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194
Effects of Thyroid Hormone on a-Actin and Myosin Heavy
Chain Gene Expression in Cardiac and Skeletal Muscles of
the Rat: Measurement of mRNA Content Using Synthetic
Oligonucleotide Probes
THOMAS A. GUSTAFSON, BRUCE E. MARKHAM, AND EUGENE MORKIN
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Effects of thyroid hormone on a-actin and myosin heavy chain gene expression were compared in
ventricle, soleus, and extensor digitorum longus muscles of hypothyroid rats. Changes in mRNA
content were analyzed using synthetic oligonucleotide probes complementary to the unique 3' untranslated regions of four striated myosin heavy chain mRNAs and cardiac and skeletal muscle a-actin
mRNAs. The results indicate that daily treatment with 3,5,3'-triiodo-L-thyronine (2 /n.g/100 g body
weight) increased a-myosin heavy chain mRNA content in heart muscle by 500% and decreased /3myosin heavy chain mRNA by 65% within 48 hours. /3-mRNA in extensor digitorum longus was
decreased by 60% at 48 hours while in soleus, /3-mRNA levels were not affected by 9 weeks of
treatment. Fast Ha mRNA was present in small amounts in hypothyroid soleus and increased by 150%
and 200% after 7 and 9 weeks of thyroid hormone administration, respectively. Fast lib mRNA also
was found in hypothyroid soleus and a small increase (60%) was observed after 1 day of treatment. In
extensor digitorum longus, Fast lib mRNA increased by 200% and Fast Ha mRNA decreased by 50%
after 1 week of treatment. When larger daily doses of thyroid hormone (15 /n.g/100 g body weight) were
administered, similar changes in mRNA levels were observed, except that /3-mRNA content of soleus
muscle was decreased slightly (25%). Expression of the cardiac form of a-actin was induced transiently in ventricle, but the skeletal form of a-actin mRNA in soleus and extensor digitorum longus did not
change significantly after thyroid hormone treatment. Skeletal a-actin mRNA was not found in
ventricle, and cardiac a-actin mRNA was not detected In skeletal muscles. Thus, cardiac a-actin
mRNA levels are transiently stimulated in ventricle by thyroid hormone treatment while all of the
myosin heavy chain genes expressed in adult striated muscles are affected in a complex, tissue-specific
manner. (Circulation Research 1986;59:194-201)
M
YOSIN heavy chain (MHC) isoforms in striated muscles are encoded by a highly conserved multigene family.1 At least six
MHCs are known to be expressed in striated muscles
of the rat, including embryonic and neonatal skeletal
muscle forms, fast oxidative glycolytic (Fast Ha), and
fast glycolytic (Fast lib) skeletal forms, and two cardiac types, a and /3. The /3-MHC form is also expressed
in slow skeletal muscles.2 Although the coding regions
of these genes show a high degree of homology, the 3'untranslated sequences are not as closely conserved.3
The MHC genes show distinct patterns of tissuespecific expression that are hormonally and developmentally regulated.2"7 The effects of thyroid hormone
on expression of a- and /3-MHC forms in the ventricle
have been studied most extensively (see review in
Morkin et al. 8 ). In the rat ventricle, there are three
myosin isoforms, referred to as V,_3, in order of decreasing electrophoretic mobility on nondenaturing
From the Departments of Internal Medicine and Pharmacology,
University of Arizona College of Medicine, Tucson, Arizona.
Supported by grants from the Arizona Affiliate of the American
Heart Association, the National Institutes of Health (HL-20984)
and the Gustavus and Louise Pfeiffer Research Foundation.
Address for reprints: Eugene Morkin, Department of Internal
Medicine, University of Arizona College of Medicine, Tucson, A2
85724.
