Loss of SM-B myosin affects muscle shortening velocity and maximal force development.

We used an exon-specific gene-targeting strategy to generate a mouse model deficient only in the SM-B myosin isoform. Here we show that deletion of exon-5B (specific for SM-B) in the gene for the heavy chain of smooth muscle myosin results in a complete loss of SM-B myosin and switching of splicing to the SM-A isoform, without affecting SM1 and SM2 myosin content. Loss of SM-B myosin does not affect survival or cause any overt smooth muscle pathology. Physiological analysis reveals that absence of SM-B myosin results in a significant decrease in maximal force generation and velocity of shortening in smooth muscle tissues. This is the first in vivo study to demonstrate a functional role for the SM-B myosin isoform. We conclude that the extra seven-residue insert in the surface loop 1 of SM-B myosin is a critical determinant of crossbridge cycling and velocity of shortening.

Regulation of alpha-smooth muscle actin and CRBP-1 expression by retinoic acid and TGF-beta in cultured fibroblasts.

We have reported that Cellular Retinol Binding Protein-1 (CRBP-1) is expressed de novo during skin wound healing by a proportion of fibroblastic cells which then differentiate into myofibroblasts and express alpha-smooth muscle actin. In fibroblasts cultured from different tissues we have shown that alpha-smooth muscle actin expression, mainly controlled by Transforming Growth Factor-beta (TGF-beta), is also regulated by retinoic acid and that CRBP-1, known to be a retinoic acid-responsive gene, is modulated by TGF-beta. The aim of the present study has been to investigate the relationships between retinoic acid and TGF-beta in regulating the expression of CRBP-1 and alpha-smooth muscle actin in cultured rat subcutaneous tissue fibroblasts. We have observed that the TGF-beta-induced, but not the retinoic acid-induced, alpha-smooth muscle actin expression is associated with a modulation of endogenous TGF-beta and TGF-beta receptors, suggesting that the action of retinoic acid on alpha-smooth muscle actin expression is not mediated by TGF-beta. The expression of CRBP-1 is regulated at the transcriptional level by TGF-beta and retinoic acid but not synergistically, suggesting a possible common pathway. However, retinoic acid, but not TGF-beta, increases the transcription of a transiently transfected chimeric construct containing the retinoic acid response element of the CRBP-1 promoter, indicating that TGF-beta does not influence CRBP-1 through the retinoic acid pathway. Our results indicate that distinct pathways regulate the genes involved in the appearance and evolution of the myofibroblastic cells. The characterization of these pathways will be helpful for the design of drugs influencing wound healing.

Modulation of myofibroblast and smooth-muscle phenotypes in the lung.

Considerable progress has been made in defining the phenotype of contractile cells of the lung during development, in the adult and during the remodeling process. The high degree of phenotypic heterogeneity of subgroups of these cells, such as SMCs, is appreciated. Recent studies also have explored the relationship between phenotype and cell function, though this remains an important area for research in the coming years. Similarly, though our understanding of the regulation of cell phenotype is expanding rapidly, much remains to be done, particularly at the level of gene regulation. New transgenic models, coupled with gene-promotor analyses in transgenic animals and in cultured cells should allow rapid progress. Studies of the regulation of specific contractile and cytoskeletal proteins at the gene level by specific cytokines and extracellular-matrix elements will be particularly important.

Smooth-muscle myosin heavy-chain SM-B isoform expression in developing and adult rat lung.

The smooth-muscle cells composing the vasculature and airways of the lung display a variety of contractile protein phenotypes. To date, however, it has remained unclear how these phenotypes might contribute differentially to contractile activity. To address this issue, we made monospecific rabbit polyclonal antibodies against the difference peptide for the SM-B smooth-muscle myosin heavy chain (SMMHC) and used these to investigate the distribution of the SM-B isoform in lung. SM-B has a seven-amino acid insert in the head region that is known to result in a higher actin-activated adenosine triphosphatase activity and in vitro motility. During development, reactivity is first seen in the trachea and bronchi of saccular lung at the time of birth, when other SMMHC isoforms also are present. Immunoreactivity spreads distally through the airways as development proceeds, reaching the level of alveolar septae in the adult. Although the smaller vessels of the pulmonary vasculature react strongly with the SM-B antibody, reactivity is infrequently observed in large pulmonary vessels. Adult tracheal smooth muscle is highly and more uniformly reactive, commensurate with its relatively high maximal velocity of shortening. The differential expression of the SM-B isoform in vascular and airway smooth muscles demonstrated in this study may provide the molecular basis for functional differences between these smooth-muscle cell types and may provide one mechanism for adapting contractility in response to physiologic stresses in the lung.

