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Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55905
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Sieck, Gary C., Louise E. Wilson, Bruce D. Johnson, and
Wen-Zhi Zhan. Hypothyroidism alters diaphragm muscle development. J. Appl. Physiol. 81(5):
1965-1972, 1996.
The impact of hypothyroidism (Hyp) on
myosin heavy chain (MHC) isoform expression, maximum specific force
(Po), fatigability, and maximum
unloaded shortening velocity
(Vo) was
determined in the rat diaphragm muscle (Dia) at 0, 7, 14, 21, and 28 days of age. Hyp was induced by treating pregnant rats with
6-n-propyl-2-thiouracil (0.05% in
drinking water) beginning at gestational day
10 and was confirmed by reduced plasma levels of
3,5,3
-triiodothyronine and thyroxine. MHC isoforms were
separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by densitometry. Isometric
Po and fatigue resistance of the
Dia were measured in vitro at 26°C, and
Vo was determined at 15°C with the slack test. Compared with control muscles,
expression of MHC-slow was higher and expression of adult fast MHC
isoforms was lower in Hyp Dia at all ages. The neonatal isoform of MHC continued to be expressed in the Hyp Dia until day
28. At each age,
Po and fatigability were reduced
and Vo was slower
in the Hyp Dia. We conclude that Hyp-induced alterations in MHC isoform expression do not fully predict the changes in Dia contractile properties.
myosin heavy chain; contractile properties; maturation; specific
force; fatigue; shortening velocity
INTRODUCTION MYOSIN is both a structural and an enzymatic protein
and is responsible for the transduction of chemical energy to
mechanical work. The head of the myosin heavy chain (MHC) interacts
with actin to form cross bridges in force generation and functions as
an adenosinetriphosphatase in cross-bridge cycling and muscle shortening (14). During early postnatal development of the diaphragm (Dia) and other skeletal muscles, a neonatal isoform of MHC (MHC-neo) is expressed, either alone or in combination with MHC-slow or MHC-2A
isoforms (17). Postnatal transitions in MHC isoform expression lead to
the singular expression of MHC isoforms within most muscle fibers that
corresponds with the histochemical classification of adult fiber types
(17, 23, 24, 27).
In both adult and neonatal skeletal muscle, a correlation exists
between MHC isoform expression and maximum shortening velocity. Single
skinned fibers, expressing the MHC-slow or MHC-neo isoforms, have
slower maximum shortening velocities than those comprising adult fast
MHC isoforms (1, 2, 21, 22, 29, 30). Among fibers expressing the
various adult fast MHC isoforms, there is a tendency toward a rank
order of maximum shortening velocities with MHC-2B > MHC-2X > MHC-2A, but some investigators (1, 2) have reported that these
differences are not significant. In fiber bundles, maximum shortening
velocity correlates with the composite of MHC isoforms expressed (5, 6,
16).
Although controversial, a correlation between muscle fiber phenotype
and maximum specific force [Po;
force normalized for fiber cross-sectional area (CSA)] has also been
reported. For example, in skinned single fibers of the rat Dia,
Eddinger and Moss (8) reported that histochemically classified type II
fibers, putatively expressing adult fast MHC isoforms, generated ~1.5 times the force of type I fibers, putatively expressing the MHC-slow isoform. In agreement, Bottinelli and colleagues (1, 2) reported that,
in rat skeletal muscle, single skinned fibers expressing adult fast MHC
isoforms generated a greater Po
than fibers expressing the MHC-slow isoform. In the cat Dia, Sieck
(25) also found that the
Po of fast-twitch motor units,
comprising type II muscle fibers, was greater than that of slow-twitch
motor units comprising type I fibers. Recently, in the developing rat
Dia, we found a correlation between the relative expression of adult
fast MHC isoforms and both the maximum shortening velocity and
Po of muscle fiber bundles (16).
As the relative expression of adult fast MHC isoforms increased with
early postnatal development, maximum shortening velocity became faster
and Po increased. Watchko and Sieck (31) also found a correlation between postnatal
changes in Dia fatigue resistance and the relative expression of adult fast MHC isoforms.
