MHC polymorphism in rodent plantaris muscle: effects of
mechanical overload and hypothyroidism
Vincent J.
Caiozzo,
Fadia
Haddad,
Mike
Baker,
Sam
McCue, and
Kenneth M.
Baldwin
Departments of Physiology and Biophysics and Orthopaedics,
College of Medicine, University of California, Irvine, California 92717
 |
ABSTRACT |
In a previous study, it was shown that a
combined treatment of hyperthyroidism and hindlimb suspension
effectively converted the slow-twitch soleus muscle to a fast-twitch
muscle. The objective of this study was to test the hypothesis that
hypothyroidism [absence of triiodothyronine
(
T3)] and mechanical overload (OV) would convert the plantaris (Plan) muscle from a fast- to a slow-twitch muscle. Single-fiber analyses demonstrated that the normal rodent Plan
muscle was composed of ~13 different fiber types as defined by myosin
heavy chain (MHC) isoform content. The largest proportion of fibers
(~35%) coexpressed the fast type IIX and IIB MHC isoforms (i.e.,
type IIX/IIB fibers). In this context, the combined intervention of
T3 and OV produced a significant reduction in the
relative proportion of the fast type IIB MHC isoform and a concomitant increase in the slow type I MHC isoform. These transitions were manifested by a large decrease in the proportion of type IIX/IIB fibers
and a large increase in fibers coexpressing all four MHC protein
isoforms. The mechanical consequences of these transitions, however,
were modest, producing a 15% decrease in maximal shortening velocity.
The findings of this study demonstrate that
T3 + OV does produce a partial shift toward a slower phenotype;
however, the high degree of polymorphism found in the Plan muscle
represents a unique design that appears to minimize the functional
consequences of these significant MHC transitions.
single fiber; messenger RNA; rat; hybrid fibers; protein
 |
INTRODUCTION |
MYOSIN HEAVY CHAIN (MHC) isoform composition in rodent
skeletal muscle is controlled by a number of factors, including thyroid hormone (triiodothyronine, T3) and loading state (2, 3, 5,
7-9, 15, 18-21, 23, 25, 26). In general, hyperthyroidism (+T3) and hindlimb suspension (i.e., mechanical unloading;
HS) upregulate the fast MHC protein isoform content while concomitantly downregulating the content of the slow MHC protein isoform (2). In
contrast, hypothyroidism (
T3) and mechanical
overload (OV) produce opposite effects (9, 25, 26).
In an attempt to more rigorously examine the commonality or lack
thereof between the effects of +T3 and HS on MHC isoform composition, we recently performed extensive quantitative analyses comparing the effects of these two interventions on the MHC isoform composition of single fibers in the soleus (Sol) muscle (2, 3, 15).
These analyses yielded two key findings that clearly differentiated the
effects of +T3 from HS in the Sol muscle. First, +T3 affected a larger proportion of slow-twitch type
I fibers than did HS. This finding suggests that there are
subpopulations of slow-twitch type I fibers in the Sol muscle that
differ in their sensitivity to thyroid vs. loading state. Second,
although +T3 and HS produced significant pools of fibers
that coexpressed various combinations of slow and fast MHC isoforms
(i.e., so-called polymorphic or hybrid fibers), +T3
produced a profile of polymorphic fibers that differed substantially
from that induced by HS (2, 3).
The comparison between the separate effects of +T3 and HS
was complemented by an additive paradigm whereby both interventions were imposed simultaneously (2, 3, 15). +T3 + HS
effectively converted the Sol muscle to a fast-twitch muscle, as
evidenced by MHC protein isoform and contractile data. At the
single-fiber level, this transformation was characterized by 1)
the de novo expression of the fast type IIB MHC isoform and 2)
a large pool of fibers (~60% of the total population) that
coexpressed all four adult MHC isoforms (2, 3).
One possible interpretation of this additive/synergistic effect
produced by +T3 + HS is that the effect of thyroid state on MHC isoform expression might be dependent on loading state. To further
explore this possibility, the present study employed an approach
analogous to that described above but contrasted, instead, the effects
of
T3, mechanical overload (OV), and
T3 + OV on the MHC isoform expression in the
fast-twitch plantaris (Plan) muscle. In a previous study (5), we noted
that
T3 had little effect on the native myosin
isoform composition of the fast-twitch Plan muscle. If loading state of
the muscle modulates the effect of thyroid state, then we hypothesize
that
T3 + OV should produce alterations in MHC
isoform expression greater than those produced by
T3 alone.
The findings of this study demonstrate that the normal rodent Plan
muscle contains a complex spectrum of muscle fiber types (i.e., ~13
different pools of fibers as defined by MHC isoforms) previously
unrecognized. The largest proportion of fibers coexpressed the fast
type IIX and IIB MHC isoforms (~35% of the total population). Although
T3 had little effect on the
distribution of MHC isoforms, OV produced a significant reduction in
the proportion of type IIX/IIB fibers and a concomitant increase in the
proportions of type IIA/IIX and I/IIA/IIX fibers.