Age dependence of smooth muscle myosin expression by cultured rat aortic smooth muscle cells.

Vascular smooth muscle cells (SMC) in vivo are highly heterogeneous phenotypically, particularly during development and in the adult during periods of remodeling. Much remains to be learned, however, regarding regulation of the SMC phenotype at the gene level. Here, we studied smooth muscle myosin heavy chain (SMMHC) expression at the transcriptional and mRNA levels in SMC cultured from newborn, adult, and old animals, which express different patterns of differentiation markers. We also examined regulation of SMMHC gene expression by TGF-beta, a cytokine known to be involved in the differentiation process. The activity of SMMHC promoter constructs, the expression of which is smooth-muscle-specific, was greatest in SMC from newborn animals and least in cells from old animals. Thus, differences in the degree of differentiation of SMC from these three sources may at least in part be due to transcriptional events. SMC from the three animal sources each contained mRNAs for the SM-1A and SM-2A tail but not those for the SM-1B and SM-2B head isoforms. Total SMMHC mRNA levels reflected similar differences as found at the transcriptional level. SM-2A mRNA as a proportion of total SMMHC mRNA was greatest in SMC from newborn animals, consistent with their higher degree of differentiation. TGF-beta up-regulated both transcription and mRNA levels but did not change the proportions of SMMHC mRNAs. Though the levels of transcriptional activity and mRNA were widely different in untreated cells, the degree of TGF-beta stimulation was approximately the same in all cases.

Myofibroblasts in diffuse alveolar damage of the lung.

The myofibroblast is an ultrastructurally and metabolically distinctive connective tissue cell identified as a key participant in tissue remodeling in human granulation tissue, organ fibrosis, and the fibroblastic host response to malignant neoplasms. In this study of myofibroblasts in human lung diffuse alveolar damage (DAD), we identified 36 autopsy cases in which DAD could be histologically documented. DAD is known to progress from initial injury through an exudative, proliferative, and terminal fibrotic phase. In the exudative phase (16 cases), myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA) are found in the septa and less frequently in hyaline membranes. In the proliferative phase (18 cases), many myofibroblasts in septa, hyaline membranes, and intra-alveolar fibroplasia express alpha-SMA. The alpha-SMA phenotype should be used in additional studies of myofibroblast differentiation, replication, and apoptosis. A better understanding of the biology of this cell type should offer new therapy for patients with DAD.

Lung smooth muscle differentiation.

The vascular and visceral smooth muscle tissues of the lung perform a number of tasks that are critical to pulmonary function. Smooth muscle function often is compromised as a result of lung disease. Though a great deal is known about regulation of smooth muscle cell replication and cell and tissue contractility, much less is understood regarding the phenotype of the contractile protein machinery of lung smooth muscle cells. This review focuses on the expression of cytoskeletal and contractile proteins of lung vascular and airway smooth muscle cells during development, in the adult and during vascular and airway remodeling. Emphasis is placed on the expression of the heavy chain of smooth muscle myosin, as well as the regulation of its gene. Important areas for future research are discussed.

Myosin heavy chain isoform expression in rat smooth muscle development.

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.

Smooth muscle actin and myosin expression in cultured airway smooth muscle cells.

In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos fibers.

