Department of Physiology and Biophysics, University of California, Irvine, California 92697
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the weight-bearing
hindlimb soleus muscle of the rat, ~90% of muscle fibers express the
-myosin heavy chain (
-MHC) isoform protein. Hindlimb suspension
(HS) causes the MHC isoform population to shift from
toward the
fast MHC isoforms. Our aim was to establish a model to test the
hypothesis that this shift in expression is transcriptionally regulated
through specific cis elements of the
-MHC promoter. With the
use of a direct gene transfer approach, we determined the activity of
different length
-MHC promoter fragments, linked to a firefly
luciferase reporter gene, in soleus muscle of control and HS rats. In
weight-bearing rats, the relative luciferase activity of the longest
-promoter fragment (
3500 bp) was threefold higher than the
shorter promoter constructs, which suggests that an enhancer sequence
is present in the upstream promoter region. After 1 wk of HS, the
reporter activities of the
3500-,
914-, and
408-bp
promoter constructs were significantly reduced (~40%), compared with
the control muscles. However, using the
215-bp construct, no
differences in promoter activity were observed between HS and control
muscles, which indicates that the response to HS in the rodent appears
to be regulated within the
408 and
215 bp of the promoter.
-myosin heavy chain promoter; direct gene transfer; hindlimb
suspension; dual luciferase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SKELETAL MUSCLE FIBERS are generally classified as fast
glycolytic, fast oxidative, and slow oxidative, which describe the speed of shortening, energy metabolism, and fatigue resistance of the
muscle fibers (11, 25). These classifications of muscle fibers are in
part related to the type of myosin heavy chain (MHC) that is expressed
(24). Typically, there are four MHC isoforms expressed in adult
skeletal muscles: one slow type, designated as type I or ; and three
fast types, designated as IIA, IIX, and IIB (24). Each isoform has
slightly different biochemical properties that impact the shortening
properties of the fiber (1). Different MHC isoform(s) are expressed in
the various muscle fiber types depending on the functional
characteristics of the muscle type. For example, the weight-bearing
hindlimb soleus muscle expresses primarily the
-MHC isoform (15),
whereas the nonweight-bearing fast-twitch tibialis anterior (TA) muscle
expresses a predominance of the IIB MHC isoform (13).
Despite the specific pattern of isoform expression in certain fiber
types, there is a high degree of MHC phenotype plasticity that occurs
in response to variations in contractile activity, neural input, and
thyroid hormone status (2, 6, 12, 29). In the soleus muscle, for
example, the expression of -MHC is responsive to changes in
circulating thyroid hormone levels, manipulations in load, and
denervation (3, 12, 15). Specifically, when hyperthyroidism or
hypothyroidism is induced in the rat,
-MHC mRNA expression in the
soleus is downregulated or upregulated, respectively, relative to the
euthyroid state in rats (12). Unloading of the weight-bearing soleus
muscle by hindlimb suspension (HS) or microgravity causes a reduction
in the expression of
-MHC and a shift in the MHC isoform population
toward the faster MHC types (3, 15). Likewise, the suppression of
contractile activity through denervation results in decreased levels of
-MHC and increased relative levels of IIX and IIA MHCs (12). This
plasticity of the
-MHC phenotype is believed to be regulated in part
by transcriptional processes (15, 29). However, the specific
transcriptional mechanisms by which exogenous signals, such as
unloading, influence the expression of the
-MHC gene are not known.
Putative cis-acting elements within the promoter sequence of
the -MHC gene and corresponding transcription factors have been the
subject of numerous studies on the regulation of the
-MHC gene (7,
10, 14, 18, 30, 32). Most studies have focused on the highly conserved
proximal region of the promoter that contains, in addition to the
ubiquitous basal transcription machinery, specific positive and
negative regulatory sites (7, 18, 30, 32). It has been suggested by
findings from transgenic (18, 32) and muscle cell culture (30)
experiments that three positive elements:
e2 or MCAT-binding site
(
285/
269); the CCAC box (
245/
233); and the
e3 element (
210/
188) are essential and sufficient for muscle-specific gene activation (18, 30). In addition to
these positive motifs, a repressor element that flanks the
e2
element upstream has been identified, and it appears to play a distinct role in
-MHC expression in skeletal and cardiac muscle cells (7, 9,
10). When this repressor element,
e1 (
330/
300), is
deleted or mutated, a significant increase in reporter activity is
observed (7, 9). The role, if any, that these positive and
negative elements in the gene promoter play in the regulation of
-MHC expression in response to the loading state of soleus muscle
has not been determined.