Received December 10, 1985; accepted May 13, 1986.
pyrophosphate-containing gels. 9 These three forms are
comprised of two types of MHC, a and /3. The V, and
V3 forms contain two a-MHCs and two /3-MHCs, respectively, while the V2 form contains one MHC of
each type.10 Thyroid hormone has been shown to regulate the expression of ventricular myosin isoenzymes
by causing an accumulation of a-MHC mRNA and
inhibiting expression of /3-MHC mRNA." Recently,
similar alterations in the expression of these isoforms
have been reported after thyroid hormone treatment of
cultured fetal rat heart cells,12 indicating a direct action
of the hormone on this target tissue.
Alterations in myosin composition of various muscles may be physiologically significant, since the relative proportions of the isoforms seem to be directly
related to the intrinsic speed of contraction.13 14 Thyroid hormone treatment has been shown to increase the
proportion of the V, isoenyzme and the maximal velocity of shortening in isolated ventricular muscle (see
review in Morkin et al). 8 Similarly, in skeletal muscle
the shortening velocity of whole muscles and individual fibers has been shown to be closely related to the
proportion of fast-to-slow MHCs.15-17
Alterations in thyroid status also have been reported
to effect myosin isoforms in skeletal muscles. An increase in the percentage of fast (Type II) fibers in both
fast- and slow-twitch skeletal muscles has been found
after thyroid hormone administration. 161819 Con-
Gustafson el al
Control of a-Actin and MHC Gene Expression by T 3
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versely, slow (Type I) fibers have been shown to increase in rat skeletal muscles after thyroidectomy.18
Furthermore, during the development of skeletal and
cardiac muscles the normal transition of embryonic to
adult MHC isoenzyme patterns has been shown to be
inhibited by hypothyroidism and accelerated by hyperthyroidism.20'21 The question naturally arises as to
whether thyroid hormone produces these changes in
skeletal muscles by altering the expression of MHC
genes as is observed in ventricular muscle.
a-Actins also represent a family of highly conserved
proteins. In the adult rat, two major isoforms, a-cardiac and a-skeletal actin, are expressed in heart and
skeletal muscles, respectively. The hearts of neonatal
rats have been known to contain significant amounts of
a-skeletal actin mRNA, and conversely, a-cardiac actin has been shown to be expressed in neonatal skeletal
muscle.22 Thus, like the MHC gene family, the a-actin
family appears to be developmentally regulated in the
rat, but it is not clear whether thyroid hormone plays a
role in regulating the expression of these genes.
In this report, we have compared the effects of thyroid hormone treatment at two dosage levels on MHC
and a-actin mRNA expression in ventricle, soleus, and
extensor digitorum longus (EDL) muscles of the rat.
To facilitate analysis of these effects, we have developed a rapid, simple dot-blot assay using synthetic
[32P]-labelled oligonucleotide DNA probes constructed
to be complementary to the unique 3' flanking sequences of four of the six major striated muscle myosin
mRNAs and the two sarcomeric a-actin mRNAs of the
rat. The results indicate that expression of all of the
MHC genes is regulated by thyroid hormone in a highly complex, tissue-specific manner. Analysis of the
expression of the a-actin genes after administration of
3,5,3'-triiode-L-thyronine (T3) shows a transient increase in a-cardiac actin mRNA content in the ventricle, but no a-actin isoform transitions are observed in
ventricle, EDL, or soleus muscles.
Materials and Methods
Animal Experiments
Thyroidectomized male Sprague-Dawley rats
weighing approximately 200 g were obtained commercially and maintained on rat chow. Methimazole (200
mg/1) and calcium gluconate (1.5 g/1) were added to
the drinking water. Animals were weighed twice
weekly and considered to be hypothyroid only after
cessation of growth was observed. Stock solutions of
L-T3 were prepared in 0.1 N NaOH at a concentration
of 2 mg/ml and diluted in physiological saline prior to
administration. Animals received either 2 or 15 /Ag
Tj/100 g body weight daily by intraperitoneal injection. The lower dose was found to restore normal
growth to thyroidectomized rats and to result in a euthyroid myosin isoenzyme pattern.23 During treatment
with the higher dosage, normal increases in body
weight were also observed. Animals were killed by
decapitation and the tissues quickly removed and
placed in cold phosphate buffered saline. The tissues
then were weighed, and RNA was prepared.