The genes controlling MHC expression are responsive to thyroid hormone
(15). Accordingly, altered thyroid status can affect the normal
developmental transitions in MHC isoform expression (10, 11). For
example, it has been reported that hypothyroidism (Hyp) delays the
appearance of adult fast native myosin in developing rat Dia (7), but
the effect of Hyp on the relative expression of the different
individual MHC isoforms is unknown. In hindlimb muscles of the adult
rat, Hyp increases the relative expression of the MHC-slow isoform, and
there is a corresponding slowing of maximum shortening velocity (6). In
the adult rat Dia, we found that, although Hyp induces a small but
significant increase in the expression of the MHC-slow isoform, the
changes in Dia contractile properties are very pronounced. This led us
to conclude that, in the adult rat, Hyp-induced changes in Dia
contractile properties cannot be solely attributed to altered MHC
isoform composition (12). In developing muscle, Hyp may interact with both the ongoing transitions in MHC isoform expression and the changes
in muscle contractile properties. Presently, there is very little
information regarding the interactions of Hyp with developmental
plasticity of either MHC isoform expression or contractile properties
of the Dia.
The purpose of the present study was threefold:
1) to determine the impact of Hyp on
the normal postnatal transitions of MHC isoform expression in the rat
Dia; 2) to determine the impact of Hyp on the
Po, fatigue resistance, and
maximum shortening velocity of the developing Dia; and
3) to evaluate the relationships
between the Hyp-induced alterations in MHC isoform expression and the changes in contractile properties.
METHODS Pregnant adult Sprague-Dawley rats (10 days gestation) were assigned to
either a control (Con) or a Hyp group. Thyroid hormone deficiency was induced by adding
6-n-propyl-2-thiouracil (PTU) to the
drinking water (final concentration 0.05%) beginning at 10 days
gestation and continuing until the pups were weaned at 21 days of age.
Thereafter, the weaned rats were provided with food and water (0.05%
PTU in the Hyp group) ad libitum. After parturition, the rats were
weighed weekly. The animals were studied at 0, 7, 14, 21, and 28 days
of age (days 0-28) because
during this initial 4-wk period, there are marked developmental
transitions in MHC isoform expression in the rat Dia (16, 18).
At selected ages, animals were anesthetized by an intraperitoneal
injection of pentobarbital sodium and killed by exsanguination. The Dia
was rapidly removed, and segments from the midcostal region were used
for either MHC analysis or the study of contractile properties. Blood
samples were analyzed for serum 3,5,3 Sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis. The methods for separating and
identifying different MHC isoforms have been previously described (16,
27). Briefly, segments of Dia (10-30 mg) were scissor minced on
ice for 40 min in a high-salt solution (300 mM NaCl, 100 mM
NaH2PO4,
50 mM
Na2HPO4, 1 mM
Na4P2O7,
and 10 mM EDTA) at pH 6.5. The extracts were centrifuged (13,000 g for 30 min) at 4°C. The
supernatant was recovered and diluted 1:10 in 1 mM EDTA and 0.1%
2-mercaptoethanol and stored overnight at 4°C to allow
precipitation of the myosin filaments. The sample was centrifuged
(13,000 g for 30 min) at 4°C to
form a myosin pellet. The supernatant was discarded, and the myosin pellet was dissolved directly in SDS sample buffer (at a final dilution
of 1:200). The denatured sample was further diluted 1:200 with SDS
sample buffer before it was loaded onto gels. Gel preparation was based
on a modification of the procedure by Sugiura and Murakami (28). A 3.5% acrylamide concentration (pH 6.8) was used in the stacking gel, whereas the resolving gel (8 × 10 cm in size, 0.75 mm thick; Hoefer SE250) consisted of a gradient of 5-8%
acrylamide (pH 8.8) with 25% (vol/vol) glycerol. All samples were run
at a constant current of 20 mA/gel until the tracking dye reached the
bottom of the gel (~1.75 h). After completion of the gel run, the
gels were removed from the plates and silver stained according to the
procedure of Oakley et al. (20). After staining, the gels were imaged
on a computerized image-processing system, and the relative expression
of different MHC isoforms was quantified densitometrically. Figure
1 shows an example of the electrophoretic separation of different MHC isoforms in the Dia at different ages.