T3 + OV produced a further reduction in the proportion of type IIX/IIB
fibers accompanied by a large proportion of fibers that coexpressed all
four MHC isoforms (~30% of the total population of fibers). This
latter finding is consistent with the hypothesis that the effect of
thyroid state on MHC isoform expression is dependent on loading state. Of the three different interventions employed in this study, only the
T3 + OV group produced a significant reduction
(
15%) in maximal shortening velocity
(Vmax,muscle). In this context, however, the high
degree of polymorphism found in the Plan muscle may represent a unique
design feature that minimizes the functional consequences of large
changes in MHC isoform composition.
 |
METHODS |
Animal care and experimental groups.
All experiments described in this study were approved by our
Institutional Animal Care and Use Committee before they were performed.
Female Sprague-Dawley rats (~250-300 g) were randomly assigned
to four groups designated 1) control (Con, n = 8),
2) hypothyroid (
T3, n = 8),
3) OV (n = 7), and 4)
T3 + OV (n = 7). All animals were housed individually and given food
and water ad libitum. Animals were killed after 6 wk of a given treatment.
Hypothyroidism was induced by daily intraperitoneal injections of
propylthiouracil (PTU; 12 mg/kg body wt). We have demonstrated, in
previous studies, that heart weight and heart weight normalized to body
mass decrease under hypothyroid conditions (5, 6, 9). Hence, the
effectiveness of PTU in creating a hypothyroid condition in the present
study was verified using heart weight and heart weight-to-body weight ratios.
OV of the left and right Plan muscles was accomplished by anesthetizing
the animal with acepromazine (4.5 mg/kg) and ketamine (75 mg/kg) and
then removing the Sol and gastrocnemius muscles. Previous studies by a
number of investigators (11, 13, 17, 23, 25) have shown that this
technique produces substantial hypertrophy of the Plan muscle.
After 6 wk of a given intervention, animals were anesthetized as
described above, and then contractile measurements were made on the
left Plan muscles. After these measurements, the left and right Plan
muscles were harvested from each animal and weighed. The midportion of
each left Plan muscle (~10-15 mm) was cut transversely and
divided into two segments. One of these segments was used for
determining the MHC isoform composition of single fibers. The second
segment was placed into cooled glycerol and stored (
20°C)
until it was used for determination of whole muscle MHC isoform
composition. The right Plan muscle of each animal was quickly frozen by
tongs cooled in liquid nitrogen. These samples were stored at
70°C until they were used for determination of the whole
muscle MHC mRNA isoform distribution via the Northern blot technique.
Contractile measurements.
Contractile measurements were made in accordance with methods described
by us previously (2, 3, 5). The distal tendon of a Plan muscle was
isolated and freed from all connective tissue along the medial and
lateral borders. The nerve and vasculature were left intact.
Contractions of the Plan muscle were produced by stimulating the
sciatic nerve with a bipolar stainless steel electrode. The stimulation
parameters were controlled using a stimulator with an isolation unit
(model S-48; Grass Instruments, Quincy, MA). All muscles were
stimulated using a pulse duration of 0.2 ms and a voltage that was two
times greater than that needed to produce a maximal twitch.
All contractile measurements were made using a computer-controlled
ergometer system (model 310; Cambridge Instruments, Watertown, MA). A
computer and digital-to-analog board (model DAS-16G;
MetraByte-Keithley, Taunton, MA) were used to control the afterload
imposed on the muscle. Additionally, the computer and an
analog-to-digital board (model DAC-06, MetraByte-Keithley) were used to
collect force and muscle length data. Force production and muscle
length data were each collected at a rate of 1,000 Hz.
The optimal muscle length was defined as the muscle length that yielded
the greatest tetanic isometric tension. All subsequent measurements
were made at this muscle length. After the determination of optimal
muscle length, the isometric twitch properties of the muscle were
determined. These properties included measurements of isometric twitch
tension, time to peak tension, and one-half relaxation time.
The force-velocity relationship of a given muscle was determined using
~12-15 different afterload conditions that ranged from ~2 to
100% of maximal isometric tension (Po). The force-velocity data of a muscle were fitted mathematically using a linearized version
of the Hill equation (2, 3, 5). This equation was also used to estimate
maximal shortening velocity of the muscle (i.e.,
Vmax,muscle). The muscle was allowed 1 min of rest
between each afterload condition. Immediately after the completion of contractile measurements, the Plan muscles were removed and processed as described above.
Hill's statistical model of the force-velocity relationship and
the importance of MHC isoform distribution at the single-fiber level.