195
Isolation of RNA
Total cellular RNA was isolated from ventricular
and skeletal muscles using the guanidinium isothiocyanate-hot phenol method.24 All plastic and glassware was pretreated with 0.1 % diethylpyrocarbonate
at 37° C for 30 minutes and autoclaved. The final RNA
pellet was resuspended in distilled H2O and stored at
-70°C.
Preparation of Synthetic Probes
Synthetic oligonucleotide probes 20 bases in length
were prepared in an Applied Biosystems DNA Synthesizer (Model 380A) and purified by electrophoresis.
The probes were labelled at the 5' end using T4 polynucleotide kinase23 to a specific activity of 2 to 4 x 108
cpm//xg. Labelled probe was separated from unincorporated [•y-32P]-ATP by filtration through small columns of Bio-Gel P-2 (Bio-Rad), precipitated in 70%
ethanol at - 70° C, and then resuspended in deionized
H2O.
The sequences of the four MHC oligonucleotide
probes used in this study have been reported earlier.7
The sequences of the two additional probes used here
are as follows (5' to 3'): a-cardiac actin,
TGCACGTGTGTAAACAAACT; and a-skeletal actin, GCAACCATAGCACGATGGTC. 22 M
Dot Blot Assays
RNA samples at a concentration of 0.5 to 2.0 mg/ml
were denatured by heating at 65°C for 15 minutes and
cooled on ice. Aliquots of 4 \x\ were spotted in triplicate directly onto dry nitrocellulose filters that had
been pretreated with 3.0 M NaCl, 0.3 M Na citrate as
described earlier.27 After spotting, filters were baked at
80°C for 2 hours. Denaturation of RNA samples by
glyoxylation or formaldehyde treatment prior to spotting did not result in any increase in the sensitivity of
the assay (data not shown). Prehybridizations, hybridizations, and wash steps were carried out essentially as
described earlier,28 with some modifications. Prior to
prehybridization, the filters were wet in deionized H2O
and prehybridized at 45°C for at least 8 hours in buffer
containing 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium dodecyl sulfate
(SDS), 0.1 % Ficoll, 10% dextran sulfate, 0.9 M NaCl,
0.18 M Tris-HCl, pH 8, 6 mM EDTA, and 0.5%
Nonidet P-40. Hybridization was carried out by heating the probe-containing solution to 65° C for 10 minutes and adding it directly to the prehybridization fluid
at a concentration of approximately 3 ng/ml in a volume of at least 5 ml per bag and about 2 ml per filter.
After hybridization for 18-20 hours the filters were
washed with four 10-minute changes of 0.9 M NaCl,
0.09 M sodium citrate at room temperature (22-23°C).
A final 1-minute wash at 50° C in the same solution
was performed, and the filters were allowed to air dry.
Filters were wrapped in plastic and autoradiography
was carried out with intensifying screens at — 70° C for
4 hours to 2 days. Afterwards, individual spots were
cut from the filters and the radioactivity was determined by liquid scintillation counting in Aquasol (New
Circulation Research
196
Vol 59, No 2, August 1986
England Nuclear Corp., Boston, Mass.). Aliquots of
total RNA prepared from rat liver were spotted at the
same concentrations as the muscle RNAs. These spots
were counted and the values were considered to represent nonspecific background hybridization.
Northern Blot Analysis
For Northern blot analysis, 10 fig of total cellular
RNA were size fractionated on 1.1% agarose gels containing 40 mM morpholinopropanesulfonic acid, pH
7.4, 10 mM Na acetate, 1 mM EDTA, and 2.2 M
formaldehyde.29 The RNA was electrophoretically
transferred to Nytran membranes (Schleicher and
Schuell, Inc.) using the procedures recommended by
the manufacturer. Filters were baked for 2 hours and
hybridized with the synthetic probes using the conditions described above.