Isometric and isotonic contractile
measurements. The methods for determining isometric and
isotonic contractile properties of the Dia have been previously
described (16). Briefly, muscle segments (one for isometric and one for
isotonic properties) were mounted in glass tissue chambers containing
mammalian Ringer solution aerated with 95%
O2 and 5%
CO2 and maintained at either 26 or 15°C. The PO2 (~430 Torr),
PCO2 (~38 Torr), and pH (~7.40)
of the Ringer solution were periodically monitored throughout the
experiment. D-Tubocurarine
(0.012 mM) was added to the bath to prevent activation of intramuscular
nerve fibers. The costal margin origin of the fibers was fixed with a
surgical clamp mounted in series with a micropositioner near the base
of the tissue chamber. A small piece of aluminum foil was glued to the
central tendon and then attached to a force transducer (model 300B,
Cambridge Technology) via a fine wire (isometric) or a glass pipette
(isotonic). These connections provided a noncompliant attachment of the
muscle bundle to the force transducer and prevented tearing of the
central tendon. The muscle bundles were stimulated directly (Grass
model S-88 stimulator and current amplifier) with rectangular current
pulses (1.0-ms duration) delivered through platinum plate electrodes
placed on either side of the muscle (~1 cm apart). To assure
supramaximal stimulation, the current was increased by 25% over the
current necessary to obtain peak twitch force responses (250-300
mA). Muscle fiber length was adjusted incrementally with a
micropositioner until maximal isometric twitch force responses were
obtained [i.e., optimal fiber length
(Lo)]. Lo was then
measured with digital calipers.
Isometric contractile properties were measured at 26°C. Muscle
fiber bundles were stimulated at varying frequencies ranging from 1 to
100 Hz, delivered in 1-s-duration trains, to determine Po. Fatigue resistance of the
muscle fiber bundle was evaluated with repetitive stimulation at 40 Hz
in 330-ms-duration trains repeated each second for a 2-min period. A
fatigue index (FI) was calculated as the ratio of the residual force
after 2 min to the initial force.
Isotonic contractile properties were measured at 15°C in a separate
muscle fiber bundle from each animal. Maximum unloaded shortening
velocity (Vo)
was determined with the "slack" test (9) in which muscle length
was rapidly shortened in a series of four to six steps ranging from 5 to 15% of Lo
while the muscle was maximally activated. Due to the rapid length
change (dL), the muscle bundle was
unloaded, force fell to zero, and the muscle shortened at maximum
velocity as the slack in the muscle was taken up. The time required for
force to redevelop (dt) was then
used to calculate
Vo
(dL/dt).
Vo was normalized
for Lo and
expressed as the relative change in optimal muscle length per second
(in ml/s). The lower bath temperature (i.e., 15 vs. 26°C) was used because of improved accuracy in measuring
Vo.
The stimulation paradigm and imposed length changes were controlled by
a computer program. Force and length signals of the Cambridge dual-mode
servo-control module were digitized at 1,000 Hz and stored on a
computer disk file. After measurement of
Po and
Vo, the
stimulated muscle segments were weighed, and CSA was estimated based on
the following formula: CSA = muscle weight (in
g)/[Lo (in
cm) · density (1.056 g/cm3)]. The
estimated CSA of the fiber bundle was then used to determine specific
force (i.e., force/CSA) of the muscle.
