Myosin heavy chain isoform expression in rat smooth muscle
development
Sheryl L.
White,
Ming Yuan
Zhou,
Robert B.
Low, and
Muthu
Periasamy
Department of Molecular Physiology and Biophysics, University of
Vermont College of Medicine, Burlington, Vermont 05405; and Molecular
Cardiology Laboratory, Cardiovascular Research Center, University of
Cincinnati, Cincinnati, Ohio 45267
 |
ABSTRACT |
Smooth muscle
myosin heavy chains (MHCs), the motor proteins that power smooth muscle
contraction, are produced by alternative splicing from a single gene.
The smooth muscle MHC gene is capable of producing four isoforms by
utilizing alternative splice sites located at the regions encoding the
carboxy terminus and the junction of the 25- and 50-kDa tryptic
peptides. These four isoforms, SM1A, SM1B, SM2A, and SM2B, are a
combination of one of two heavy chains containing different
carboxy-terminal tails (1 or 2) without (A) or with (B) an additional
motif in the myosin head. In the present study, using RNA analysis and
isoform-specific antibodies, we demonstrate the expression patterns of
MHC isoforms during development in rat smooth muscle tissues. RNase
protection analysis indicates that the mRNAs for SMA and SMB isoforms,
which differ by a 21-nucleotide insertion in the region encoding the S1
head region of the myosin molecule, are differentially expressed during
development in a highly tissue-specific manner. Smooth muscle MHC
transcripts are first detectable in developing rat smooth muscle
tissues at 17 days of fetal development. The SMB mRNA is shown to be
expressed in smooth muscle from fetal bladder, intestine, and stomach
and from neonatal aorta; however, it is not expressed in cultured smooth muscle cells from rat aorta. The SMA mRNA is also present at all
stages of development in the smooth muscles examined; however, it is
much less abundant than SMB mRNA in most fetal smooth muscles. We show
here that the SMB isoform, which contains a unique seven-amino acid
insertion at the junction of the 25- and 50-kDa tryptic peptides, is
present in conjunction with SM1 and SM2 tails on immunoblots of smooth
muscle from stomach, intestine, bladder, and uterus and is expressed
during development in a pattern distinct from that of the SM1 and SM2
tail isoforms.
gene expression; stomach; intestine; bladder; uterus; aorta
 |
INTRODUCTION |
MYOSIN, A MAJOR CONTRACTILE protein in muscle cells,
acts as a molecular motor to convert chemical energy to cellular
contraction. The myosin molecule is a hexamer composed of two heavy
chains and two pairs of light chains. The myosin heavy chain (MHC)
contains the site for ATP hydrolysis and actin binding. A large
multigene family encoding distinct isoforms of MHCs has been identified in striated muscle, smooth muscle, and nonmuscle tissues (17, 19). The MHC isoform content has been shown to be an important determinant of contractile characteristics in striated muscle (5, 26,
29).
Our initial studies have shown that two smooth muscle MHC isoforms (SM1
and SM2) are produced by alternative splicing from a single gene (3).
The SM1 mRNA encodes 43 unique amino acids at the carboxy terminus and
has an apparent molecular mass of 204 kDa. The SM2 isoform, which is
nearly identical to SM1, is produced by the inclusion of an
alternatively spliced exon and contains nine unique carboxyl amino
acids; it has an apparent molecular mass of 200 kDa. More recently, we
and others have shown that additional isoform diversity is generated by
alternative splicing in the region encoding the junction of the 25- and
50-kDa tryptic peptides of the MHC head (1, 12, 14, 30). In the rat,
inclusion of a 21-nucleotide (nt) exon results in an isoform with seven
unique amino acids (QGPSFAY) at the junction of the 25- and 50-kDa
tryptic peptides. This isoform, referred to here as SMB, was
differentially expressed at the mRNA level in smooth muscle tissues and
was most abundant in visceral smooth muscle tissues (30). The smooth
muscle MHC mRNA lacking this 21-nt exon, referred to here as SMA, is
the predominant MHC message expressed in aortic and vena caval vascular
smooth muscles. Alternative splicing in the region of the transcript
encoding the junction of the 25- and 50-kDa tryptic peptides, in
combination with splicing at the 3'-end of the MHC mRNA, can
therefore produce four smooth muscle MHC variants (SM1A,
SM1B, SM2A, and SM2B), as diagrammed in Fig.