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Results
Untreated thyroidectomized rats showed no significant change in heart weight:body weight ratio
throughout the duration of these experiments (Figure
1). Both low and high dosages of thyroid hormone
caused a rapid increase in this ratio, which was evident
by the second day of treatment. While the amount of
ventricular weight gain during the first week of treatment was similar at both dosages, the higher dosage
ultimately resulted in greater myocardial growth.
With all of the probes used in this study the amount
of radioactivity bound increased linearly as increasing
amounts of total cellular RNA were applied to the
filters up to about 8 fig per spot (Figure 2). In these
experiments, total cellular RNA was prepared from
tissues known to express each myosin isoform and
spotted on nitrocellulose filters in amounts ranging
from 0.5 to 8.0 fig. After hybridization and wash
Time
FIOURE 1. Changes in heart weight/body weight ratio of hypothyroid rats before and after treatment with two doses ofT3.
Untreated hypothyroid animals (*); animals treated with 2
fig/100 g day-i (A); and 15 fig/100 g body weight day - ' (•).
Data points represent mean ± SEM for four to six animals. T3
= 3,5,3' -triiodo-L-thyronine.
0 12
FIGURE 2. Standard curves for dot-blot assay of MHC and exact in mRNA. The a-MHC probe was hybridized to RNA prepared from hypothyroid ventricle 24 hours after a single intraperitoneal injection ofT3 (2 fig/100 g body weight). Thefi-MHC
probe was hybridized to RNA from hypothyroid ventricle. The
Fast IIa probe was hybridized to RNAfrom soleus after 7 weeks
of T3 administration at this dosage. The Fast lib probe was
hybridized to RNAfrom EDL after 1 week ofT3 treatment at the
same dosage. Cardiac and skeletal a-actin probes were hybridized to RNA from hypothyroid ventricle and soleus muscle,
respectively. Dot blots were quantitated as described in "Materials and Methods." Values are in means ± SEM of triplicate
determinations. In some cases, the SEM is within the diameter
of the symbol. • = radioactivity bound to total cellular RNA
from various rat muscles. A = nonspecific background radioactivity obtained after hybridization of the probes with similar
amounts of total RNA extracted from liver. MHC = myosin
heavy chain; T3 = 3,5,3'-triiodo-L-thyronine; EDL = extensor
digitorum longus.
steps, the dot blots were quantitated using the procedures outlined in "Materials and Methods." Specifically bound counts were obtained as the difference between total bound radioactivity and counts bound
nonspecifically to an equivalent amount of total cellular RNA from liver.
The specificity of the hybridization of synthetic oligonucleotide probes to MHC mRNAs was verified by
Northern blot analysis with RNA from ventricle and
skeletal muscles of hypothyroid and T3-treated animals. Hybridization to blots of size-fractionated total
RNA showed that all MHC probes hybridized to a
single band with a mobility of about 7,100 nucleotides
(Figure 3). This corresponds to the size reported for
MHC mRNA.30 Duplicate samples of RNA prepared
from untreated hypothyroid ventricles showed very little hybridization to the a-MHC probe (Figure 3A, lane
1) and a large amount of hybridization to the /3-MHC
Gustafson et al
1
197
Control of a-Actin and MHC Gene Expression by T 3
A. Ventricle
2
3
B. EDL
4
6
7
8
C. Soleus
9
10
-MHC
-28S
-18S
Fast
lla
Fast
lib
Fast
lla
Fast
lib
0
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FIGURE 3. Demonstration of MHC probe specificity by Northern blot analysis. About 10 ng of total RNA were size fractionated and
transferred to nitrocellulose as described in "Materials and Methods." (A) Duplicate blots were hybridized to either the a- orfi-MHC
probes. RNA in lanes I and 3 was prepared from ventricle of untreated hypothyroid rats; RNA in lanes 2 and 4 was prepared from
ventricles after 1 week ofTj treatment (2 fig/100 g daily). (B) Duplicate blots of RNA isolated from hypothyroid EDL were hybridized
to Fast lla MHC probe (lane 5), Fast lib MHC probe (lane 6), andfi-MHC probe (lane 7). (C) Duplicate blots of RNA prepared from
hypothyroid soleus muscle hybridized to Fast lla MHC probe (lane 8), Fast lib MHC probe (lane 9), andfi-MHC probe (lane 10). All
autoradiographs were obtained by 20—36 hours exposure with intensifying screens at — 70° C, except lanes 8 and 9, which were
exposed for % hours. The relative mobilities of the 18S and 28S ribosomal RNAs are indicated. EDL = extensor digitorum longus;
MHC = myosin heavy chain; Tj = 3,5,3'-triiodo-L-thyronine.