The main goal of the present study was to establish a model to examine
-MHC gene regulation in an in vivo setting using the approach of
direct gene injection. One aim was to verify that the injected
-MHC
promoter-linked reporter construct was expressed in a muscle
type-specific manner by comparing reporter activity in soleus and TA
muscles. Also reported herein is a deletion analysis to determine which
segments of the
-MHC promoter sequence are essential for gene
expression in the soleus muscle during normal weight bearing, and which
are relevant to the altered
-MHC expression in response to HS. The
findings derived from this initial study indicate that a significant
distal enhancer sequence is present between
3500 and
2500
bp, which is required for full promoter activity. Conversely, the
specific response to unloading appears to be regulated through a more
proximal region of the promoter, between
408 and
215 bp.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Design and Sequence of Experiments
The experiments described herein were conducted in three phases. Phase I was designed to verify the application of the direct gene injection technique in the study ofFor each phase, female Sprague-Dawley rats (Taconic Farms, Germantown,
NY) weighing 95-125 g were anesthetized (ketamine acepromazine, 10-20 mg/100 g) during all surgical and gene injection procedures. A skin incision was made to expose the muscle of interest, and 20 µl
PBS containing an equimolar [equivalent to 10 µg 3500-bp MHC-firefly luciferase (FLuc)] mixture of two
supercoiled DNA plasmids was injected into the muscles using a 29-gauge
needle attached to a 0.5-ml insulin syringe. To improve plasmid uptake, the muscles were preinjected with 25% sucrose in 20 µl PBS based on
the method of Wolff et al. (33).
All experiments were for a duration of 7 days, which was verified in
pilot experiments to be within the time-course window of maximum
reporter expression (33). After 7 days, muscle tissues were excised
after pentobarbital sodium (100 mg/kg) euthanasia, and the samples were
quick frozen and stored at 80°C. All animals undergoing HS
were prepared as such immediately after plasmid injections. The HS
model employed a tail-traction method using a noninvasive casting
procedure described by Caiozzo et al. (4). The tail casting included a
swivel attachment that was hooked to the top of the cage, allowing the
rat to move freely about the cage using only its front legs. All
animals in the study were allowed food and water ad libitum, and all
procedures were approved by the institutional Animal Care and Use Committee.
Reporter Plasmid Constructs
The plasmidsTo correct for variation in gene transfer efficiency in the direct
injection technique, a plasmid containing a viral promoter, such as the
CMV (cytomegalovirus) promoter, linked to a different reporter gene, is
typically coinjected with the test plasmid. However, in our hands, the
CMV promoter subcloned into a renilla luciferase expression vector
(CMV-RLuc; Promega) proved unsatisfactory as a control vector. Although
repeated experiments were performed, CMV-RLuc activity was persistently
variable (Fig. 1A) and
did not reflect transfection efficiency. For example, CMV-RLuc activity was often significantly lower than the test plasmid (3500
-MHC-FLuc) activity. Therefore, we chose instead to use the promoter
sequence of another muscle sarcomeric protein, the 2-kb human skeletal
-actin (gift from S. Swoap), linked to a renilla luciferase reporter as the control reporter plasmid. Unlike the MHC, skeletal
-actin is
the only actin isoform expressed in all types of adult rodent skeletal
muscle, and, therefore, is not fiber type specific. Figure 1B
shows that the skeletal
-actin reporter plasmid was effective in the
normalization of transfection efficiency because the level of firefly
luciferase activity correlated well to the level of renilla luciferase
activity in control muscles. Although it is expected that
as an integral muscle protein, skeletal
-actin expression may be
decreased somewhat in response to HS-induced atrophy, we regard it to
still be useful as a means for correcting for transfection efficiency.
The reduction in
-MHC promoter expression in response to HS-induced
atrophy was normalized relative to the response of the actin promoter.
This procedure enabled us to compare the activities of the different
-MHC fragments in response to both weight-bearing and unloading
states.
|
Unless stated otherwise, all of the following data that are expressed
as "firefly luciferase (or FLuc) activity" represent the relative
FLuc activity determined by the ratio: -MHC promoter-driven FLuc
activity divided by skeletal
-actin-driven RLuc activity.