1.

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Fig. 1.
Schematic diagram of smooth muscle myosin heavy chain isoforms.
A: encoded regions involved in
splicing of alternative exons and unique amino acids encoded by exons.
B: 4 possible isoforms generated by
alternative splicing at regions encoding carboxy terminus and junction
of 25- and 50-kDa tryptic peptides. *, Stop codon.
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The functional significance of the four smooth muscle MHC isoforms
remains to be determined. Attempts to correlate the SM1 and SM2 isoform
composition in smooth muscles with different physiological properties
have yet to establish a clear relationship (18, 23, 24). However,
studies of the SMB isoform demonstrate that it has approximately
twofold higher ATPase activity and propels actin filaments two to three
times faster in in vitro motility assays (14, 21). These
findings, in conjunction with the observation that smooth muscle types
with faster contractile properties, such as bladder, contain large
proportions of the SMB isoform mRNA, suggest that isoform
diversity at the junction of the 25- and 50-kDa tryptic peptides may be
an important determinant of smooth muscle contractile performance.
Previous studies of SM1 and SM2 isoforms demonstrate that they are
differentially expressed in developing smooth muscles and in cultured
smooth muscle cells. The SM1 isoform is expressed first in fetal rabbit
development, whereas the SM2 isoform expression begins in late fetal or
early neonatal stages of development (6, 15). This pattern of
expression is also observed in rat lung development (31). In adult
smooth muscle the SM1-to-SM2 isoform ratios are tissue specific. The
expression of SM1 and SM2 isoforms is also differentially regulated in
cultured smooth muscle cells. Primary cultures of aortic smooth muscle
cells continue to express SM1 but express little or no SM2 myosin
(2, 13).
In the present study, using RNA analysis and isoform-specific antibody
probes, we further explore MHC isoform diversity in mammalian smooth
muscle. The objectives of the current study were 1) to determine the developmental
expression of the SMA and SMB isoforms,
2) to determine whether cultured
smooth muscle cells express SMA or SMB mRNA, and
3) to examine the expression of the SMB protein in adult and developing rat smooth muscles, particularly with respect to its association with the SM1 and SM2 isoforms.
RNase protection analysis indicates that the SMA and SMB isoform
transcripts are differentially expressed during development in a
tissue-specific manner. Additionally, the expression of SMA and SMB
transcripts is differentially regulated in cultured smooth muscle
cells, where only the SMA transcript is expressed. Western blotting
results provide evidence that the SMB isoform is expressed in
conjunction with SM1 and SM2 tails in adult rat stomach, bladder, intestine, and uterus and that these smooth muscle MHC proteins are
differentially expressed during development in rat aorta, stomach, and
bladder. The SM1 isoform is first detectable at 17 days of fetal
development, whereas the expression of SM2, detectable at 20 days of
fetal development in bladder, is delayed until after birth in aorta and
stomach. These results indicate that the developmental pattern of
smooth muscle MHC isoform expression is complex and highly tissue
specific. These studies provide new information regarding MHC isoform
expression in rat smooth muscle tissues, an important step toward
understanding the molecular basis for smooth muscle functional
diversity.
 |
METHODS |
RNA isolation and analysis.
Tissues were isolated from fetal, neonatal, and adult rats after
euthanasia, frozen in liquid nitrogen, and then stored at
80°C for RNA and protein procedures. Total RNA was extracted by the guanidinium thiocyanate procedure (7). RNA (20 µg/sample) was
then hybridized to
[32P]cRNA made by
transcribing antisense RNA with T7 RNA polymerase from a cDNA fragment
containing the 21-nt divergent region, as previously described (30).
Hybridizations, RNase digestions, and electrophoresis were performed in
accordance with the RNase protection method described previously (30).
RNase protection assays were repeated once with the same RNA samples,
then repeated twice with independently isolated RNA.
Tissue extracts, electrophoresis, and immunoblotting.