Statistics. All data are reported as
means ± SE. A two-way analysis of variance was used to evaluate the
data, with age and experimental condition as grouping variables. When
appropriate, an unpaired t-test was
used as a post hoc analysis to compare Con and Hyp groups. Correlations
between postnatal changes in the relative expression of all adult fast
MHC isoforms (percentage of total MHC expression) and changes in
Po, FI, and
Vo were
calculated. These correlations are reported as
r2 values instead
of r values because both positive and
negative correlations were observed. In addition, multiple stepwise
linear regression was used to determine the contribution of each MHC isoform to the correlations between postnatal MHC isoform transitions and changes in contractile properties. Statistical significance of
group differences and regressions were tested at
P < 0.05.
RESULTS In the pups treated with PTU, Hyp was confirmed by the marked reduction
in serum T3 and
T4 levels, which were below
detectable levels of the assay in the Hyp group (Table
1). In the Con animals, serum T3 levels progressively
increased with postnatal development (P < 0.05; Table 1),
whereas T4 levels peaked on
day 14. From day
14 onward, the body weights of Hyp animals were
significantly lower than those of Con animals
(P < 0.05; Table
2). In the Hyp group, body weights
stabilized on day 14 with no
subsequent growth (Table 2). This compared with the rapid growth of Con
animals that doubled their body weights during this 2-wk period
(P < 0.05; Table 2).
Table 1.
Age-related changes in serum T3 and T4 levels
in Con and Hyp rats
Table 2.
Age-related changes in body weights of Con and Hyp rats
-triiodothyronine (T3) and thyroxine
(T4) levels by radioimmunoassay.
Fig. 1.
Myosin heavy chain (MHC) isoform composition of diaphragm (Dia) was
determined by densitometric analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Representative gels
for control (Con) and hypothyroid (Hyp) animals at different ages are
shown. D0, D7, D14, D21, and D28, days 0, 7, 14, 21, and 28 of age,
respectively; MHC-neo, neonatal isoform of MHC.
[View Larger Version of this Image (48K GIF file)]
Age
Con
Hyp
T3
T4
T3
T4
Day 0
<15
3.0 ± 0.1
<15
<1
Day 7
32.5 ± 2.0*
4.7 ± 0.2*
<15
<1
Day 14
35.2 ± 5.3*
6.5 ± 0.4*
<15
<1
Day 21
70.1 ± 4.6*
3.9 ± 0.1*
<15
<1
Day 28
90.8 ± 3.7*
5.4 ± 0.1*
<15
<1
Values are means ± SE in ng/dl for 3,5,3
-triiodothyronine
(T3) and in µg/dl for thyroxine (T4). Con,
control; Hyp, hypothyroid.
*
Significant age-related difference.
Age
Con
Hyp
Day 0
6.4 ± 0.2
6.1 ± 0.5
Day 7
15.5 ± 0.2
15.3 ± 0.3
Day 14
29.6 ± 0.5
21.5 ± 0.2*
Day 21
48.1 ± 0.5
21.3 ± 0.8*
Day 28
64.4 ± 1.4
23.7 ± 0.2*
Values are means ± SE in g.
*
Significant difference from
Con.
Postnatal MHC isoform transitions. The MHC-neo isoform comprised ~69% of all MHC isoforms expressed in the Con rat Dia at birth but completely disappeared by day 28 (Table 3). During the same period, there was an increase in the relative expression of the MHC-slow isoform from ~11 to ~28% and of the MHC-2A isoform from ~23 to ~35% (P < 0.05; Table 3). From day 0 to day 7 in Con animals, there was an abrupt decrease in the expression of the MHC-neo isoform with a concomitant increase in MHC-2A isoform expression (P < 0.05; Table 3). Thereafter, the expression of the MHC-neo isoform continued to decrease rapidly, but the expression of the MHC-2A isoform remained relatively stable. Between day 7 and day 14, the decrease in MHC-neo isoform expression was matched by an increase in the expression of the MHC-slow isoform (P < 0.05; Table 3), which stabilized after day 14. Between day 14 and day 28, the disappearance of the MHC-neo isoform was primarily matched by an increase in MHC-2X isoform expression, which initially appeared at day 14 and continued to increase until day 28 (P < 0.05; Table 3). Expression of the MHC-2B isoform did not emerge until day 21, and thereafter, the relative expression of the MHC-2B isoform remained relatively low (Table 3). As a result of these developmental transitions in MHC isoform expression, the relative expression of adult fast MHC isoforms in Con animals increased from ~23% on day 0 to ~72% on day 28 (P < 0.05).