Reporter Expression Assays
Frozen muscle tissues were homogenized in ice-cold lysis buffer from Promega using a glass homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was reserved for the luciferase activity assay using the Promega dual luciferase assay kit, which is designed for sensitive detection of both firefly and renilla luciferase activities in a single extract aliquot. Activities were measured as total light output (as measured by a Monolight 2010-C luminometer) per muscle per second and were expressed as relative light units (RLUs). Background levels, based on luciferase activities of noninjected tissue, were subtracted from the activities of test samples. In this assay system, RLU activity was directly proportional to the amount of tissue aliquot analyzed.Gel Mobility Shift Assay
Isolation of skeletal muscle nuclei.
Skeletal muscle nuclei were isolated according to the method described
previously (14, 27). Nuclei isolated from soleus muscles (5-6
muscles were pooled per sample) were stored at 80 in a nuclei
storage buffer consisting of 25% glycerol, 1 mM
dithiothreitol, 20 mM Tris (pH 7.8), 1 mM EDTA, 1 mM
MgCl2, 0.2 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml
aprotinin, and 2.5 µg/ml leupeptin for subsequent nuclear extraction.
A 30-µl aliquot was saved for determining the DNA content.
DNA determination. DNA concentration in the total homogenate as well as in the nuclei suspension was determined by fluorometry using a minifluorometer (TK0100; Hoeffer Scientific, San Francisco, CA). Bis-benzimide H-33258 was used as the fluorescent dye (20), and calf thymus DNA was used as a standard.
Nuclear extraction.
Nuclei material designated for extraction (equivalent to 300 µg DNA)
were pelleted down by centrifugation at 2,000 g for 5 min. The
supernatant was discarded, and the nuclei were suspended in 300 µl of
nuclei storage buffer (see Isolation of skeletal muscle
nuclei) containing 0.42 M KCl, and fresh protease
inhibitors were added. After 30-min incubation on ice
with gentle agitation, the nuclei were pelleted down by centrifugation
at 5,000 g for 5 min, and the supernatant was diluted in
storage solution (1:4) to lower the KCl concentration to 100 mM. The
nuclear extract was stored in aliquots at 80°C until
subsequent use for gel mobility shift assays. Using this approach, the
nuclear extract amount was normalized to the DNA content, and each
microliter of extract was equivalent to 0.25 µg of nuclear DNA.
Gel mobility shift assay.
Gel mobility shift assays were used to examine and quantify binding of
nuclear extract protein to the e2 cis-regulatory element of
the rat type I MHC gene promoter (30). This approach allows one to
detect changes in transacting factor content and/or changes in pattern
of shifted bands resulting from binding of one or more factors.
Statistical Analysis
Statistical analysis was performed using the Graphpad Prism 2.0 statistical software package. Values are means ± SE. Differences among groups were determined by ANOVA followed by the Newman-Keuls posttest. Differences between the means of two experimental groups were assessed by an unpaired, two-tailed t-test. P < 0.05 was taken as the level of statistical significance. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Direct Gene Injection Is an Effective Approach to Study MHC Isoform Regulation
Muscle type-specific expression of the -MHC
promoter.
Our initial studies confirm that the method of direct gene injection is
a useful approach to examine
-MHC gene regulation in skeletal muscle
by demonstrating that the promoter-linked reporter,
3500
-MHC-FLuc, was expressed in a muscle fiber type-specific pattern
similar to that of endogenous
-MHC mRNA expression (15). Figure
2 (top) shows that the
level of activity of the
3500
-MHC-FLuc was high in the
control soleus muscle. The fast-twitch muscle TA also
expressed the reporter gene, but at a level ~3% of the level
observed in soleus, similar to the endogenous
-MHC mRNA expression
in TA muscle (13). As an additional control to ensure that the
luciferase expression in muscle was due to the specificity of the
-MHC promoter, we injected the basic pGL3 reporter plasmid, which is
the same as the test plasmid but does not contain the
-MHC promoter
sequence fragment. The activity of basic pGL3 in soleus muscle was just
above background levels and measured 0.75% of the activity of the
3500-bp promoter-driven reporter (data not shown).
|
Comparison of and IIB MHC promoter activities in
fast- and slow-twitch muscles.