Tissues were powdered in liquid nitrogen and homogenized at 4°C in
pyrophosphate extraction buffer, as previously described (2). Samples
were electrophoresed through 3.8% polyacrylamide (0.05%
bis-acrylamide) using a Laemmli buffer system (2). Proteins were
electroblotted onto nitrocellulose membranes, as described elsewhere
(28). Bound antibodies were detected using the enhanced chemiluminescence (ECL) kit, as described by the manufacturer (Amersham). The SM1 and SM2 peptide-specific antibodies were generated as described previously (31) and used at a 1:2,000 dilution. The SMB
antibody (a generous gift of Dr. Arthur Rovner) was generated against
the following peptide:
glutamine-glycine-proline-serine-phenylalanine-alanine-tyrosine-glycine-glutamic acid-leucine-glutamic acid-cysteine. This peptide was then coupled to
keyhole limpet hemocyanin at the cysteine residue and injected into
rabbits for antibody production (HTI Bio-services). The SMB antibody
was characterized initially by examining the preimmune and postimmune
serum reactivity on minigel immunoblots containing extracts from
bladder, stomach, and aorta, as well as baculovirus-expressed SMA and
SMB heavy meromyosins. The postimmune serum reacted specifically with
proteins migrating at 204-200 kDa (data not shown). The SMB antibody was used at a 1:2,000 dilution for Western blot procedures. The smooth muscle MHC antibody is a polyclonal antibody generated in
rabbits against bovine aorta (a generous gift of Dr. R. Adelstein) that
recognizes all smooth muscle myosins and was used at a 1:50,0000 dilution. Experiments were repeated three times for the adult muscle
tissues and the developmental series.
Densitometric analysis was conducted on autoradiograms generated by
Western blotting followed by ECL detection. Autoradiograms were scanned
and analyzed with quantitation software (Quantity One, PDI).
Primary smooth muscle cell culture.
Smooth muscle cells from neonatal Sprague-Dawley rat thoracic aorta
were isolated and cultured as previously described (2). Total RNA was
isolated from confluent cells by the guanidinium thiocyanate procedure
(7). RNase protection experiments were repeated twice with two separate
samples of RNA.
 |
RESULTS |
SMA and SMB transcripts are differentially expressed
during development in a tissue-specific manner.
To determine whether SMA and SMB mRNA expression is
developmentally regulated, RNase protection analysis was performed on rat smooth muscle tissues obtained beginning with fetal embryos at 16 days of development (Fig. 2). Full
protection of the cRNA probe, which contains the 21-bp alternative
exon, results in a 490-nt band, indicating the presence of the
SMB message. Partial protections of 201 and 268 nt, resulting from
digestion at the 21-nt insertion, indicate the presence of SMA message.
Minor bands above and below partial protections are common with this
method and may be the result of under- or overdigestion of the cRNA
probe by RNases.

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Fig. 2.
Smooth muscle myosin heavy chain (MHC) isoform SMB mRNA is
differentially expressed during development. In presence of SMB
transcripts, 577-nucleotide (nt) cRNA probe produces a 490-nt fragment.
Partial protections of 268 and 201 nt are produced in presence of SMA
transcripts. Twenty micrograms of total RNA per sample were hybridized
to antisense cRNA probe, digested with RNase, then electrophoresed on a
denaturing sequence gel. Skeletal, brain, and transfer RNAs are
included as negative controls, since these contain negligible/no smooth
muscle MHC mRNA. mRNA samples were obtained at 16, 17, and 18 days from
visceral smooth muscle tissue taken from abdominal cavity.
A: RNase protection analysis of total
RNA from smooth muscle obtained from fetal, neonatal, and adult bladder
and aorta. B: RNase
protection analysis of total RNA from fetal, neonatal, and adult
intestine and lung smooth muscle. X174 end-labeled DNA was utilized
as a molecular weight marker. C: RNase
protection analysis of total RNA from fetal, neonatal, and adult
stomach smooth muscle. mRNAs from aorta, vein, and lung were included
as positive controls.
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The SMB transcripts are first detectable at 17 days of fetal
development in RNA isolated from smooth muscle obtained from whole
abdominal viscera (Fig. 2A). The SMB
mRNA predominates in the 17- and 18-day visceral smooth muscle tissues,
although detectable levels of SMA mRNA are also found.
The SMB mRNA is the predominant transcript in fetal, neonatal, and
adult bladder (Fig. 2A). In
contrast, SMB transcripts are clearly expressed in 1-wk neonatal rat
aorta but decrease at later stages until the SMB transcript is nearly
undetectable in the adult aorta. The transition in mRNA composition
from predominantly SMB to equal or higher levels of SMA mRNA during
development, observed in aorta, is also observed in smooth muscle from
stomach and intestine.