Hill (16) developed a statistical model that described the dependence
of the whole muscle force-velocity relationship on the
force-velocity relationships of the individual fibers of that muscle.
In this regard, we attempted to predict the effects of
T3 + OV on Vmax,muscle by using
Hill's statistical model of the force-velocity relationship in
conjunction with 1) force-velocity data of single fibers with
known MHC isoform compositions (1) and 2) the distribution of
MHC isoforms at the single-fiber level, as determined in the present study.
The Hill equation can be written as
|
(1)
|
where
Po is maximal isometric tension, P is isotonic tension,
V is shortening velocity, and a and b are
constants with dimensions of force and velocity, respectively. The Hill
equation (27) can be normalized relative to Po and
Vmax as follows
By
use of substitutions of P/Po = P',
V/Vmax = V', and
a/Po = b/Vmax = 1/G, Eq. 2 can be rewritten as follows
|
(3)
|
Whereas Hill used a statistical distribution of 82 fibers with
10 distinct populations differing in maximal shortening velocity (Vmax,fiber), the statistical distributions of
fibers used in the present study were based on the actual MHC isoform
distributions observed at the single-fiber level in the populations of
Con and
T3 + OV fibers. The V' for any
given pool of fibers can be determined by dividing the shortening
velocity of the whole muscle (Vmuscle) by the
maximal shortening velocity for that given group or pool of fibers
(i.e., Vmax,fiber). The contribution of each pool
of fibers to overall force production can be determined by multiplying the value obtained from Eq. 3 by the number of fibers in each pool. For the purposes of this model, the
Vmax,fiber values of pure slow-twitch type I and
fast-twitch type IIA, IIX, and IIB fibers were assumed to be 0.64, 1.40, 1.45, and 1.8 fiber lengths per second (1). The
Vmax,fiber for any given fiber type was determined
by
where
fx is the fraction of that given MHC isoform in the
fiber and Vmax,x is the maximal
shortening velocity associated with that given MHC isoform.
Isolation of single fibers.
After removal, the muscle segment used for single-fiber MHC isoform
analyses was placed immediately into a glycerol relaxing solution (50%
glycerol, 2 mM EGTA, 1 mM MgCl2, 4 mM ATP, 10 mM imidazole,
100 mM KCl, pH 7.0). It was then quickly cut into small strips and
stored in glycerol relaxing solution overnight at
20°C. To
obtain single fibers, the muscle strips were placed into a small
dissection chamber that contained the glycerol relaxing solution.
Approximately 45 single fibers from each muscle (total 1,379, ~345
fibers/group) were then isolated using microsurgical forceps (Super
Fine Dumont tweezers, Biomedical Research Instruments, Rockville, MD)
and a dissection microscope (Technival 2, Jena, Germany) with
backlighting. Each fiber was transferred to individual polypropylene
microcentrifuge tubes (500 µl) that contained 30 µl of a running
buffer [62.5 mM Tris (pH 6.8), 1.0% (wt/vol) SDS, 0.01%
(wt/vol) bromphenol blue, 15.0% (vol/vol) glycerol, and 5.0%
(vol/vol)
-mercaptoethanol]. Each sample was heated (70°C for 2 min) and placed in a sonicator for 60 min. This method is a
modification of that initially described by Giulian et al. (12) and
published by us previously (2, 3). Approximately 15 µl of each sample
were then loaded into the well of a gel, and electrophoresis was
performed as described below.
Discontinuous PAGE separation of MHC isoforms.
MHC protein isoforms were separated using techniques described
previously (2, 3). The separating gel consisted of 8% acrylamide,
0.16% bis-acrylamide, 30% glycerol, 0.4% SDS, 0.2 M Tris (pH 8.8),
and 0.1 M glycine. This solution was degassed for ~15 min.
Polymerization was then initiated by addition of N,N,N',N'-tetramethylenediamine (0.05%
final concentration) and ammonium persulfate (0.1% final
concentration) to the separating gel solution. The separating gel was
poured, layered with ethyl alcohol, and given ~30 min to polymerize.
The stacking gel solution contained 4% acrylamide, 0.08%
bis-acrylamide, 30% glycerol, 70 mM Tris (pH 6.7), 4 mM EDTA, and 0.4 % SDS. This solution was also degassed for 15 min before addition of
N,N,N',N'-tetramethylenediamine (0.05%
final concentration) and ammonium persulfate (0.1% final concentration). It was then layered onto the separating gel. The running buffer contained 0.1 M Tris, 0.15 M glycine, and 0.1% SDS.