probe (lane 3). After 1 week of daily treatment of T3 at
a dose of 2 ^ig/100 g body weight, this pattern of
hybridization was markedly altered. Hybridization of
the a-MHC probe increased dramatically (lane 2)
whereas the duplicate filter (lane 4) showed much less
hybridization of the /3-MHC probe as compared to
RNA from the hypothyroid control (lane 3). Some
nonspecific binding to 28s and 18s ribosomal RNA
was observed. Since this background was only seen in
lanes where MHC mRNA was abundant, the nonspecific binding may have been produced by hybridization
of the probes to partially degraded MHC mRNAs
whose migration through the gel has been retarded by
interaction with the more abundant rRNA species. The
absence of extensive hybridization by these probes to
total RNA prepared from liver is consistent with this
interpretation. As indicated in Figure 1, nonspecifically bound counts always were less than 10% of specifically bound counts and did not interfere with estimations of specific MHC mRNA levels.
Northern blot analysis showed hybridization of both
the Fast Ua (lane 5) and the Fast lib MHC probes (lane
6) with RNA prepared from hypothyroid EDL (Figure
3B). There also was a surprisingly large degree of
hybridization to the slow /3-MHC probe (lane 7). The
strong signal observed with the /3-MHC probe presumably is caused by a switch in the hypothyroid EDL
from fast- to slow-type MHCs.
Northern blot analysis of RNA from hypothyroid
soleus (Figure 3C) showed a strong hybridization to
the /3-MHC probe after 16 hours of exposure of the
autoradiogram (lane 10). After longer exposure (96
hours), probes for Fast Ua MHC and Fast lib MHC
showed faint hybridization with a band of similar mobility (lanes 8 and 9). Probes for fetal- and embryonicMHC skeletal mRNAs did not hybridize with MHC
mRNAs either before or after T3 treatment. When analyzed by Northern blot hybridization, both cardiac and
skeletal a-actin probes hybridized to a single band of
total RNA with a mobility of approximately 1,650
nucleotides (data not shown).
Daily administration of T3 (2 ^ig/100 g body weight)
to hypothyroid rats resulted in changes in the MHC
mRNA content of muscle types examined (Figure 4).
In ventricle, there was a rapid increase in a-MHC
mRNA, which began 6-12 hours after treatment, and
by 1 day it was increased by 500% over the control
value. After 1 week of treatment, a-MHC mRNA had
declined from peak values and thereafter remained at
about 300% of control. During the first two days of
treatment, the levels of /3-MHC mRNA decreased by
about 65% and then remained fairly constant.
Another pattern of MHC gene regulation by T3 was
observed in EDL, a predominately fast-twitch muscle
(Figure 4). Levels of all three mRNAs expressed in
this muscle appeared to be regulated by T3 administration, but in very different ways. As observed in the
ventricle, /3-MHC mRNA was repressed by about 40%
after 48 hours of treatment and by 50% after 1 week.
Fast Ha MHC mRNA was decreased to about 50% of
Circulation Research
198
Ventricle
500
400
300
200
I0O
0
EDL
Soleus
a
U i i i
20
0
20
Q)
"40
Ol -60
J -80
"~ 250
200
ISO
100
50
0
-50
Vol 59, No 2, August 1986
i
II
i i i
o
2OO
FIGURE 4. Dot-blot analysis of the effect of low-dose
thyroid hormone administration (2 ng/100 g daily) on
hybridization of synthetic oligonucleotide MHC probes
with RNAfrom ventricle and skeletal muscles. The values
represent the change in filter-bound radioactivity associated with RNA from animals that received T3 as compared to untreated hypothyroid animals. Each point represents the mean ± SEM of triplicate determinations on
individual muscles from 4 to 8 animals. EDL = extensor
digitorum longus; MHC = myosin heavy chain; T3 =
3,5,3''-triiodo-L-thyronine.