In developing intestinal smooth muscle, SMB mRNA is abundant and
appears to increase throughout development (Fig.
2B). The SMA mRNA is also expressed
throughout development; however, the abundance of SMA mRNA increases
abruptly during the 1st wk of postnatal development. Approximately
equal proportions of the SMA and SMB transcript have been shown to be
present in adult intestine (30). A similar profile of SMB and SMA
expression is also observed in the developing rat stomach smooth muscle
(Fig. 2C). In this tissue the SMB
mRNA predominates during fetal and early neonatal stages. However,
during the 1st wk of postnatal development, expression of the SMA
transcript is increased. In the adult rat stomach the SMA mRNA has been
shown to be the most abundant (30).
In the developing lung, SMA and SMB isoform mRNAs are present in fetal
tissue (Fig. 2B). In this tissue the
SMA mRNA appears to be equally as abundant as the SMB mRNA or more
abundant than the SMB mRNA in fetal and neonatal stages of development.
Because of the relatively low levels of smooth muscle MHC mRNA
(reflective of the low proportion of smooth muscle cells to other cell
types in the lung), it is difficult to discern any significant
differences in the pattern of smooth muscle MHC mRNA expression during
development. Adult lung appears to express small amounts of SMB mRNA,
as well as the SMA mRNA, which has been shown to be the predominant
transcript (30).
Cultured aortic smooth muscle cells express the SMA isoform
exclusively.
Cultured smooth muscle cells have previously been shown to
differentially regulate expression of the SM1 and SM2 mRNAs (2, 4).
Cultured aortic smooth muscle cells continue to express SM1, but the
expression of SM2 mRNA is curtailed or greatly reduced in culture. We
wished to determine whether the SMA or SMB mRNA expression is also
differentially regulated in cultured smooth muscle cells and, if so,
which smooth muscle MHC transcripts are expressed in cultured cells.
RNase protection analysis of RNA obtained from cultured primary aortic
smooth muscle cells indicates that only the SMA mRNA is present (Fig.
3). RNase protection analysis of cultured
tracheal smooth muscle cells and rat lung mesenchymal cells also
indicates exclusive expression of SMA mRNA (data not shown). Because
neonatal aorta and adult trachea express SMB mRNA and protein in vivo, the lack of SMB mRNA in cultured cells suggests that the expression of
SMA and SMB transcripts is differentially regulated under cultured conditions. Expression of the SMB mRNA is downregulated in the cultured
cells, whereas SMA mRNA expression is maintained.

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Fig. 3.
RNase protection analysis of total RNA from cultured smooth muscle
cells. Probe (8U) size and protections expected are identical to those
described in Fig. 1 legend: 490 nt for SMB and 268 + 201 nt for SMA.
X174 end-labeled DNA was utilized as a molecular weight marker.
Total RNA (20 µg) from cultured primary rat aorta smooth muscle cells
(rASMC) contains only SMA mRNA. Adult bladder RNA was used as a
positive control for presence of SMB mRNA; tRNA was used as negative
control.
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SMB isoform is associated with the SM1 or SM2 tail in adult smooth
muscle.
To determine whether the SMB protein is associated with SM1
and/or SM2 tails in rat smooth muscle, the SMB isoform was
examined at the protein level by utilizing peptide-specific antibodies to probe smooth muscle tissue proteins separated by gel electrophoresis and blotted to nitrocellulose. Reaction of the immunoblot with the SMB
antibody resulted in detection of a doublet of bands in stomach,
bladder, intestine, and uterus (Fig.
4A).
Approximately equivalent amounts of SMB isoform were detected in the
204-kDa (SM1) and 200-kDa (SM2) bands in bladder smooth muscle. In the stomach and uterine extracts, however, staining by SMB antibody appeared more intense in the 204-kDa band. In contrast, in the intestinal sample the SMB staining appeared more intense in the 200-kDa band. These results suggest that the SMB isoform variant is not
always equally distributed between SM1 and SM2 isoforms. The blot was
also reacted with SM1 and SM2 antibodies (Fig. 4, B and
C) to demonstrate the presence of
these isoforms in tissues that lack staining for the SMB isoform,
such as aorta.