Myofibril samples obtained from the whole muscle segments were
denatured in a sample buffer solution that contained 5%
-mercaptoethanol, 100 mM Tris-base, 5% glycerol, 4% SDS, and
bromphenol blue. Approximately 1 µg of protein was then loaded into
each well. An SG-200 vertical slab gel system (CBS Scientific, Del Mar,
CA) was used for electrophoresis. Gels were run for ~24 h and at 270 V. This method separated the fast type IIA, IIX, and IIB and the slow
type I MHC isoforms (order of migration). MHC protein isoform bands
obtained from the whole muscle segments were visualized using Coomassie
blue G-250. Those obtained from single fibers were stained using a
silver stain kit (Bio-Rad, Richmond, CA). A personal densitometer
(Molecular Dynamics, Sunnyvale, CA) was used to scan and quantify the
MHC isoform bands.
Northern blot analyses of MHC mRNA isoform content.
The Northern blot analyses used in this study were similar to those
described by us previously (4, 14, 15). Total cellular RNA was isolated
using the RNAzol method (TEL-TEST, Friendswood, TX). Total RNA (5 µg)
was electrophoretically fractionated, transferred to a nylon membrane,
and ultraviolet fixed to immobilize the RNA on the membrane. The
membrane was dried at 80°C for 30 min and then stored dry at
4°C until subsequent hybridization.
Oligonucleotides were purchased from Chemgene (Waltham, MA). The
5'-ends of the oligonucleotide probes were labeled with
[
-32P]ATP (1-2 × 109
cpm/µg). Northern blots were prehybridized (~2 h, 5°C less than the melting temperature of the probe) in 10× Denhardt's
solution, 6× saline-sodium-phosphate-EDTA, 1% SDS, sonicated
salmon sperm DNA (50 µg/ml), and yeast tRNA (50 µg/ml).
Hybridization was performed overnight at the prehybridization
temperature in 6× saline-sodium phosphate-EDTA and 1% SDS at a
probe concentration of 1-2 × 106 cpm/ml. The
blots were then washed and exposed to an X-omat autoradiographic film
(Kodak) with intensifying screen (DuPont) at
70°C for
1-3 days, depending on the signal intensity. After signal
detection by autoradiography, the probes were washed off the blots by
boiling for 10-15 min in 1% SDS. The Northern blot membranes were
rehybridized with an excess of a 32P end-labeled 18S
oligoprobe that hybridizes to 18S rRNA. Band intensities on the
autoradiogram were quantitated using a laser scanning densitometer
(Molecular Dynamics), and each specific MHC absorbance was normalized
to its corresponding 18S signal on the Northern blots.
Statistical analyses.
All statistical analyses were performed using a computer program
(Systat, Evanston, IL). The protein MHC, mRNA MHC, and contractile data
were analyzed using a two-way ANOVA. The data for each separate MHC
isoform were analyzed independent of the other isoforms. If a
significant F ratio was obtained, Tukey's honestly significant difference test was employed to determine significant differences between groups. Differences between distributions of fiber types (e.g.,
HA distributions are dependent on
T3 and HS status) were determined using
2
analysis. Only those
2 tests that were statistically
significant are reported in RESULTS. Statistical tests were
considered significant when P
0.05.
 |
RESULTS |
Physical characteristics and muscle mass.
The heart weights (absolute and relative to body mass) of the animals
assigned to the
T3 and
T3 + OV
groups were significantly less than those of the animals assigned to
the Con and OV groups (Table 1). This
finding suggests that the administration of PTU was effective in
creating a
T3 condition.
Muscle weights are reported in Table 1. The weights of the Plan muscles
in the OV (+63%) and
T3 + OV (+39%) groups were significantly greater than those of the Con group. This finding demonstrates that synergistic ablation was very effective in producing compensatory hypertrophy in the OV and
T3 + OV groups.
Whole muscle MHC protein and mRNA isoforms.
The whole muscle MHC protein isoform data are shown in Fig.
1. The predominant MHC protein
isoforms found in the Con Plan muscles were the fast type IIX (~37%
total MHC pool) and IIB MHC isoforms (~45% total MHC pool).
Hypothyroidism had little effect on the distribution of MHC protein
isoforms. In contrast, OV and
T3 + OV produced
substantial changes. OV significantly reduced the fast type IIB MHC
isoform and concomitantly upregulated the expression of the slow type I
and fast type IIA MHC isoforms.
T3 + OV produced an
MHC isoform profile that was slower than that produced by OV alone.
This slower profile was characterized by a larger pool (~20% total
MHC pool) of the slow type I MHC protein isoform.

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Fig. 1.
Effect of absence of triiodothyronine ( T3),
mechanical overload (OV), and T3 + OV on whole
muscle myosin heavy chain (MHC) protein isoform composition of
plantaris (Plan) muscles. A: type I; B: type
IIA; C: type IIX; D: type IIB. Values are means ± SE.