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ISO
100
SO
0
SO
Time
control levels by 1 week. By contrast, Fast lib MHC
mRNA increased during this time to about 200% of
hypothyroid values.
Interesting differences also were noted between expression of the /3-MHC gene in ventricle and soleus, a
slow-twitch skeletal muscle that expresses predominantly /3-MHC mRNA (Figure 4). Rapid repression of
/3-MHC mRNA by T3 dosing is observed in ventricle,
whereas in soleus this mRNA is not repressed significantly even by 9 weeks of treatment. Furthermore, in
soleus, T3 administration stimulates production of Fast
Ila MHC mRNA, which has been shown to be produced in a small number of soleus fibers in euthyroid
rats.31 After 7 weeks of daily treatment, Fast Ha is
increased by 150% and by 200% after 9 weeks. Binding of a second fast MHC mRNA form, designated as
Fast lib, showed smaller increases (60—80%) over the
same period.
With minor exceptions, daily administration of larger doses of T3 (15 /u.g/100 g body weight) produced
similar effects on MHC gene expression (Figure 5). aMHC mRNA was induced only in ventricle and
reached levels comparable to those achieved with lower doses. At the higher dosage of T3, /3-MHC mRNA
was repressed to the same degree in ventricle as with
the lower dosage but was inhibited to a greater degree
in EDL (75% inhibition versus 50%). In soleus, /3MHC gene expression was slightly more sensitive to
the higher dose of T3, as evidenced by a 30% reduction
in /8-MHC mRNA after 1 week of treatment. The Fast
MHC mRNAs showed similar patterns of regulation.
Fast lib MHC mRNA was increased in EDL by 300%,
and Fast Ha MHC mRNA was repressed by 60% after
1 week. In soleus, Fast Ila MHC mRNA was induced,
but only after 1 week of treatment.
Daily administration of the lower dose of T3 (2
/xg/100 g body weight) caused accumulation of acardiac actin mRNA to levels 100% and 150% above
the control after 1 and 2 days of treatment, respectively, but returned toward control levels after 1 week of
treatment (Figure 6). Very little hybridization of the askeletal actin probe with ventricular RNA was observed, and this did not change after T3 injection (data
not shown). Skeletal a-actin levels did not change
significantly in either soleus or EDL muscles, and cardiac a-actin mRNA did not appear to be expressed in
these muscles.
Discussion
Recently, we have demonstrated the usefulness of
synthetic oligonucleotide DNA probes in a simple dotblot assay for studying mRNA expression in a highly
conserved multigene family.7 Evidence was presented
to suggest that a low dose of T3 was able to alter MHC
expression in ventricle and soleus muscle in a tissue
specific manner. Here we have extended our preliminary findings by examining the effects of two dosage
levels of T3 on the expression of the MHC mRNAs and
skeletal and cardiac a-actin mRNAs. The tissues examined included ventricle, soleus (a predominantly
slow-twitch muscle), and EDL (a predominantly fasttwitch muscle). The results obtained in this study warrant further discussion in terms of the highly complex
and tissue-specific actions of thyroid hormone on
MHC and a-actin gene expression.
Although the assay used to measure MHC mRNA
levels does not differentiate between changes in transcriptional activity and alterations in mRNA stability,
the reciprocal regulation of the same MHC mRNAs in
different tissues and the rapid induction of a-MHC
Gustafson et al
Control of a-Actin and MHC Gene Expression by T 3
Ventricle
EDL
199
Soleus
FIGURE 5. Dot-blot analysis of the effects of high-dose
thyroid hormone administration (15 fj.g/100 g) on hybridization of synthetic oligonucleotide MHC probes
with RNAfrom ventricle and skeletal muscles. The assay
was performed as described in Figure 3. Each point
represents the mean ± SEM of triplicate determinations
on individual muscles from 4 to 6 animals. EDL = extensor digitorum longus.