Epidermal growth factor (EGF) is a potent mitogen for human mesothelial cells, and autophosphorylation of the EGF receptor (EGF-R) occurs in these cell types after exposure to asbestos, a carcinogen associated with the development of mesothelioma. Here, the intensity and distribution of EGF-R protein was documented by immunocytochemistry in a human mesothelial cell line (MET5A) exposed to various concentrations of crocidolite asbestos and man-made vitreous fibers (MMVF-10). Whereas cells in contact with or phagocytizing shorter asbestos fibers (<60 microm length) or MMVF-10 at a range of concentrations showed no increase in EGF-R protein as determined by immunofluorescence, elongated cells phagocytizing and surrounding longer fibers (> or =60 microm) showed intense staining for EGF-R. In contrast, human A549 lung carcinoma cells showed neither elongation nor increased accumulation of EGF-R protein in response to long fibers. Patterns of aggregation and increases in EGF-R protein in mesothelial cells phagocytizing long asbestos fibers were distinct from diffuse staining of phosphotyrosine residues observed in asbestos-exposed cultures. These studies indicate that aggregation of EGF-R by long fibers may initiate cell signaling cascades important in asbestos-induced mitogenesis and carcinogenesis.

Identification of promoter elements involved in cell-specific regulation of rat smooth muscle myosin heavy chain gene transcription.

In order to identify cis-acting regulatory elements involved in smooth muscle cell-specific gene regulation, we have cloned a 4. 7-kilobase pair fragment of the promoter for the rat smooth muscle myosin heavy chain, a protein expressed in differentiated smooth muscle cells. Sequence analysis of a 1.7-kilobase pair portion of this clone reveals potential binding sites for known transcription factors. A comparison of the primary sequence between the rat and rabbit smooth muscle myosin heavy chain promoters reveals numerous conserved consensus binding sites. Transient transfection analysis of promoter deletion constructs in rat aorta and tracheal smooth muscle cells, L8 myoblast cells, and rat pulmonary aorta endothelial cells suggests that a region of the promoter located between -1,249 and -1,317 base pairs is important for the restriction of gene expression to smooth muscle cells. Electrophoretic mobility shift analysis of a highly conserved region located between -1,317 and -1, 085 base pairs reveals specific DNA-protein complexes formed in smooth muscle cell extracts, which can be competed with an oligonucleotide containing a nuclear factor 1 binding site.

Glucagon and phenylephrine combination vs glucagon alone in experimental verapamil overdose.

OBJECTIVE: To evaluate glucagon and phenylephrine in combination as a treatment for the hemodynamic effects of verapamil overdose. METHODS: Pentobarbital-anesthetized and instrumented dogs were overdosed using a previously developed verapamil overdose model (15 mg/kg IV over 30 minutes). The animals were maintained and observed for 90 minutes or until death. Cardiac output (CO), heart rate (HR), and mean arterial pressure (MAP) were monitored. Following the 30-minute verapamil infusion (toxicity), the control animals received no treatment; the glucagon animals received a 5-mg glucagon bolus and a drip of 5 mg/90 minutes; and the glucagon/phenylephrine animals received the same glucagon therapy plus a phenylephrine drip titrated to 180 micrograms/min over 15 minutes. The groups were compared using analysis of variance: the experimental variables were group and time; the response variables were changes from toxicity for the hemodynamic parameters. Post-hoc comparisons were done with alpha set at 0.05. RESULTS: A significant change in CO was seen in the glucagon group (delta = 2.6 L/min) and the glucagon/phenylephrine group (delta = 1.9 L/min) compared with the control group (delta = 0.8 L/min). The change in CO was significantly larger for the glucagon animals compared with the glucagon/phenylephrine animals. The change in MAP for the glucagon/phenylephrine group (delta = 24 mm Hg) was significant compared with the control group (delta = 14 mm Hg). The MAP change for the glucagon group (delta = 19 mm Hg) was not significantly different from that of either the control or the glucagon/phenylephrine group. The change in glucagon HR (delta = 6 beats/min) was significant compared with the control group (delta = -4 beats/min) and the glucagon/phenylephrine group (delta = -4 beats/min). CONCLUSION: The glucagon/phenylephrine therapy improved MAP compared with the control, but reduced CO and HR compared with glucagon alone. Glucagon/phenylephrine therapy is not as effective as glucagon alone in reversing the hemodynamic effects of experimental verapamil overdose.

Increased collagen synthesis and decreased collagen degradation in right ventricular hypertrophy induced by pressure overload.