OV and T3 + OV produced substantial decreases in the
fast type IIB MHC protein isoform content. OV and T3 + OV produced significant increases in slow type I and fast type IIA
MHC protein isoforms. T3 + OV produced a larger
increase in slow type I MHC isoform than did OV. a P
< 0.05 compared with control (Con). b P
< 0.05 compared with T3.
C P < 0.05 compared with
OV.
|
|
The whole muscle MHC mRNA data are shown in Fig.
2. The trends observed at the mRNA level
for the slow type I, fast type IIA, and fast type IIB MHC isoforms were
fairly consistent with that seen at the protein level. For instance, OV
and
T3 + OV produced significant reductions in the
fast type IIB MHC mRNA isoform signal, as was observed at the protein
level. However, there was a discrepancy between the mRNA and protein
data for the fast type IIX MHC isoform.

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Fig. 2.
Effects of T3, OV, and T3 + OV on
whole muscle MHC mRNA isoform signals. Values are means ± SE. OV and
T3 + OV produced substantial reductions in fast type
IIB MHC mRNA signals. T3 + OV also produced a
substantial increase in slow type I MHC mRNA signal.
a P < 0.05 compared with Con.
b P < 0.05 compared with
T3. C P < 0.05 compared with OV. A: type I; B: type IIA;
C: type IIX; D: type IIB.
|
|
Single-fiber MHC protein isoform composition.
As indicated previously (2, 3), single-fiber analyses of MHC isoform
composition can be considered valid only if two criteria are met:
1) random sampling and 2) adequate number of observations. With respect to this latter issue, ~1,300 single fibers
were analyzed in this study. The regression analyses between single-fiber and whole muscle analyses yielded a
coefficient of determination of 0.93 (Fig.
3). Importantly, the slope approximated 1.0 and the y-intercept was close to 0. Another approach that could
be used to validate the single-fiber methods used in this study would
be to simply compare distributions obtained from several different
studies. Within this context, we recently examined the developmental
time course of MHC isoform expression in the Plan muscle (10). The
patterns of single-fiber polymorphism observed at 40 days postpartum
are remarkably similar to those shown for the Con Plan muscles in Fig.
4. Collectively, these findings provide a
high degree of confidence in the single-fiber MHC isoform distributions shown in Fig. 4.

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Fig. 3.
Regression analyses between percentage of whole muscle myosin MHC
isoform composition predicted by single-fiber (total n = 1,379 fibers) analyses and actual whole muscle analyses. Mean percentage of a
given MHC isoform determined from single-fiber analyses within a given
group (e.g., Con group) was calculated as follows: fraction of a given
MHC isoform (fMHC,fiber type) within a given fiber type
was multiplied by fraction of that pool of fibers
(ffibers). Resulting value was then multiplied by
cross-sectional area for a given fiber type
(xsectfiber type), and product for a given MHC isoform
was summed across all fiber types (e.g., sumMHC I). This
value was then divided by total sum (i.e., sumMHC I + sumMHC IIA + sumMHC IIX + sumMHC IIB) and multiplied by 100. This yielded an
estimate of relative proportion of a given MHC isoform. , Relative
amount of slow type I MHC isoform from 4 different groups; ,
relative amount of fast type IIA MHC isoform from 4 different groups;
, relative amount of fast type IIX MHC isoform from 4 different
groups; , relative amount of fast type IIB MHC isoform from 4 different groups.
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Fig. 4.
Single-fiber MHC protein isoform composition of Plan muscles. All
possible combinations of MHC isoform expression are shown along
x-axis. Each bar represents proportion of a given population
relative to total population of fibers examined for that group. Solid,
dark gray, light gray, and open bars represent relative amounts of slow
type I, fast type IIA, fast type IIX, and fast type IIB MHC protein
isoforms, respectively, found within any given fiber type. Con Plan
muscles contained a large number of polymorphic fibers with predominant
population coexpressing fast type IIX and IIB MHC protein isoforms.
T3 + OV produced a significant reduction in
proportion of type IIX/IIB fibers and a large increase in fibers
coexpressing all 4 MHC protein isoforms. A: Con; B:
T3; C: OV; D: T3 + OV.
|
|
The single-fiber MHC isoform data are shown in Fig. 4. With respect to
the single-fiber distribution of the Con Plan muscles, there are four
key findings. First, only ~33% of the fibers exclusively expressed
one type of MHC isoform. In this context, it was surprising to observe
that fibers expressing solely the fast type IIB MHC isoform represented
only 10-12% of the total population of fibers. Second, the
majority of fibers (i.e., ~66% of the total population) exhibited
various forms of polymorphism. Third, collectively the Con Plan muscles
contained ~13 different types of fibers. Finally, the fast-twitch
type IIX/IIB fibers represented the largest proportion of fibers
(~33% of the total population) in the Con Plan muscles.
