Expression Of Smooth Muscle Myosin Research Articles

Identifying peristaltic pacemaker cells in the upper urinary tract.

Urine expulsion from the upper urinary tract is a necessary process that eliminates waste, promotes renal filtration and prevents nephron damage. To facilitate the movement of urine boluses throughout the upper urinary tract, smooth muscle cells that line the renal pelvis contract in a coordinated effort to form peristaltic waves. Resident pacemaker cells in the renal pelvis are critical to this process and spontaneously evoke transient depolarizations that initiate each peristaltic wave and establish rhythmic contractions. Renal pacemakers have been termed atypical smooth muscle cells due to their low expression of smooth muscle myosin and poor organization of myofilaments compared to typical (or contractile) smooth muscle cells that perform peristalsis. Recent findings discovered that pacemaker cells also express the tyrosine kinase receptor PDGFRα, enabling their identification and purification amongst other renal pelvis cell types. Improved identification methods have determined that the calcium-activated chloride channel, ANO1, is expressed by pacemaker cells and may contribute to spontaneous depolarization. A greater understanding of pacemaker and peristaltic mechanisms is warranted since aberrant contractile function may underlie diseases such as hydronephrosis, a deleterious condition that can cause significant and irreversible nephron injury.

High Altitude Hypoxia Induces Cellular Immaturity of Pulmonary Arteries in the Fetal Lamb: Assessment of Protein Biomarkers

Pulmonary Arterial Hypertension (PAH) in newborns can be mediated by intrauterine hypoxia and which can cause significant aberrations on vascular structure and function. Previous evidence suggests that long term hypoxic stress can induce phenotypic modifications to various vascular beds, including those of the lung. We are now seeking to interrogate the influence of intrauterine hypoxia on pulmonary vascular phenotypes and to test the hypothesis that intrauterine hypoxia causes the vasculature to shift from a mature to an immature phenotype. This was evaluated by examining protein expression using proteomic analyses on pulmonary arteries isolated from near term fetal lambs that were exposed to intrauterine high altitude hypoxia (3801 m) for the latter 110+ days of pregnancy or those gestated near sea level. Protein networks were created via Ingenuity Pathway Analysis (IPA) and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Intrauterine hypoxia caused a number of significant changes including reducing the expression of smooth muscle myosin, fibronectin, and collagen along with upregulation of proteins involved in nucleotide synthesis, mitosis/cell proliferation, as well as cell adhesion and migration. The observed changes are consistent with a hypoxia‐induced phenotypic shift towards cells being more synthetic, proliferative, and migratory. Such findings provide insight into the mechanistic underpinnings related to the development of pulmonary hypertension due to gestational hypoxia. This work opens new avenues of investigation to determine common signaling pathways affected by intrauterine hypoxia with the aim of discovering the fundamental mechanisms driving hypoxia‐induced remodeling and senescence.Support or Funding InformationSupported in part by NIH grants HD083132 (LZ), HD098477 (SMW), RA was a Apprenticeship Bridge to College summer research fellow through the LLU Center for Health Disparities Research with funding from P20MD006988 (M. DeLeon) from the National Institute of Health Disparities and Minority Health of the National Institutes of Health.

Smooth muscle myosin expression, isoform composition, and functional activities in rat corpus cavernosum altered by the streptozotocin-induced type 1 diabetes

Diabetes mellitus (DM) is a quite common chronic disease, and the prevalence of erectile dysfunction (ED) is three times higher in this large population. Although diabetes-related ED has been studied extensively, the actin-myosin contractile apparatus was not examined. The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and essential light chains (LC(17)) exist as several different alternatively spliced isoforms with distinct contractile properties. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed corpus cavernosum smooth muscle (CCSM). In this study, we examine whether diabetes alters SMM expression, alternative splicing, and/or functional activities, including sensitivity to BLEB. By using streptozotocin (STZ)-induced 2-mo diabetic rats, functional activities were tested in vivo by intracavernous pressure (ICP) recording during cavernous nerve stimulation and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin, and embryonic SMM (SMemb) expression by real-time RT-PCR. Results revealed that the blood glucose level of STZ rats was 407.0 vs. 129.5 mg/dl (control). STZ rats exhibited ED confirmed by significantly increased CCSM contractile response to phenylephrine and decreased ICP response. For STZ rats, SM-B, LC(17a) and SM2 isoforms, total SMM, and myocardin expression increased, whereas SM-A, LC(17b), and SM1 isoforms were decreased, with SMemb unchanged. BLEB was significantly more effective in relaxing STZ CCSM both in vitro and in vivo. Thus we demonstrated a novel diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype was supported further by total SMM expression increase. Moreover, the change in CCSM phenotype was associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED.