Muscle fiber type specificity of two MHC isoforms,
and type IIB,
was demonstrated using the direct gene transfer technique in soleus and
TA muscles (Fig. 2, bottom). When a plasmid consisting of a
2.6-kb fragment of the IIB promoter fused to a renilla luciferase reporter (IIB-RLuc) was simultaneously injected with the
3500
-MHC-FLuc plasmid into the soleus and TA muscles, a clear pattern of
tissue-specific expression was exhibited. In the soleus muscle, the
3500
-MHC-FLuc activity was significantly higher (140-fold) than that of the IIB-RLuc activity, and this pattern of expression was
reversed such that in the TA muscle, the IIB-RLuc activity was much
greater (37-fold) than the
3500
-MHC-FLuc activity. The fact
that both MHC isoform promoters were activated in a characteristic pattern in the slow- and fast-twitch muscle suggested that there was no
inherent difference in the ability of the two muscle types to express
both reporter constructs.
Deletion Analysis of -MHC Promoter Activity in
Weight-Bearing Animals
|
Activity of the -MHC Promoter in Response to
Hindlimb Unloading
|
It should be mentioned that the attenuation of reporter expression in
response to HS was associated specifically with the -MHC promoter.
The inset graph of Fig. 4 depicts an increase in the type IIB
MHC promoter-linked FLuc activity after HS. This result reflects the
antithetic plasticity of certain MHC mRNAs in response to unloading
(15), and confirms that the decrease of
-MHC is not merely the
result of a generalized depression in transcriptional activity by the
HS intervention, but is a specific effect due to muscle unloading.
Gel Mobility Shift Assay Comparing Weight Bearing and Unloaded Soleus Muscle Nuclear Extracts
Considering that the relative activity of theA gel mobility shift assay was performed that examined the specific
interactions of the e2 element with nuclear proteins extracted from
the soleus muscle of control and HS groups (Fig. 5). Two retarded bands, labeled A and B,
were competed out by an unlabeled
e2 probe, and thus were indicative
of specific interactions between nuclear proteins and the
e2
element. As determined by densitometric analysis, there was no
difference in the specific binding pattern between the control and HS
nuclear extracts. These results suggest that
e2 is probably involved
in
-MHC activation to the same degree under both conditions of
weight bearing and unloading and does not play a specific role in the
response to HS.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is well known that skeletal muscle fibers undergo changes in both
mass and MHC phenotype as an adaptive response to different contractile
activity demands. What is not known is the specific mechanism(s)
involved in establishing the MHC phenotype under normal conditions and
during an adaptation response. In the present report, we established an
in vivo gene injection model to examine the regulation of -MHC gene
expression in normal and unloaded rodent soleus muscle. Examination of
-MHC promoter-linked reporter activity enabled us to ascertain which
subregions of the promoter sequence are essential in the regulation of
-MHC gene expression in a physiological context of weight bearing.
Deletion analysis revealed that the upstream sequence between
3500 and
2500 bp was necessary for full activation of the
-MHC promoter in soleus muscle. The presence of an upstream enhancer in the region between
3500 and
2500 bp has been proposed
in our recent report on the
-MHC promoter activity in rodent cardiac muscle (34). Deletion constructs of
-MHC promoter were injected into
the left ventricle of rats, and the pattern of reporter activity that
was observed was similar to that in the soleus (Ref. 34 and Fig. 3).
Evidence that an enhancer exists in the upstream region was revealed
through a mutation experiment (34). A three base substitution in the
proximal region of
408 promoter construct completely eliminated
promoter activity in ventricular samples; however, when this same
mutation was tested in the
3500 promoter, some activity (10%)
was recovered. These data indicate that the sequence(s) in the upstream
promoter is a strong enhancer in that it accounted for about 70% of
the promoter activity, and it also compensated for the loss of an
essential downstream element (34).