As discussed previously, the whole muscle analyses (Fig. 1) suggest
that
T3 had little effect on the relative
distribution of MHC protein isoforms. The single-fiber analyses are
largely consistent with this perspective (Fig. 4); however, there were subtle differences between the population distributions of the Con and
T3 groups.
The whole muscle analyses demonstrated that OV produced a significant
reduction in the relative content of the fast type IIB MHC protein
isoform (Fig. 1). This was manifested in two different ways at the
single-fiber level: 1) there was a selective reduction in the
relative proportion of the fast type IIB MHC isoform within the IIX/IIB
pool of fibers (Fig. 4), and 2) there was a reduction in the
proportion of fast-twitch type IIX/IIB fibers (Fig. 4). In addition to
these alterations, the OV Plan muscles contained larger proportions of
type IIA/IIX and I/IIA/IIX fibers than did the Con or
T3 Plan muscles (Fig. 4).
The whole muscle MHC protein isoform analyses demonstrated that
T3 + OV produced substantial decreases in the
relative amount of the fast type IIB MHC protein isoform and a
concomitant increase in the slow type I MHC protein isoform. As shown
at the single-fiber level (Fig. 4), the reduction in the relative
amount of the fast type IIB MHC protein isoform occurred primarily as a
result of the loss of type IIB and IIX/IIB fibers. The upregulation of
the slow type I MHC protein isoform resulted from increases in the proportions of types I/IIA, I/IIA/IIX, and I/IIA/IIX/IIB fibers.
Contractile data.
The contractile data are reported in Table
2. Consistent with the muscle mass data
(Table 1), OV and
T3 + OV produced significant increases in Po. Neither
T3 nor OV had
an effect on Vmax,muscle. In contrast,
T3 + OV did produce a significant reduction
(~15%) in Vmax,muscle. Consistent with this
observation, the Hill statistical model predicted an ~11% decrease
in Vmax,muscle (see Fig. 6) for the
T3 + OV group. In accordance with the transition to
a slower phenotype,
T3 + OV also produced
significant increases in time to peak tension and one-half relaxation
time.
 |
DISCUSSION |
Four unique findings of this study are highly relevant to the field of
muscle plasticity. First, the single-fiber analyses demonstrate that
the normal Plan muscle contains a large proportion of polymorphic
fibers (i.e., fibers coexpressing MHC isoforms), with the largest
proportion (~35% of the total population) of these coexpressing the
fast type IIX and IIB MHC isoforms. Second, the findings of this study
provide further evidence to suggest that loading state plays a more
significant role than thyroid state in modulating the MHC isoform
composition of fast-twitch skeletal muscle. Third, the interactive
effects of
T3 and OV suggest that the response of
skeletal muscle to altered thyroid state is dependent on the loading
state of the muscle. Finally, the high degree of polymorphism found in
the Plan muscle may represent a unique strategy for
minimizing the functional impact of myosin isoform transitions.
Polymorphic expression of MHC protein isoforms in single fibers of
the rodent Plan muscle.
In previous studies (2-4), we examined the MHC composition of
single fibers from rodent Sol and white medial gastrocnemius muscles.
Under normal conditions, the majority of fibers in both of these
muscles express only one MHC protein isoform. In this regard, these two
muscles represent the extremes of a spectrum of possible combinations
of MHC protein isoform expression. We have also shown (2-4),
however, that perturbations of the Sol and white medial gastrocnemius
muscles, via manipulation of thyroid hormone level or mechanical
loading, lead to dramatic increases in the number of fibers
coexpressing various combinations of MHC protein isoforms (i.e.,
polymorphic fibers).
As stated above, it has become clear that a number of perturbations can
produce transitions that give rise to the polymorphic expression of MHC
isoforms within a given muscle fiber. However, the prevailing
impression has been that muscles under steady-state conditions contain
few polymorphic fibers. Clearly, on the basis of the findings of
previous investigators (22, 24) and the findings shown in Fig. 4, it is
clear that this view needs to be modified. The normal Plan muscles, in
the present study, contained at least eight different populations of
polymorphic fibers, with the largest proportion of fibers coexpressing
the fast type IIX and IIB MHC isoforms.
Control of MHC isoform expression by thyroid hormone and loading
state: is one more dominant than the other?
Two different approaches have been used to compare the relative
importance of thyroid hormone and loading state in regulating MHC
isoform expression. The first approach (2, 3, 15) simply has contrasted
the separate effects of each intervention. The second approach (9, 17,
25) has employed a competitive strategy whereby one manipulation (e.g.,
hyperthyroidism) is used to counteract the effects of the other (e.g.,
mechanical loading).