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Time
mRNA from negligible levels in the ventricle are consistent with transcriptional control. The results can be
summarized as follows (Figure 7). First, a-MHC
mRNA is regulated by thyroid hormone only in ventricle, and this gene does not appear to be expressed in
skeletal muscles. Similarly, the two Fast-MHC
mRNAs are controlled by thyroid hormone only in
skeletal muscles and are not expressed in ventricle.
Secondly, /3-MHC mRNA is induced by hypothyroidism to varying degrees in ventricle and skeletal muscles, and is repressed by thyroid hormone treatment,
although not to the same extent in all tissues. It should
also be noted that in those tissues where T3 causes
repression of MHC mRNA expression, this process is
not complete. Third, both Fast MHC mRNAs are sensitive to thyroid status in skeletal muscles, but in an
unexpectedly complex manner. For example, Fast Ila
MHC mRNA is induced in soleus by thyroid hormone
treatment, but it is repressed in EDL. Finally, a-actin
isoform transitions do not occur in response to changes
in thyroid status. However, there is a transient increase
in the amount of the cardiac isoform of a-actin mRNA
during the first two days of T3 treatment that approximately parallels the increase in a-MHC mRNA.
These findings suggest that MHC gene expression is
subject to at least two types of regulation. One type
involves the stable repression of MHC genes within a
particular muscle type. MHC genes repressed in this
manner are unresponsive to changes in thyroid status.
The second mechanism involves regulation of MHC
genes in a manner that allows the gene to respond to
changes in thyroid status with either increased or decreased expression. Processes such as tissue-specific
DNA methylation might be envisioned in the first type
of control, whereas binding of protein activator or repressor molecules to regulatory regions of the responsive genes might underlie the second type of regulation.
Control of /3-MHC gene expression provides an example of the second type of regulation. This gene is
constitutively expressed in ventricular, soleus, and
EDL muscles in the hypothyroid state. In ventricle and
EDL, the /3-MHC gene may be affected by a negative
regulatory factor that itself is under the control of thyroid hormone. This putative regulatory factor may be
absent, or present only at very low levels, in soleus
muscle and may act as a positive regulator of a-MHC
synthesis in ventricle. Alternatively, thyroid hormone
may repress the expression of a positive regulatory
factor that activates /3-MHC expression in the hypothyroid state. The apparently simultaneous activation
of the aMHC gene and repression of the /3-MHC gene
suggests the existence of a common control mechanism, but independent control of the two genetically
linked genes cannot be ruled out.
Any plausible explanation of MHC gene control
also must explain the marked difference in time of
response to T3 administration. The delayed onset of T3
action on Fast Ila MHC mRNA expression, for example, is consistent with earlier results indicating that at
least 6 weeks of high-dose thyroid hormone administration are required to increase the proportion of fast
oxidative fibers (Ha) in soleus muscle from the normal
value of 12% to 26%. 16
The slow onset of T3 action on Fast Ila MHC mRNA
expression in soleus as compared with the rapid effect
on a-MHC mRNA accumulation in ventricle might be
related to a larger number of T-, nuclear receptors in
Circulation Research
200
Ventricle
Cardiac Actin
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-SO .
Time
I 1 11
13 5 7
wk
FIGURE 6. Effects ofT3 administration on cardiac and skeletal a-actin mRNA content in the ventricle, EDL, and soleus.