The upstream region of the -MHC promoter has been virtually
unexplored as a potential regulatory region. Sequence analysis reveals
that there are several sites that are homologous to MCAT elements,
basic helix-loop-helix motifs (N-box and E-boxes), and NF-AT (nuclear
factor of activated T cells) binding site motifs in this region. These
potential upstream regulatory elements have not yet been specifically
examined. The NF-AT factor holds particular interest as a potential
regulator. Recently, NF-AT was shown to be involved in muscle phenotype
regulation, specifically, in the expression of slow isoforms, sTnI and
myoglobin (5). NF-AT activation is regulated by calcineurin, a
phosphatase enzyme that is dependent on intracellular Ca2+
concentration. The authors assert that the neural activity pattern typical of slow-twitch muscle nerves sustains a level of intracellular Ca2+ that maintains calcineurin activity and ultimately the
NF-AT factor, in an activated state. NF-AT, once activated via a
dephosphorylation mechanism, translocates to the nucleus and interacts
with a binding site in the genes of sTnI and myoglobin. These findings
implicate the NF-AT factor as a possible link between neural activity
and the expression of slow isoforms in skeletal muscle.
In addition to -MHC gene regulation in normal soleus, we are also
interested in understanding the mechanism that underlies the shift in
MHC expression in response to HS. Our research group has previously
demonstrated that after HS, there is a significant decrease in the
level of
-MHC mRNA and a concomitant increase in IIX and IIB MHC
mRNA in soleus muscle (15). Likewise, in the present report, the
3500
-MHC promoter activity was reduced, and the IIB MHC
promoter activity was increased in HS soleus, compared with control.
Deletion analysis revealed that even though shorter fragments of the
-MHC promoter generated less reporter activity than the
3500
-MHC-FLuc, an HS effect was still observed with all fragments except
the
215
-MHC-FLuc construct. Therefore, the HS
response appears to be conferred between
408 and
215 bp
of the proximal region of the promoter. In fact, this same region
appears to harbor positive regulatory element(s) essential to normal
-MHC gene expression, because, in control soleus, there was a
significant downregulation of activity when the sequence between
408 and
215 was eliminated (Fig. 3).
Other investigators, using muscle cell culture and transgenic mouse
models, have found that the proximal region is sufficient to confer
muscle-specific expression (18, 30, 32). As previously mentioned,
numerous positive regulatory elements: e2,
e3, and CCAC, have
been identified in skeletal and cardiac cell culture studies (30). The
e2 element (
285 to
269) contains a 12-bp sequence that
is identical to the simian virus 40 enhancer element, AP5/GTII (30),
and within which the muscle-specific MCAT motif is harbored. Mutation
analysis indicates that the interaction of
e2 and transcriptional
enhancer factor 1 (TEF-1), the MCAT-binding protein (8), is essential
for
-MHC expression in cardiac and skeletal cells (30). The CCAC box
(
250 to
230) represents a binding site for the Sp1
transcription factor. Although this is an ubiquitous factor, it has
been shown to act cooperatively with the
e2 in the activation of
-MHC gene in cardiomyocytes (30). The
e3 element (
210 to
188) is also a recognition site for TEF-1 (16), and deletion
analysis indicates that it is essential for
-MHC gene activation by
-adrenergic stimulation or protein kinase C activation in
cardiomyocytes (17). However, in vivo studies of
-MHC gene
regulation are inconsistent with the findings from the cell culture
experiments. In a transgenic mouse model,
-MHC
promoter-linked CAT activity was still maintained when any of the three
e2, CCAC, or
e3 elements were mutated (18). However, when all
three elements were mutated simultaneously, there was a significant
reduction in CAT activity. Therefore, these results indicate that any
of the three elements are dispensable, but all three are needed for
full gene activation. Yet, another transgenic study concluded that the
simultaneous mutation of all three elements did not abolish the
-MHC
gene activation in response to functional overload in the plantaris
muscle (31).
It should be mentioned that these disparities between the results from
muscle cell cultures and transgenic mice models may stem from the
various limitations inherent in these model systems. There is evidence
that the pattern of MHC gene expression is dependent on loading state
as well as the presence of the nerve (23), and these physiological
influences cannot be effectively mimicked in culture. Although these
influences would be maintained in a transgenic mouse model, this time-
and labor-intensive approach is largely limited to only one species
thus far. Moreover, due to the inherent problems in controlling the
copy number of transgenes, interpreting data is difficult and deletion
analysis is troublesome. For example, the level of reporter activity is
dependent on the transgene copy number, which varies in each animal.