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The normal developmental transition of MHC isoform expression in the rat Dia was markedly altered in the Hyp group (Table 3). On days 0 and 7, the relative expression of the MHC-neo isoform did not differ between the Con and Hyp animals, but there were differences in the expression of the MHC-slow and MHC-2A isoforms. At both ages, the expression of the MHC-slow isoform was greater in the Hyp animals compared with the Con animals (P < 0.05; Table 3), whereas the expression of the MHC-2A isoform was lower (P < 0.05; Table 3). This difference in the relative expression of the MHC-slow and MHC-2A isoforms progressively widened with age (P < 0.05; Table 3). The relative expression of the MHC-neo isoform was significantly greater on days 21 and 28 compared with the Con animals (P < 0.05; Table 3). The expression of the MHC-2X and MHC-2B isoforms was completely inhibited in the Hyp animals (Table 3). As a result of the effect of Hyp, the relative expression of adult fast MHC isoforms did not change (from ~15% on day 0 to ~18% on day 28).
Isometric and isotonic contractile
properties. From day 0 to day 28 in Con animals,
Po increased by approximately
twofold (from ~8.4 to ~16.4
N/cm2;
P < 0.05; Fig.
2). In Hyp animals,
Po also increased from
day 0 to day
14 (P < 0.05), but
after day 14,
Po declined
(P < 0.05; Fig. 2). At each age, the
Po generated by the Hyp Dia was
much lower than that generated by the Con Dia
(P < 0.05; Fig. 2). In Con animals,
the increase in Po during early
postnatal development correlated with an increase in the relative
expression of adult fast MHC isoforms
(r2 = 0.50;
P < 0.05; Fig.
3). In contrast, in the Hyp animals, there was no correlation between adult fast MHC isoform expression and the
postnatal increase in Po
(r2 = 0.007; Fig.
3). Stepwise linear regression indicated that, in Con animals, the
postnatal decrease in MHC-neo isoform expression provided the strongest
correlation with the postnatal increase in
Po (MHC-neo,
r2 = 0.61;
P < 0.05), although the emergence of
the MHC-2B isoform also contributed significantly (MHC-neo + MHC-2B,
r2 = 0.68;
P < 0.05). Postnatal changes in the
expression of other MHC isoforms did not further contribute
individually to the overall correlation between MHC isoform expression
and Po. In the Hyp animals, there
were no individual changes in MHC isoform expression that correlated
with the postnatal changes in Po.
), progressive increase in
Po during early postnatal
development correlated with progressive increase in relative expression
of adult fast MHC isoforms. In Hyp animals (
), no such correlation
existed. Values are means ± SE.
In Con animals, the Dia FI decreased progressively from
day 0 to day
28 (P < 0.05; Fig.
4). In contrast, in Hyp animals, the FI
declined from day 0 to
day 14 (P < 0.05) but then increased thereafter (P < 0.05; Fig. 4). In
Con animals, there was a correlation between the developmental decrease
in the Dia FI and the age-related increase in the relative expression
of adult fast MHC isoforms (r2 = 0.59;
P < 0.05; Fig.
5). In Hyp animals, no such relationship existed between the FI and the relative expression of adult fast MHC
isoforms (r2 = 0.01; Fig. 5). Stepwise linear regression indicated that, in Con
animals, the postnatal decrease in MHC-neo isoform expression provided the strongest correlation with the postnatal decrease in the
FI (MHC-neo,
r2 = 0.64;
P < 0.05). Postnatal changes in the
expression of other MHC isoforms did not further contribute
individually to the overall correlation between MHC isoform expression
and FI. In the Hyp animals, there were no individual changes in MHC
isoform expression that correlated with the postnatal changes in the
FI.