Generalized neuromuscular hypoplasia, reduced smooth muscle myosin and altered gut motility in the klotho model of premature aging

Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging-associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make-up of the small intestinal and colonic neuromuscular apparatus. Klotho expression was studied by RT-PCR and immunohistochemistry. Motility was assessed by dye transit and bead expulsion. Smooth muscle and neuron-specific gene expression was studied by Western immunoblotting. Interstitial cells of Cajal (ICC) and precursors were analyzed by flow cytometry, confocal microscopy, and three-dimensional reconstruction. HuC/D(+) myenteric neurons were enumerated by fluorescent microscopy. Klotho protein was detected in neurons, smooth muscle cells, and some ICC classes. Small intestinal transit was slower but whole-gut transit of klotho mice was accelerated due to faster colonic transit and shorter intestinal lengths, apparent only after weaning. Fecal water content remained normal despite reduced output. Smooth muscle myosin expression was reduced. ICC, ICC precursors, as well as nitrergic and cholinergic neurons maintained their normal proportions in the shorter intestines. Progeric klotho mice express less contractile proteins and develop generalized intestinal neuromuscular hypoplasia mainly arising from stunted postweaning growth. As reduced fecal output in these mice occurs in the presence of accelerated colonic and whole-gut transit, it likely reflects reduced food intake rather than intestinal dysmotility.

Differential Expression of Smooth Muscle Myosin, Smooth Muscle Actin, H-Caldesmon, and Calponin in the Diagnosis of Myofibroblastic and Smooth Muscle Lesions of Skin and Soft Tissue

The diagnosis of low-grade and pseudosarcomatous spindle cell lesions of skin and soft tissue can sometimes be problematic; in particular, distinction between fibroblastic, myofibroblastic, and smooth muscle proliferations can occasionally pose difficulties on routine histologic examination. We have applied a panel of immunohistochemical markers to a series of spindle cell lesions of skin and soft tissue to assess the utility of the differential expression of smooth muscle and myofibroblastic-associated markers. Twenty-eight cases of nodular fasciitis, 42 cases of fibromatosis, and 3 cases of myofibroblastic sarcoma were stained with antibodies against smooth muscle actin (SMA), smooth muscle myosin (SMMS), calponin, and high-molecular weight caldesmon (h-caldesmon). For comparison, 12 cases of cutaneous leiomyoma and 8 cases of leiomyosarcomas involving superficial soft tissues and fascia were studied with the same panel of antibodies. Thirty-eight of 42 cases of fibromatosis were positive for SMA, 42/42 cases were positive for calponin, 39/42 cases were negative for SMMS, and all cases were negative for h-caldesmon. All cases of nodular fasciitis were positive for SMA and calponin, and all were negative for h-caldesmon and SMMS. All cases of myofibroblastic sarcoma were positive for SMA and 2/3 cases for calponin, and were negative for SMMS and h-caldesmon. All cases of cutaneous leiomyoma and leiomyosarcoma were positive for all 4 markers tested. Our results demonstrate a remarkably consistent pattern of reactivity of muscle and myofibroblastic-associated markers in lesions predominantly composed of myofibroblastic spindle cells, characterized by positive staining for SMA and calponin and absence of reactivity for SMMS and h-caldesmon. Application of this panel of stains may be of aid in the differential diagnosis of low-grade myofibroblastic lesions such as nodular fasciitis and fibromatosis from smooth muscle tumors of skin and soft tissue. This panel may additionally be of assistance in the diagnosis of myofibroblastic sarcoma.

Effects of ML-7 and Y-27632 on carbachol- and endothelin-1-induced contraction of bovine trabecular meshwork

The trabecular meshwork is considered a smooth muscle like tissue contributing to aqueous outflow regulation and thus to regulation of intraocular pressure. An elevation in intraocular pressure is one of the greatest risk factors for most forms of glaucoma. We assume that contraction of trabecular meshwork reduces aquous humor outflow and thus enhances intraocular pressure, whereas relaxation exerts the opposite effect. The present paper supports the hypothesis of the trabecular meshwork beeing a smooth muscle-like tissue. We perform measurements of isometric force in isolated bovine trabecular meshwork strips. Contractility of this tissue is induced by carbachol or endothelin-1. The contractile force is sucessfully inhibited by ML-7, a highly specific inhibitor of myosin light chain kinase. The contraction is also reduced in the presence of the RhoA kinase inhibitor Y-27632. We further describe the protein expression of smooth muscle myosin and its regulatory kinase, the myosin light chain kinase, in human and bovine trabecular meshwork cells. Additionally, the serine phosphorylation of myosin light chain kinase is shown. These data indicate that the trabecular meshwork expresses major contractility regulating proteins which are involved in tissue function. Inhibition of the signaling pathways which lead to myosin phosphorylation causes inhibition of contractile force in trabecular meshwork. According to our concept of aqueous humor outflow regulation, trabecular meshwork relaxing substances appear to be ideal antiglaucomatous drugs, leading to increased outflow facility.