Therefore, the validity in assuming that different reporter activities
are attributable to different deletion fragments could prove
problematic. In fact, some animals with a relatively high -MHC
transgene copy number exhibited flawed gene regulation by
inappropriately expressing considerable levels of the
-MHC transgene
in the adult mouse ventricle (19). Our in vivo gene
injection technique has the advantage that it can be performed on rats,
the species in which this research group has established experimental
models and has extensively characterized MHC isoform expression in
different muscle types. Moreover, the genes can be injected in any
specific muscle at any time during a given experimental period, and
coinjecting a control plasmid normalizes the plasmid uptake and
relative expression of the transferred gene, making the
deletion/mutation analyses relatively easier to interpret.
Our results indicate that the HS response is most likely conferred
within the 408- and
215-bp fragment. The possibility that
the aforementioned positive regulatory elements play a role in this
regulation was considered. The CCAC box (
245 to
233) was
not specifically examined in this current paper because previous evidence strongly suggests that this element is pertinent only in
cardiac myocytes (30). However, this does not preclude its possible
relevance in the regulation of
-MHC expression during an unloading
response, and its role, if any, should be considered in the future. Our
gel mobility shift assay revealed that the
e2 sequence (
285
to
269) did interact with proteins from control and HS soleus
nuclear extracts, but there was no difference in the degree of binding
between the two groups. Therefore, although the
e2 element may
enhance the promoter activity in normal soleus, we concluded that it
was not specifically involved in the regulation of the HS response. The
influence of the
e3 element (
210 to
188) was
considered irrelevant because it is contained within the
215 bp.
It is of interest to note that McCarthy et al. (21) reported that the
proximal 600-bp sequence of the mouse
-MHC gene promoter was
sufficient to direct an HS response in a transgenic mouse model.
Moreover, they found that the HS effect remained undiminished despite
the simultaneous mutations of the
e2, C-rich, and
e3 elements
within both 5600- and 600-bp promoter sequences (21). Therefore, their
results imply that the three elements may not be involved at all or may
require the interaction with some other element(s) within this
subregion of the
-MHC promoter to confer the HS response.
Within the 408- and
215-bp sequence, a putative repressor
element termed
e1 (
330/
300) has been identified (7,
9). The repressor activity of
e1 was exposed through deletion
analysis experiments using the human
-MHC gene in skeletal and
cardiac muscle cell culture and rat heart gene injection models (7, 9).
It was found that when the
e1 element was deleted or mutated, a
large increase in CAT expression was detected (7, 9). It is conceivable
that a repressor signal protein is upregulated in soleus muscle in
response to unloading and then binds to the
e1 site in the
-MHC
promoter, thus suppressing
-MHC gene expression. In fact, a recent
study from McCarthy et al. (22) identified two proteins (50- and
52-kDa) derived from soleus muscle nuclear extracts of suspended rats
that show highly enriched specific binding activity with a human
e1
sequence probe (equivalent to
322 to
301 bp of the rat
-MHC
e1 sequence) in electrophoretic mobility shift assays. In
light of all of the findings at this point, we would like to test a
299
-MHC deletion construct, as well as a
408
-MHC
construct harboring a
e1 mutation in our HS model. As
both constructs will in effect hinder the signal protein(s) from
binding to the
e1 site, we would be able to ascertain whether the
e1 element is essential to confer the HS response. Gel mobility
shift assay experiments comparing binding activity of the
e1 sequence with nuclear extracts from soleus muscle of control and
suspended rats are also planned.
In summary, we have established a working model, using the gene
injection technique, to examine the regulatory role of specific promoter regions of the -MHC gene in vivo. Initial results indicated that the upstream sequence of the
-MHC promoter was necessary for
full promoter activity. However, the proximal sequence was sufficient
to confer promoter activation in soleus muscle tissue of both normal
and HS rats. Specifically, our findings indicate that a conceivable
HS-responsive site may exist in the
408- to
215-bp subregion.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant HAR-30346 and by National Space Biochemical Research Institute Grant NCC9-58.
![]() |
FOOTNOTES |
---|
Segments of this work work were done during the tenure of a fellowship from the American Heart Association, Western States Affiliate.
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: K. M. Baldwin, Dept. of Physiology and Biophysics, Univ. of California, Irvine, D-346, Med. Sci. I, Irvine, CA 92697 (E-mail: kmbaldwi{at}uci.edu).
Received 17 May 1999; accepted in final form 10 December 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barany, M.