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Fig. 4.
Western blot analysis of adult smooth muscle tissues. Total protein
(200 µg) from extracts was electrophoresed, electroblotted to
nitrocellulose, then reacted sequentially with SMB, SM1, and SM2
antibody (Ab) with stripping of blot before reaction with a different
antibody. A: SMB antibody reacts with
bands at 204 and 200 kDa (SM1 and SM2). No significant reactivity is
detectable in samples from aorta, heart, brain, skeletal muscle,
trachea, or lung. SMB protein can be detected in trachea and lung, but
only at much higher protein concentrations ( 1 mg).
B: SM1 antibody reacts with all smooth
muscle tissue samples but not with samples from heart, brain, or
skeletal muscle. C: SM2 antibody
reacts with samples from stomach, bladder, aorta, intestine, and
uterus. SM2 is not detectable in tissues from trachea or nonsmooth
muscle. Lack of reactivity in trachea is thought to be due to relative
weakness of SM2 antibody.
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An additional Western blot (Fig. 5), loaded
to observe similar amounts of myosin (rather than equal amounts of
total protein), was probed sequentially with the SMB antibody and an
antibody that recognizes all smooth muscle myosins (SMHC antibody) to
emphasize the differential expression of the SMB head isoform with
respect to the SM1 and SM2 tail isoforms. Scanning densitometry of this blot and others with independently isolated samples was
used to generate the data summarized in Table
1. Intestinal and uterine tissues have
unequal distributions of the SMB isoform between the SM1 and SM2 tail
isoforms, whereas bladder and stomach have equal or nearly equal
proportions of the SMB motif associated with each tail isoform.

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Fig. 5.
Western blot analysis of adult smooth muscle: a comparison of SMB
content to SM1 and SM2 isoform content. Adult smooth muscle extracts
were loaded at different total protein amounts to give similar amounts
of smooth muscle myosin per lane to facilitate comparisons (total
protein loaded = 50, 85, 150, 150, and 115 µg for bladder, aorta,
stomach, intestine, and uterus, respectively).
A: blot reacted with SMB antibody.
B: blot in
A after stripping and reaction with a
smooth muscle MHC (SMHC) antibody that recognizes SM1 and SM2
isoforms.
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Smooth muscle MHC proteins are differentially expressed during
development.
Previous studies of the SM1 and SM2 isoforms in rabbit indicate that
these isoforms are differentially expressed during development. The SM1
isoform expression, at RNA and protein levels, precedes that of SM2 in
developing rabbit smooth muscle (15, 16). To determine the
developmental pattern of SMB protein expression, as well as its
association with SM1 and SM2 tail isoforms, the SMB protein was
detected with the peptide-specific antibody on immunoblots of
developing smooth muscle from rat aorta, bladder, and stomach.
Western blot analysis of extracts of rat smooth muscle at various
stages of development indicates that all smooth muscle MHC isoforms are
developmentally regulated (Fig. 6). Bladder
and stomach extracts react with the SMB antibody at all stages of
development examined. Samples from 1-day and 1-wk aorta react weakly
with this antibody, including a band of relatively low molecular
weight, whereas the adult aortic samples do not react with this
antibody. The early fetal visceral smooth muscle samples (17 and 18 days) do not react with the SMB antibody, despite evidence that SMB mRNA is present at these stages of development (Fig. 2). Additionally, these results demonstrate that the SM1 isoform is present during early
and adult stages of development in all rat smooth muscle tissues
examined, with the exception of the 17-day fetal aorta, which lacks
expression of any smooth muscle MHC isoform, perhaps reflecting the
presence of few differentiated smooth muscle cells in the sample.

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Fig. 6.
Western blot analysis of fetal, neonatal, and adult smooth muscle
tissues. Immunoblot was processed as described in Fig. 3. Blot was
reacted sequentially with SMB, SM1, and SM2 antibodies.
A: SMB antibody reacts with a doublet
in all bladder samples and in postnatal stomach samples. A faint
reactivity is observed in samples from stomach at 20 days and from
neonatal aorta at 1 day and 1 wk. A lower-molecular-weight band is
observed in 1-day aorta, possibly a result of protein degradation.