Sympathetic innervation promotes vascular smooth muscle differentiation

The sympathetic nervous system (SNS) is an important modulator of vascular smooth muscle (VSM) growth and function. Several lines of evidence suggest that the SNS also promotes VSM differentiation. The present study tests this hypothesis. Expression of smooth muscle myosin (SM2) and alpha-actin were assessed by Western analysis as indexes of VSM differentiation. SM2 expression (normalized to alpha-actin) in adult innervated rat femoral and tail arteries was 479 +/- 115% of that in noninnervated carotid arteries. Expression of alpha-actin (normalized to GAPDH or total protein) in 30-day-innervated rat femoral arteries was greater than in corresponding noninnervated femoral arteries from guanethidine-sympathectomized rats. SM2 expression (normalized to alpha-actin) in neonatal femoral arteries grown in vitro for 7 days in the presence of sympathetic ganglia was greater than SM2 expression in corresponding arteries grown in the absence of sympathetic ganglia. In VSM-endothelial cell cultures grown in the presence of dissociated sympathetic neurons, alpha-actin (normalized to GAPDH) was 300 +/- 66% of that in corresponding cultures grown in the absence of neurons. This effect was inhibited by an antibody that neutralized the activity of transforming growth factor-beta2. All of these data indicate that sympathetic innervation increased VSM contractile protein expression and thereby suggest that the SNS promotes and/or maintains VSM differentiation.

Characterization of Neuropathic Bladder Smooth Muscle Cells in Culture

Clinically bladder cells used in tissue engineering techniques will come from neuropathic bladders and not normal bladders. We determined if neuropathic bladder smooth muscle (SM) cells (SMCs) retain functional differences when cultured in vitro. Primary cultures of SMCs were established from patients with a neuropathic bladder (5) and a normal bladder (5). Expression of alpha-SM actin and SM myosin heavy chain was determined using immunocytochemical staining and Western blot analysis. Baseline cell proliferation and the mitogenic response to angiotensin II was assessed by cell counting and cell viability assays. Cell contractility was determined for normal and neuropathic SMCs using an in vitro collagen lattice assay. Cell adherence was measured assessed using partial and complete trypsinization assays. Normal and neuropathic SMCs showed similar morphology in culture, and similar patterns of alpha-SM actin and SM myosin expression. Following 10 days of plating under optimal growth conditions the number of neuropathic SMCs was 170% more than normal SMCs. In response to angiotensin II neuropathic SMCs reached 54% of maximal growth capacity as opposed to 30% for normal SMCs (p <0.01). Neuropathic SMCs contracted significantly less in 10% serum and calcium ionophore (p <0.05), as determined by in vitro contractility assays. Neuropathic SMCs had 19% and 30% less adherent cells than normal SMCs (p <0.01) following isotonic solution washes and trypsinization, respectively. These results demonstrate that cultured neuropathic bladder SMCs possess and maintain different characteristics than normal SMCs in vitro. The potential clinical implications of using these cells in conjunction with tissue engineering techniques for the promotion of bladder regeneration requires further investigation.

CArG elements control smooth muscle subtype-specific expression of smooth muscle myosin in vivo.

Expression of smooth muscle myosin heavy chain (SM-MHC) is tightly controlled depending on the differentiated state of smooth muscle cells (SMCs). To better understand the mechanisms that regulate transcription of the SM-MHC gene in vivo, we tested the function of several conserved CArG elements contained within the -4200 to +11,600 region of this gene that we had previously shown to drive SMC-specific expression in transgenic mice. CArG1 in the 5'-flanking sequence was required for all SMCs, while CArG2 and a novel intronic CArG element were differentially required in SMC subtypes. Of particular note, mutation of the intronic CArG selectively abolished expression in large arteries. A promoter construct containing three repeats of a conserved 227-bp intronic CArG-containing region was sufficient to direct transcription in vascular SMCs in transgenic mice, although this construct was also expressed in skeletal and cardiac muscle. These results support a model in which transcriptional regulation of SM-MHC is controlled by multiple positive and negative modular control regions that differ between SMCs and non-SMCs and among SMC subtypes. We also demonstrated that the CArG elements of the endogenous SM-MHC gene were bound by SRF in chromatin.

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-β, 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-β 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-β stimulation was approximately the same in all cases.

Lipid lowering promotes accumulation of mature smooth muscle cells expressing smooth muscle myosin heavy chain isoforms in rabbit atheroma.