OBJECTIVE: The aim was to examine the effect of pressure overload in rabbits on ventricular collagen metabolism and procollagen gene expression. METHODS: Right ventricular hypertrophy was induced by banding the pulmonary artery such that the diameter of the vessel was reduced by 50%, and animals killed in groups after two and 14 days. Collagen synthesis and degradation of newly synthesised collagen were assessed following a single intravenous injection of 3H-proline with a flooding dose of non-radioactive proline, given 3 h before the animals were killed. Northern and slot blot analyses were performed to measure procollagen alpha 1(I) mRNA. RESULTS: The fractional collagen synthesis rate increased sixfold in the right ventricle only 2 d after pulmonary artery banding (p < 0.001), then fell to just over double the control value by 14 d (p < 0.05 from control). The proportion of newly synthesised collagen degraded decreased from 50.7(SD 12.8)% to 26.8(15.8)% in 2 d (p < 0.05) and remained at this level. The procollagen alpha 1(I) mRNA level increased by more than fourfold in the right ventricle 2 d after the onset of pressure overload, and was less than three times control levels at 14 d. CONCLUSIONS: The development of right ventricular hypertrophy is associated with a rapid increase in collagen production, with regulation at multiple sites in the biosynthetic pathway. This regulation occurs at both transcriptional and post translational levels.

Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

Cyclic mechanical deformation stimulates human lung fibroblast proliferation and autocrine growth factor activity.

Cellular hypertrophy and hyperplasia and increased extracellular matrix deposition are features of tissue hypertrophy resulting from increased work load. It is known, for example, that mechanical forces play a critical role in lung development, cardiovascular remodeling following pressure overload, and skeletal muscle growth. The mechanisms involved in these processes, however, remain unclear. Here we examined the effect of mechanical deformation on fibroblast function in vitro. IMR-90 human fetal lung fibroblasts grown on collagen-coated silastic membranes were subjected to cyclical mechanical deformation (10% increase in culture surface area; 1 Hz) for up to 5 days. Cell number was increased by 39% after 2 days of deformation (1.43 +/- .01 x 10(5) cells/membrane compared with control, 1.03 +/- 0.02 x 10(5) cells; mean +/- SEM; P < 0.02) increasing to 163% above control by 4 days (2.16 +/- 0.16 x 10(5) cells compared with 0.82 +/- 0.03 x 10(5) cells; P < 0.001). The medium from mechanically deformed cells was mitogenic for IMR-90 cells, with maximal activity in the medium from cells mechanically deformed for 2 days (stimulating cell replication by 35% compared with media control; P < 0.002). These data suggest that mechanical deformation stimulates human lung fibroblast replication and that this effect is mediated by the release of autocrine growth factors.

In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin isoform expression in developing and remodeling rat lung.

The tissue distribution of myosin isoforms was examined in developing smooth muscle of rat lung. Antisera employed included a general smooth muscle myosin antibody (aSMMG) and two smooth muscle myosin isoform specific antisera (aSM1 and aSM2). In the pseudoglandular, canalicular, and saccular lung, the isoform-specific aSM1 antiserum was very lightly reactive only with large airway and not reactive with vascular smooth muscle, whereas aSM2 was unreactive with any lung cells. During these same stages, the aSMMG serum reacted well with the mesenchymal coat around the larger airways, declining in intensity as the tube size diminished. Vascular smooth muscle elements had only moderate reactivity at this time. In the adult, aSM1 marked airway smooth muscle as well as the tips of the alveolar septae. Vascular reactivity was seen in both arterial and venous elements. An identical distribution of reactivity was seen for aSMMG. aSM2 reactivity appeared confined primarily to airway smooth muscle and was absent from all but the largest vascular structures. Companion Western blot analyses confirmed the presence of SM1 in fetal and mature tissues as well as the relative lack of SM2 in all but the fully differentiated airways. Lung injury due to intratracheal instillation of bleomycin is characterized by a proliferation of mesenchymal cells similar to immature smooth muscle cells. These cells express smooth muscle forms of actin but lacked the mature smooth muscle myosin isoforms. In summary, differentiation of smooth muscle in the lung proceeds with progressive replacement of nonmuscle isoforms of myosin with differentiation-specific forms. In this regard, the maturation of vascular muscle tissue lags behind that of nonvascular (visceral) muscle structures.(ABSTRACT TRUNCATED AT 250 WORDS)