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
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Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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
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Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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. phi 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.

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. phi 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.

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.

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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Table 1.   Percentage of smooth muscle MHC isoforms in adult smooth muscle tissue extracts

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.

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.

                              
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Table 2.   Developmental pattern of smooth muscle MHC expression: summary of Western blot results

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -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.

    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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(2):C581-C589
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