Total cellular RNA was preparedfrom ventricle at various times
after daily administration of 2 fxg T3ll00 g body weight (top)
and hybridized with the synthetic cardiac a-actin probe. The
synthetic skeletal a-actin probe also was hybridized to RNA
preparedfrom EDL (middle) and soleus muscles (bottom). EDL
= extensor digitorumlongus.Tj = 3,5,3' -triiodo-L-thyromne.
ventricle as compared with skeletal muscles.8 Alternatively, the delay in accumulation of the Fast Ha MHC
mRNA in soleus muscle may be related to slower accumulation of an intermediate signal in the steps between
binding of T3 to its nuclear receptors and stimulation of
specific genes.23 The time required to accumulate an
essential regulatory factor also might explain the delay
in /3-MHC mRNA repression after T3 dosing in soleus
and EDL as compared with ventricle.
It should be noted that soleus muscle has not been
reported to contain Type lib fibers in either the euthyroid or hyperthyroid state by histological criteria.15-l6
Possibly, the histochemical properties designated as
a
VENTRICLE
SOLEUS
EDL
—
FAST
FAST
IIA
IIB
—
—
o
OfiDIAC
ACTIN
Vol 59, No 2, August 1986
Type Ila may reflect the combined ATPase activities of
the two myosin isoforms within the same fiber.
Different effects of thyroid hormone on the same
protein in different tissues have been reported earlier.
For example, synthesis of phosphoenolpyruvate kinase
has been found to be increased in the liver of hyperthyroid animals, whereas decreased synthesis was found
in the kidney, despite similar levels of nuclear occupancy in both organs.32 Also, the specific activity of
mitochondrial a-glycerolphosphate dehydrogenase
(aGPDH) has been shown to be affected differently by
thyroid hormone in fast and slow skeletal muscles and
cardiac muscle of the rat.33 Increases in the activity of
aGPDH were found in soleus and heart after administration of high doses of T3, but no changes were observed in the rectus femoris, a fast muscle. The induction of various components of the /3-adrenergic
receptor system have also been shown to be differentially regulated by thyroid hormone in a tissue-specific
manner. 3435 In most tissues, including heart and adipose tissue, hyperthyroidism increases, and hypothyroidism decreases, synthesis of these proteins, but in
liver the opposite pattern is observed. Many hypotheses have been proposed to account for these tissuespecific responses including differences in the entry or
conversion of thyroid hormones at the tissue level*
and quantitative and/or qualitative differences in the
nuclear T3 receptors. To date, however, little evidence
has been presented to support these hypotheses.
Although the a-actin mRNAs have been shown to
undergo developmental isoform transitions in cardiac
and skeletal muscles in the neonatal rat,22 the present
results indicate that isoform transitions are not regulated by thyroid hormone in adult ventricle or skeletal
muscles. On the other hand, a transient accumulation
of a-cardiac actin was observed in ventricle following
thyroid hormone administration, suggesting that the
hormone also may modulate the level of expression of
other muscle-specific genes. The stimulation of a-actin in ventricle would be consistent with the accumulation of actin related to cardiac growth (Figure 1). It is
unclear whether this is caused by direct actions of
thyroid hormone on the actin gene or by a more general
stimulus to growth that might involve a number of
muscle protein genes. These effects of thyroid hormone on the expression of muscle-specific proteins
other than MHC in the myocardium deserve further
study.
SKELETAL
ACTIN
—
—
nc
—
nc
FIGURE 7. Schematic representation of changes in expression of MHC and a-actin mRNAs after administration ofT3 to hypothyroid rats. The arrows represent the
direction and relative degree of the change observed.
The dashes represent a lack of expression of the particular mRNA. The asterisk denotes a transient change in
mRNA levels. NC = no significant change in the mRNA
levels; EDL = extensor digitorum longus; T} = 3,5,3'triiodo-L-thyronine.
Gustafson et al
Control of a-Actin and MHC Gene Expression by T 3
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KEY WORDS • myosin heavy chain • a-actin
• gene regulation • thyroid hormone • dot
blot assay
Effects of thyroid hormone on alpha-actin and myosin heavy chain gene expression in
cardiac and skeletal muscles of the rat: measurement of mRNA content using synthetic
oligonucleotide probes.
T A Gustafson, B E Markham and E Morkin
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Circ Res. 1986;59:194-201
doi: 10.1161/01.RES.59.2.194
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