As shown in this and other studies (17, 25), the loading state imposed
on the Plan muscle appears to play a more important role than thyroid
state (e.g., hypothyroidism) in modulating MHC protein and mRNA isoform
profiles (Figs. 1 and 4). In the present study we found that
T3 produced only minor changes in the single-fiber distribution of MHC isoforms. OV, in contrast, produced a significant reduction in the relative amount of the fast type IIB MHC protein isoform that was manifested by 1) a reduction in the proportion of fast-twitch type IIX/IIB fibers and 2) a selective reduction in the relative amount of the fast type IIB MHC isoform within the type
IIX/IIB fibers. Consistent with the concept that loading state exerts a
stronger influence over MHC expression in the rodent fast-twitch Plan
muscle, Swoap et al. (25) reported that OV significantly attenuated the
effects of hyperthyroidism on the fast type IIB MHC isoform.
Interestingly, these findings cannot be generalized to include rodent
slow-twitch skeletal muscle. In the rodent Sol muscle, the data (2, 3,
9, 15) suggest that thyroid state exerts stronger control over MHC
isoform expression than loading state. The findings of competitive
experiments are also consistent with this interpretation (9).
The competitive effects of thyroid state
(+T3/
T3) and increased neuromuscular
activity have also been examined. Kirschbaum et al. (19) examined the
antagonistic effects of chronic low-frequency stimulation (CLFS) and
thyroid state on MHC expression in rodent tibialis anterior and
extensor digitorum muscles. Their findings suggest that thyroid state
is only partially effective in blunting the effects of CLFS.
Collectively, the studies noted above suggest that, from a comparative
perspective (i.e., thyroid vs. loading state), 1) loading state
plays a more significant role in controlling the expression of MHC
isoforms in fast-twitch skeletal muscle, 2) thyroid state is a
more powerful modulator of MHC isoform expression in slow-twitch skeletal muscle, and 3) the antagonistic effects of thyroid
state are dependent on the competitor (e.g., loading state or CLFS). In
making these generalizations, however, it must be noted that the
dominance of one intervention (i.e., thyroid vs. loading state) over
the other appears to be isoform specific and time dependent. For
instance, in the present study, the major difference between the
effects of
T3 and OV was the greater repression of
the fast type IIB MHC isoform induced by OV. With respect to time
dependence, we recently observed (2) that +T3 had a greater
impact than HS on the expression of the slow type I and fast type IIA
MHC isoforms after 4 wk of intervention, whereas very little difference was found at earlier time points of 1 and 2 wk.
Interactive effects between thyroid hormone and loading states
appear to be isoform specific.
As shown in Figs. 1 and 2, the interactive effects of
T3 and OV, at the whole muscle level, were isoform
specific and appeared to be restricted primarily to the slow type I MHC
isoform. Importantly, however, this finding supports the concept that
the effect of the thyroid hormone is dependent on the loading state of
the muscle.
T3 + OV produced an increase in the slow
type I MHC isoform signal that appeared to be the sum of the individual
effects of
T3 and OV (i.e., an additive effect).
Analyses at the single-fiber level, however, revealed a much more
complex interaction between these two stimuli. Specifically, the
reduction in the proportion of type IIX/IIB fibers and the concomitant
increase in the proportion of type I/IIA/IIX/IIB fibers produced by
T3 + OV were much greater than that predicted from
the individual effects of
T3 and OV. Despite these
synergistic effects on the proportions of type IIX/IIB and type
I/IIA/IIX/IIB fibers,
T3 + OV was only partly
successful in converting the fast-twitch Plan muscle to a slow-twitch
muscle. This interactive effect is in dramatic contrast to the effect of +T3 + HS on the slow-twitch Sol muscle, whereby the
slow-twitch Sol muscle was effectively converted to a fast-twitch
muscle (2, 3).
In previous studies (11, 23) employing immunohistochemical techniques,
it has been noted that OV produces a two- to threefold increase in the
percentage of fibers exclusively expressing the slow type I MHC
isoform. Although we did not observe a similar increase in the
percentage of such fibers, we did find that OV produced an ~2.5-fold
increase in the proportion of fibers (monomorphic + polymorphic)
expressing the slow type I MHC isoform (Con ~16%, OV ~40%). This
finding raises the possibility that many of the fibers classified as
pure type I fibers in previous studies (11, 23) may have, in fact, been
polymorphic fibers that expressed the slow type I MHC isoform in
various combinations with other MHC isoforms. As noted above,
the interactive effect of OV and
T3 on the
expression of the slow type I MHC isoform was complex. Within this
context, it should be noted that ~70% of the fibers in the
T3 + OV group expressed some level of the slow type
I MHC isoform as identified by electrophoretic analyses. This finding is consistent with immunohistochemical analyses performed using a
monoclonal antibody (BAD5) specific for the slow type I MHC isoform
(Fig. 5). Hence, although
T3 + OV did not produce dramatic increases in the
relative proportion of the slow type I MHC isoform at the whole muscle
level (compared with OV alone), it significantly expanded the pool of
fibers that expressed this MHC isoform.

