kN/m2,
n = 57;
P < 0.02, Mann-Whitney). The higher
percentage of type IIA fibers and the lower percentage of type I fibers
in SHR soleus may explain in part previously reported observations with
intact soleus muscles from young SHRs (less than 10 wk old), which
developed 15% lower tetanic forces compared with their counterparts
from WKY rats (2, 4). The difference in the maximal
Ca2+-activated specific force
between type I and type IIA fibers was not statistically significant
for the older rats of either strain.
In summary, the present study demonstrates that although for a given
fiber type there are no significant differences between SHR and WKY rat
soleus muscles with respect to any of the
Ca2+-activation properties
examined, the soleus muscles from SHRs and WKY rats are at different
states of transformation as reflected by major differences in MHC
composition and fiber type populations.
 |
ACKNOWLEDGEMENTS |
We thank Domenica Trifilo for excellent technical assistance and
the Animal House at Baker Medical Research Institute (Melbourne, Australia) for assistance with the animal work. We acknowledge the
contribution made by Donna Matthews in the preliminary stage of this study.
 |
FOOTNOTES |
This work was supported by the National Health and Medical Research
Council (Australia).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: G. M. M. Stephenson, School of Life
Sciences and Technology, Victoria Univ. of Technology, PO Box 14428, MCMC, Melbourne, Victoria 8001, Australia.
Received 13 May 1998; accepted in final form 7 December 1998.
 |
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