), progressive decline in Dia fatigue resistance
during early postnatal development correlated with progressive increase
in relative expression of adult fast MHC isoforms. In Hyp animals
(), no such correlation existed. Values are means ± SE.
In Con animals, the
Vo of the Dia
increased nearly fourfold between day
0 and day 28 (P < 0.05; Fig.
6). In Hyp animals, the Vo also increased
with age (P < 0.05), but the
age-related change in
Vo was only
~2.5-fold (Fig. 6). Therefore, while
Vo of the Hyp Dia
was much slower than that of the Con muscle at each age, this
difference became more pronounced with age
(P < 0.05; Fig. 6). In Con animals,
the progressive increase in Dia
Vo with early postnatal development correlated with the increase in the relative expression of adult fast MHC isoforms
(r2 = 0.79;
P < 0.05; Fig.
7). In Hyp animals, there was no
correlation between the postnatal increase in Dia
Vo and the
relative expression of adult fast MHC isoforms
(r2 = 0.002; Fig.
7). Stepwise linear regression indicated that, in Con animals, the
postnatal decrease in MHC-neo isoform expression provided the strongest
correlation with the postnatal increase in
Vo
(r2 = 0.75;
P < 0.05). Postnatal changes in the
expression of other MHC isoforms did not contribute individually to the
overall correlation between MHC isoform expression and
Vo. In the Hyp
Dia, the progressive increase in
Vo correlated
only with the decrease in MHC-neo expression (MHC-neo,
r2 = 0.58;
P < 0.05).
), progressive increase in
Vo of Dia
correlated with progressive increase in relative expression of adult fast MHC isoforms. In Hyp animals (), no such correlation existed. Values are means ± SE.
DISCUSSION
The results of the present study indicate that Hyp markedly alters the normal postnatal transitions in MHC isoform expression in the rat Dia. Expression of adult fast MHC isoforms was inhibited by Hyp, whereas MHC-slow isoform expression was increased and disappearance of MHC-neo expression was delayed. In normal Con animals, the Po and Vo increased progressively during the first 4 wk of life, whereas the FI progressively declined. In Hyp animals, the Po and FI displayed no consistent change with postnatal development, whereas the Vo increased progressively, albeit at a slower rate, compared with Con animals. In Hyp animals, the Dia Po and Vo were significantly lower than those in Con animals at each age, whereas the FI remained higher. In Con animals, postnatal changes in the Po, FI, and Vo correlated with transitions in MHC isoform expression, but this was not generally the case in Hyp animals. In Con animals, the progressive decrease and eventual disappearance of the MHC-neo isoform provided the strongest predictive correlations for postnatal changes in both the Po and FI. Such relationships were not observed in Hyp animals where MHC-neo isoform expression persisted until day 28. However, in Hyp animals, the gradual decline in MHC-neo expression, albeit attenuated in comparison with Con animals, contributed to the progressive increase in Vo. We conclude that Hyp markedly affects the postnatal transitions in MHC isoform expression in the Dia and that these alterations in MHC isoform expression contribute, at least in part, to the dramatic changes in contractile properties that also occur with Hyp. However, the altered MHC isoform expression in the Hyp Dia provides no predictive power for the changes in contractile properties that occur.
In a recent study in the adult rat Dia, Gosselin et al. (12) found that a 3-wk exposure to Hyp caused only a slight increase in MHC-slow isoform expression but caused a marked reduction in both the Po and Vo. In the adult, they concluded that, although alterations in MHC isoform expression do occur with Hyp, these alterations could not completely account for the changes in contractile properties induced by Hyp. These results contrasted with those of Caiozzo et al. (6), who found that a 20-wk exposure to Hyp induced alterations in MHC isoform expression in the rat plantaris and soleus muscles that they concluded fully accounted for the decrease in Po and Vo that were also observed. As in the present study, these authors reported that Hyp appeared to suppress the expression of adult fast MHC isoforms in both muscles while increasing the relative expression of the MHC-slow isoform. It is possible that the apparent discrepancies between the effects of Hyp on the adult rat Dia that we observed and those reported by Caiozzo et al. for the plantaris and soleus muscles might relate to the duration of exposure to Hyp. In the present study, the developing rats were exposed to Hyp throughout the embryological and early postnatal periods of muscle differentiation. Accordingly, compared with adults, the effects of Hyp on MHC isoform expression were much more pronounced in the developing rats. However, the effects of Hyp on Dia contractile properties were comparable between adults and younger animals (12).