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Fig. 5.
Transverse section (10 µm thick) of T3 + OV Plan
muscle. Section was probed using a monoclonal antibody (BAD5) specific
for slow type I MHC isoform. Note large percentage of fibers (~65%)
that stained positive for presence of slow type I MHC isoform. On basis
of single-fiber electrophoretic analyses presented in Fig. 4, it is
clear that most of these fibers coexpressed slow type I MHC isoform in
various combinations with other MHC isoforms.
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|
Single-fiber MHC polymorphism may represent a unique design for
minimizing the functional impact of MHC isoform transitions.
The intrinsic shortening speed of a muscle is often described by
measuring its maximal shortening velocity (i.e.,
Vmax,muscle). Of the three different interventions
employed in this study, only
T3 + OV produced a
statistically significant reduction in Vmax,muscle. This decrease, however, was modest (~15%), despite a substantial reduction in the relative proportion of the fast type IIB MHC isoform
(45% Con
18%
T3 + OV) and a concomitant
increase in the slow type I MHC isoform (3% Con
21%
T3 + OV). As noted by us previously (3), the
Vmax,muscle is determined by the force-velocity
relationships of each fiber or group of fibers making up the whole
muscle. In this regard, the Vmax,muscle is largely
dependent on the interaction of two key factors: 1) the MHC
isoform composition of the muscle and 2) the distribution of
the isoforms at the single-fiber level. In this context, alterations in
Vmax,muscle must be dependent on 1) the
specific isoforms affected by a given intervention and 2) the
pattern of these alterations within individual fibers. To better
appreciate the potential impact of the transitions in the slow type I
and fast type IIB MHC isoforms induced by
T3 + OV,
Hill's statistical model of the force-velocity relationship was
utilized in conjunction with 1) force-velocity data of single
fibers published previously (1) and 2) actual distributions of
MHC isoforms observed at the single-fiber level (see
T3 + OV data in Fig. 4). Tables
3 and 4 list
the proportions of various fiber types and their corresponding
Vmax,fiber values. By using Eq. 3 and
multiplying by the proportion of any group of fibers, it is possible to
determine the contribution of each group of fibers to total force
production at any given shortening velocity
(Vmuscle). As shown in Fig.
6, this integrated model predicted a
decrease in Vmax,muscle of ~11%, which is
consistent with the 15% decline actually observed. Collectively, the
actual measurements of Vmax,muscle and the
theoretical estimates of Vmax,muscle strongly
suggest that the highly polymorphic nature of the Plan muscle
represents a design feature that minimizes the effects of substantial
transitions in MHC isoform composition on the intrinsic speed of a
muscle (e.g., Vmax,muscle). This then minimizes the effect of MHC transitions on the ability of the Plan muscle to produce
power and mechanical work under any given loading condition. In also
considering the hypertrophic response, it is clear that
T3 + OV dramatically increased the ability of the
Plan muscle to produce power and mechanical work. This was achieved by
expanding the force-generating capacity of the muscle (i.e.,
hypertrophic response) without drastically compromising its speed
properties.

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Fig. 6.
Theoretical force-velocity curves determined from integration of
Hill's statistical model with single-fiber MHC data and previously
published maximal fiber velocity (Vmax,fiber) data
from Bottinelli et al. (1). Single-fiber data used to generate these
relationships are from distributions shown in Fig. 4. FL, fiber length;
P, isotonic tension; Po, maximal isometric tension.
|
|
Summary.
Collectively, the single-fiber data reported in this study offer new
and exciting perspectives on muscle plasticity. The high degree of MHC
polymorphism and the large number of different fiber types found in the
Plan muscle are clearly surprising in light of present perspectives
about fibers expressing only one MHC isoform under steady-state
conditions. In this regard, it will be interesting to survey a broad
group of muscles to determine the applicability of these findings. OV
clearly exerts an effect on the single-fiber MHC isoform distribution
that is greater than that induced by
T3. This effect
was primarily constrained, however, to three types of fibers (IIA/IIX,
IIX/IIB, and I/IIA/IIX). Finally, the distribution of MHC isoforms
across a large number of fiber types (~13) represents a unique design
criterion that appears to minimize, at the whole muscle level, the
functional consequences of significant MHC isoform transitions.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-30346.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: V. J. Caiozzo,
Medical Sciences I B-152, Dept. of Orthopaedics, College of Medicine,
University of California, Irvine, CA 92717 (E-mail:
vjcaiozz{at}uci.edu).
Received 11 February 1999; accepted in final form 16 November
1999.
 |
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