B: SM1 antibody detects myosin in
17-day visceral smooth muscle as well as all other smooth muscle
samples, except sample from aorta at 17 days. Lack of reactivity in
sample from aorta at 17 days may be result of relatively little smooth
muscle MHC in this tissue at this stage of development.
C: SM2 antibody reacts with all
bladder samples but does not react with samples from aorta at <1 wk
or sample from stomach at 20 days. Lack of reactivity in early samples
from aorta and stomach indicated that expression of SM2 isoform lags
behind that of SM1 isoform.
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The developmental expression of the SM2 isoform, however, clearly
differs from that of SMB or SM1 isoforms. The SM2 isoform is first
detectable in 1-wk aorta and 1-wk stomach, a significant delay in
expression compared with the SM1 and SMB isoforms. However, in bladder
smooth muscle, SM2 is abundant at 20 days of fetal development.
The developmental pattern of smooth muscle MHC isoform expression as
determined in this study is summarized in Table
2. These results indicate that each isoform
is developmentally regulated and that the pattern of smooth muscle MHC
isoform expression is tissue dependent.
 |
DISCUSSION |
The functional significance of smooth muscle MHC isoform diversity has
yet to be fully resolved. Early attempts to correlate SM1 and SM2
composition to smooth muscle function have been inconclusive, perhaps
because of the inability to distinguish among the four isoforms (18,
23, 24). However, studies of the SMA and SMB variants demonstrate that
the SMB isoform has a twofold higher ATPase activity and faster in
vitro motility of the actin filaments than the SMA isoform (14, 21).
The proportion of SMA and SMB MHC in a tissue is likely to be an
important determinant of contractile activity. A decrease in the SMB
content in hypertrophied rat bladder was associated with a reduction in
maximal shortening velocity (25). A study of small muscular arteries
also suggests that blood vessels with a higher SMB content have higher
maximal shortening velocities (8). Splicing in the myosin head has also
been demonstrated in nonmuscle myosins and may represent a conserved
mechanism for generating functional diversity in myosins (27). Sequence
variation in the junction of the 25- and 50-kDa tryptic peptides has
been demonstrated to affect ATP turnover rate in molluscan myosins (20). Therefore, it is likely that alternative splicing in the head
region of smooth muscle myosin is of considerable functional importance.
In the present study we demonstrate that the SMA and SMB smooth muscle
MHC isoforms are developmentally regulated in a smooth muscle cell
type-specific manner. Transcripts for the SMB, as well as SMA, isoform
are first detected at 17 days postcoitus. In fetal development the SMB
transcript is present in all smooth muscles examined. The SMA mRNA is
also present during all stages of development; however, its expression
is relatively low in fetal stages and increases in neonatal stages in
smooth muscle from intestine, stomach, and aorta.
The expression of SMA and SMB mRNA during development is highly tissue
specific. In the developing rat bladder, SMB mRNA expression is
relatively robust, whereas comparatively little SMA mRNA is expressed.
This pattern is established early (19 days postcoitus) in development
and persists into the adult stage. The developmental pattern of smooth
muscle expression in aortic smooth muscle is entirely different. In
this tissue, SMB mRNA is abundant in neonatal aorta, but its expression
diminishes with development, until only trace amounts of SMB transcript
are detected in normal adult aorta. This switch from SMB to SMA mRNA is
intriguing and suggests that neonatal aortic smooth muscle may possess
contractile properties distinct from those of adult aortic smooth
muscle.
Other developmental patterns of smooth muscle MHC mRNA expression are
also observed. In stomach and intestine, SMB mRNA expression is high
throughout development. However, the relative abundance of SMA mRNA
increases during the 1st wk of postnatal development until its
abundance is equal to or greater than that of the SMB transcript. The
significance of the upregulation of SMA mRNA during postnatal
development in these tissues is unknown and requires further
investigation.
Cultured aortic smooth muscle cells do not express detectable levels of
SMB mRNA under the culture conditions used. Immunofluorescent staining
of cultured aortic and tracheal smooth muscle cells with the SMB
antibody also shows that the cells lack detectable levels of the SMB
protein, although all cells were stained by a monoclonal smooth muscle
-actin antibody and most cells were also stained by the
peptide-specific polyclonal SM1 myosin antibody (data not shown).