ATPase activity of myosin correlated with speed of muscle shortening.
J Gen Physiol
50:
197-218,
1967
2.
Bishop, DL,
and
Milton RL.
The effects of denervation location on fiber type mix in self-reinnervated mouse soleus muscles.
Exp Neurol
147:
151-158,
1997[ISI][Medline].
3.
Caiozzo, VJ,
Baker MJ,
Herrick RE,
Tao M,
and
Baldwin KM.
Effect of spaceflight on skeletal muscle: mechanical properties and myosin isoform content of a slow muscle.
J Appl Physiol
76:
1764-1773,
1994
4.
Caiozzo, VJ,
Baker MJ,
McCue SA,
and
Baldwin KM.
Single-fiber and whole muscle analyses of MHC isoform plasticity: interaction between T3 and unloading.
Am J Physiol Cell Physiol
273:
C944-C952,
1997
5.
Chin, ER,
Olson EN,
Richardson JA,
Yang Q,
Humphries C,
Shelton JM,
Wu H,
Weiguang Z,
Bassel-Duby R,
and
Williams RS.
A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type.
Genes Dev
12:
2499-2509,
1998
6.
Diffee, GM,
McCue SA,
LaRosa A,
Herrick RE,
and
Baldwin KM.
Interaction of various mechanical activity models in regulation of myosin heavy chain isoform expression.
J Appl Physiol
74:
2517-2522,
1993[Abstract].
7.
Edwards, JG,
Bahl JJ,
Flink IL,
Milavetz J,
Goldman S,
and
Morkin E.
A repressor region in the human -myosin heavy chain gene that has a partial position dependency.
Biochem Biophys Res Commun
189:
504-510,
1992[ISI][Medline].
8.
Farrance, IKG,
Mar JH,
and
Ordahl CP.
M-CAT binding factor is related to the SV40 enhancer binding factor, TEF-1.
J Biol Chem
267:
17234-17240,
1992
9.
Flink, IL,
Edwards JG,
Bahl JJ,
Liew C-C,
Sole M,
and
Morkin E.
Characterization of a strong positive cis-acting element of the human -myosin heavy chain gene in fetal rat heart cells.
J Biol Chem
267:
9917-9924,
1992
10.
Flink, IL,
and
Morkin E.
Alternatively processed isoforms of cellular nucleic acid-binding protein interact with a suppressor region of the human -myosin heavy chain gene.
J Biol Chem
270:
6959-6965,
1995
11.
Gordon, T,
and
Pattullo MC.
Plasticity of muscle fibers and moter unit types.
Exerc Sport Sci Rev
21:
331-362,
1993[Medline].
12.
Haddad, F,
Arnold C,
Zeng M,
and
Baldwin KM.
Interaction of thyroid state and denervation on skeletal myosin heavy chain expression.
Muscle Nerve
20:
1487-1496,
1997[ISI][Medline].
13.
Haddad, F,
Herrick RE,
Adams GR,
and
Baldwin KM.
Myosin heavy chain expression in rodent skeletal muscle: effects of exposure to zero gravity.
J Appl Physiol
75:
2471-2477,
1997[Abstract].
14.
Haddad, F,
Qin A,
McCue SA,
and
Baldwin KM.
Thyroid receptor plasticity in striated muscle-types: effects of altered thyroid state.
Am J Physiol Endocrinol Metab
274:
E1018-E1026,
1998
15.
Haddad, F,
Qin A,
Zeng M,
McCue SA,
and
Baldwin KM.
Interaction of hyperthyroidism and hindlimb suspension on skeletal myosin heavy chain expression.
J Appl Physiol
85:
2227-2266,
1998
16.
Kariya, K,
Farrance IKG,
and
Simpson PC.
Transcriptional enhancer factor-1 in cardiac myocytes interacts with an 1-adrenergic- and
-protein kinase C-inducible element in the rat
-myosin heavy chain promoter.
J Biol Chem
268:
26658-26662,
1993
17.
Kariya, K,
Karns LR,
and
Simpson PC.
An enhancer core element mediates stimulation of the rat -myosin heavy chain promoter by an
1-adrenergic agonist and activated
-protein kinase C in hypertrophy of cardiac myocytes.
J Biol Chem
269:
3775-3782,
1994
18.