In the present study, Hyp was associated with a significant reduction in body weight beginning on day 14, similar to a previous report (11). It is likely that the protein synthesis rate was reduced in the Hyp animals and that this might have affected the Po by a reduction in myofibrillar density. In the present study, it was not possible to use weight-matched untreated rats as control animals because a decrease in caloric intake itself alters thyroid status in neonatal rats and induces changes in MHC isoform expression (3).
The genes controlling myosin expression are known to respond to thyroid hormone but in a muscle-specific manner (19). For example, it has been previously reported that in the rat the transition from MHC-neo to fast MHC isoform expression is delayed in the extensor digitorum longus (EDL) muscle (11), but the transition from MHC-neo to MHC-slow isoform expression in the soleus muscle occurs more rapidly (4, 11). The EDL and soleus muscles in the rat are predominantly composed of type IIb (MHC-2B) and type I (MHC-slow) fibers, respectively, and the effects of Hyp on genetic control of MHC isoform expression in these muscles may not reflect that occurring in a mixed muscle such as the Dia. However, as in the EDL, we found that the early postnatal transition between MHC-neo and adult fast MHC isoform expression was inhibited by Hyp while the transition from MHC-neo to MHC-slow was promoted. These results are in general agreement with previous work (7, 19), where it was found that Hyp delays the appearance of adult fast isomyosins in the developing rat Dia. However, in these previous studies, the developmental transitions in the expression of individual MHC isoforms were not directly examined.
Changes in thyroid hormone levels may play a role in the normal sequential transition of MHC isoform expression in the rat Dia because, in Con rats, T4 serum levels peaked around the same time as the rapid transition from MHC-neo to adult fast MHC isoforms. This possible correlation between serum T4 levels and developmental myosin transitions was also observed in hindlimb muscles of the rat (11). It should be noted, however, that the serum T3 levels progressively increased with postnatal development. T3 has a more potent effect on target cells than T4, and elevated T3 levels are associated with increased energy requirements and rapid growth. It is possible that peripheral conversion of T4 to T3 is required to support the rapid transitions of MHC isoforms. Accordingly, the divergence of Dia contractile properties between Hyp and Con animals became more pronounced after day 14.
The mechanism(s) by which thyroid hormone influences the developmental transitions in MHC isoform expression is unknown. Alterations in the activation of the myogenic helix-loop-helix transcription factors Myo-D and myogenin may occur (13). The changing innervation patterns during early postnatal development may also contribute to the postnatal transitions in MHC isoform expression. During the first 2 postnatal wk in the rat, innervation of the Dia transforms from polyneuronal innervation, where a muscle fiber may be innervated by more than one motoneuron, to the adult pattern of innervation, where each muscle fiber is innervated by only one motoneuron (26).
In summary, Hyp markedly altered the early postnatal transitions in MHC isoform expression in the rat Dia, resulting in a marked increase in MHC-slow isoform expression, a decrease in the expression of adult fast MHC isoforms, and a persistent expression of the MHC-neo isoform. Hyp also dramatically affected Dia contractile properties, causing a substantial reduction in both the Po and Vo. It is likely that the Hyp-induced alterations in MHC isoform expression during early postnatal development contributed, at least in part, to the dramatic changes in Dia contractile properties.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. Y. S. Prakash for comments on the manuscript and assistance in some of the studies.
FOOTNOTES
Address for reprint requests: G. C. Sieck, Anesthesia Research, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: gcs{at}Siecklab.Mayo.edu).
Received 6 February 1996; accepted in final form 28 June 1996.
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