Because neonatal aortic and adult tracheal smooth muscles normally
express SMB mRNA and protein, the absence of SMB message or protein
indicates that the expression of this isoform is downregulated in
culture. Cultured aortic cells are thought to undergo changes in
phenotype in culture, modulating from a contractile to a
less-differentiated, proliferative phenotype. The proliferative
phenotype is sometimes referred to as a fetal phenotype, suggesting
that the cells are regressing from an adult to a fetal differentiation
state. The results from this study suggest that this may be an
oversimplification, inasmuch as the SMB isoform is not expressed in
cultured cells, whereas it is expressed in neonatal aortic smooth
muscle cells, which are not considered to be fully differentiated. In a
recent study of smooth muscle cells from chicken aorta and gizzard,
cultured cells were shown to maintain expression of the SMB mRNA by the use of Matrigel as a plating matrix for the cells (10). These results
indicate that the extracellular matrix plays a role in maintenance of
smooth muscle MHC isoform expression in cultured cells.
We examined the smooth muscle MHC isoform content of smooth muscle
extracts via Western blot analysis in an effort to correlate the smooth
muscle MHC mRNA data to MHC protein composition of smooth muscle and to
determine whether the SMB isoform is associated with one or both tail
isoforms. Previous studies, based on cloning data, suggested that the
SM1 tail can be combined with the A or B head isoform (30). In the
chicken, antibody to the insert-containing (SMB) isoform demonstrated
that it is associated with SM1 and SM2 in chicken gizzard extract (14).
Here we show that the SMB isoform is associated with the SM1 and SM2
isoforms in several mammalian smooth muscles. A peptide-specific
antibody to the insert amino acids in the SMB isoform reacts with SM1
and SM2 proteins in immunoblots of tissue from stomach, bladder,
intestine, and uterus. As predicted, on the basis of RNase protection
data, the antibody does not detect SMB protein in adult aortic extract. Our results also suggest that the proportion of SMB isoform associated with SM1 and SM2 tails may be tissue dependent. Scanning densitometry results indicate that approximately equal proportions of the SMB head
are associated with SM1 and SM2 tails in bladder and stomach smooth
muscles. However, in intestinal smooth muscle more SMB appears to be
associated with the SM2 isoform, whereas in uterine smooth muscle more
SMB appears to be associated with the SM1 tail. Further studies are
needed to clarify these observations, including a more detailed
examination of isoforms in various regions of the intestine and in
uterus at various stages of estrus and pregnancy. The functional
significance of the differential expression of SMB in combination with
SM1 or SM2 tails remains to be determined; however, it may be another
means of producing functional diversity in smooth muscle tissues.
The tissue- and development-specific regulation of the SMB isoform was
also examined at the protein level using the SMB peptide-specific antibody. The bladder extracts show an abundance of SMB protein in
association with SM1 and SM2 tails from 19 days postcoitus throughout
development. The antibody faintly stained the 1-day and 1-wk neonatal
rat aortic extracts, indicating the presence of the SMB isoform,
although not in the quantity one might predict on the basis of the
RNase protection data. In stomach extracts, expression of SMB protein
is somewhat weak in fetal and 1-day neonatal tissues. On the basis of
RNase protection data, one might expect relatively high levels of SMB
isoform that would decrease somewhat during later developmental stages
in stomach tissue. The discrepancies observed between the RNase
protection data and the Western blot results in samples from aorta and
stomach could be due to the relative sensitivities of the mRNA and
protein assays. Alternatively, the smooth muscle MHC mRNA levels may
not directly correlate with isoform protein levels in some tissues and
may reflect additional posttranscriptional control of smooth muscle MHC
expression.
In conclusion, we have presented data demonstrating the differential
expression of the SMA and SMB isoforms in mammalian smooth muscle
during development and in cultured rat aortic smooth muscle cells. In
addition, we have demonstrated the distribution of the SMB head
isoforms with SM1 and SM2 tail isoforms in adult and developing smooth
muscle tissues. These studies provide important basic information
necessary for understanding the molecular basis for smooth muscle
functional diversity.
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ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant RO1-HL-38355-II to M. Periasamy.
 |
FOOTNOTES |
Address for reprint requests: S. L. White, Dept. of Molecular
Physiology and Biophysics, University of Vermont, Given Medical Bldg.,
Rm. E201, Burlington, VT 05405.
Received 14 January 1997; accepted in final form 1 May 1998.
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