Knotts, S,
Rindt H,
Neumann J,
and
Robbins J.
In vivo regulation of the mouse myosin heavy chain gene.
J Biol Chem
269:
31275-31282,
1994
19.
Knotts, S,
Rindt H,
and
Robbins J.
Position independent expression and developmental regulation is directed by the myosin heavy chain gene's 5' upstream region in transgenic mice.
Nucleic Acids Res
23:
3301-3309,
1995[Abstract].
20.
Labarca, C,
and
Paigen K.
A simple, rapid, and sensitive DNA assay procedure.
Anal Biochem
102:
344-352,
1980[ISI][Medline].
21.
McCarthy, JJ,
Fox M,
Tsika GL,
Gao L,
and
Tsika RW.
-MHC transgene expression in suspended and mechanically overloaded/suspended soleus muscle of transgenic mice.
Am J Physiol Regulatory Integrative Comp Physiol
272:
R1552-R1561,
1997
22.
McCarthy, JJ,
Vyas D,
Tsika GL,
and
Tsika RW.
Segregated regulatory elements direct -myosin heavy chain expression in response to altered muscle activity.
J Biol Chem
274:
14270-14279,
1999
23.
Pette, D,
and
Vrbova G.
Adaptation of mammalian skeletal muscle fibers to chronic electrical stimulation.
Rev Physiol Biochem Pharmacol
120:
115-202,
1992[ISI][Medline].
24.
Schiaffino, S,
and
Reggiani C.
Myosin isoforms in mammalian skeletal muscle.
J Appl Physiol
77:
493-501,
1994
25.
Schiaffino, S,
and
Reggiani C.
Molecular diversity of myofibrillar proteins: gene regulation and functional significance.
Physiol Rev
76:
371-423,
1996
26.
Swoap, SJ.
In vivo analysis of the myosin heavy chain IIB promoter region.
Am J Physiol Cell Physiol
274:
C681-C687,
1998
27.
Swoap, SJ,
Haddad F,
Bodell P,
and
Baldwin KM.
Effect of chronic energy deprivation on cardiac thyroid hormone receptor and myosin isoform expression.
Am J Physiol Endocrinol Metab
266:
E254-E260,
1994
28.
Swoap, SJ,
Haddad F,
Bodell P,
and
Baldwin KM.
Control of -myosin heavy chain expression in systemic hypertension and caloric restriction in the rat heart.
Am J Physiol Cell Physiol
269:
C1025-C1033,
1995
29.
Swoap, SJ,
Haddad F,
Caiozzo VJ,
Herrick RE,
McCue SA,
and
Baldwin KM.
Interaction of thyroid hormone and functional overload on skeletal muscle isomyosin expression.
J Appl Physiol
77:
621-629,
1994
30.
Thompson, WR,
Nadal-Ginard B,
and
Mahdavi V.
A MyoD1-independent muscle-specific enhancer controls the expression of the -myosin heavy chain gene in skeletal and cardiac muscle cells.
J Biol Chem
266:
22678-22688,
1991
31.
Tsika, GL,
Weidenman JL,
Gao L,
McCarthy JJ,
Sheriff-Carter K,
Rivera-Rivera ID,
and
Tsika RW.
Induction of -MHC transgene in overloaded skeletal muscle is not eliminated by mutation of conserved elements.
Am J Physiol Cell Physiol
271:
C690-C699,
1996
32.
Weidenman, JL,
Tsika GL,
Gao L,
McCarthy JJ,
Rivera-Rivera ID,
Vyas D,
Sheriff-Carter K,
and
Tsika RW.
Muscle-specific and inducible expression of 293-base pair -myosin heavy chain promoter in transgenic mice.
Am J Physiol Regulatory Integrative Comp Physiol
271:
R688-R695,
1996
33.
Wolff, JA,
Williams P,
Acsadi G,
Jiao S,
Jani A,
and
Chong W.
Conditions affecting direct gene transfer into rodent muscle in vivo.
Biotechniques
11:
474-485,
1991[ISI][Medline].
34.
Wright, CE,
Haddad F,
Bodell PW,
and
Baldwin KM.
In vivo regulation of the myosin heavy chain gene in rodent heart: role of thyroid hormone and evidence for an upstream enhancer.
Am J Physiol Cell Physiol
276:
C883-C891,
1999