Smooth muscle cells (SMCs) in the atherosclerotic intima characteristically differ from those in the arterial media, for example, by reduced expression of SMC differentiation/maturation markers such as smooth muscle myosin heavy chain isoforms (SM1 and SM2). This study tested the hypothesis that lipid lowering promotes maturation of intimal SMCs in 33 rabbits subjected to balloon injury and cholesterol feeding (0.3%) for 4 months (Baseline group, n=15); some of which then were switched to a low-cholesterol diet for 8 months (Low group at 8 months, n=3) or 16 months (Low group at 16 months, n=10). The remaining rabbits continued to consume a high-cholesterol diet for 16 months (High group, n=5). We monitored SMC phenotype by expression of immunoreactive alpha-smooth muscle actin, SM1, and SM2. alpha-Actin is an early marker, and SM1 and SM2 are late markers for SMC differentiation/maturation. Only fully differentiated or mature SMCs express SM2. Data are reported as the percentage of the alpha-actin-positive intimal area occupied by smooth muscle myosin-positive SMCs determined by color image analysis of immunostained sections. Levels of SM1 and SM2, highly expressed by SMCs in the normal aortic media (n=5) decreased in the aortic intima of the Baseline and High groups, indicating a less mature phenotype. In contrast, SM1 and SM2 increased in the Low (16 months) group, indicating that intimal SMCs exhibit a more mature phenotype after lipid lowering. Electron microscopy also showed the presence of mature intimal SMCs with abundant myofilaments. Furthermore, lipid lowering reduced levels of platelet-derived growth factor-B in the arterial intima, a factor known to suppress smooth muscle myosin expression. These data demonstrate that lipid lowering favors accumulation of mature SMCs in the atherosclerotic intima in association with reduced levels of platelet-derived growth factor-B expression. Intimal SMCs in the Low group also displayed reduced expression of matrix metalloproteinases-3 and -9 compared with the Baseline and High groups. These findings shed new light on the effects of lipid lowering at the level of the vascular wall, which may influence the biology of the atheroma.

The monoclonal antibody GB 42 ? a useful marker for the differentiation of myofibroblasts

The expression patterns of a variety of cytoskeletal antigens were studied in normal human tissues (placenta, umbilical cord, myometrium, colon, mammary gland, testis, skeletal muscle, myocardium) as well as in abnormal human tissues (palmar fibromatosis, fibrocystic disease of the mammary gland, mammary carcinoma). The immunohistochemical binding patterns of the monoclonal antibody GB 42 were compared to those of commercial antibodies directed against vimentin, desmin, smooth muscle myosin, pan actin, alpha-smooth muscle actin and gamma-smooth muscle actin. Methods applied comprised immunohistochemistry on cryostat sections and paraffin sections. Immunogold immunocytochemistry was performed on Lowicryl sections. The patterns of GB 42-binding were confirmed biochemically by SDS-PAGE and Western-blotting, and quantitative amino acid analysis. Our data suggest that the monoclonal antibody GB 42 recognizes an actin isoform which is identical to, or closely related to, gamma-smooth muscle actin. Unlike the commercially available antibody against gamma-smooth muscle actin, GB 42 does not cross-react with alpha-skeletal or alpha-cardiac actins. The GB 42-antigen is expressed in smooth muscle cells, myoepithelial cells and in later stages of differentiation of myofibroblasts, in all the tissues investigated. Throughout the development of smooth muscle cells and myofibroblasts, the appearance of the GB 42-antigen occurs after the expression of vimentin, desmin and alpha-smooth muscle actin, but prior to the expression of smooth muscle myosin. GB 42 is a reliable marker for higher stages of differentiation of smooth muscle cells and myofibroblasts.

Parallel Expression Level of Dystrophin and Contractile Performances of Rat Aortic Smooth Muscle

We have recently shown that the expression of dystrophin was in line with the conservation of contractility in primary and subcultured vascular smooth muscle cells (VSMC). In this report, we provide experimental evidence that shows the existence of a gradient in dystrophin expression from the aortic arch to the region just above the diaphragm, in line with a parallel gradient of contractility. On the contrary, there were no differences in caldesmon and smooth muscle myosin expression, as previously shown for vimentin and α-smooth muscle actin [Osborn, M., Caselitz, J., and Weber, K. (1981) Differentiation 20, 196-202]. Overall, this is the first demonstration, in a normal adult smooth muscle, of a differential expression of dystrophin, in line with its contractile performances. In addition, these results suggest that the experimental modulation of dystrophin expression in cultured VSMC in line with their contractility was the manifestation in vitro of an actual physiological property of those cells in vivo.

Neural and smooth muscle development in the chicken gizzard.

The development of the autonomic ganglia of Auerbach's plexus and gizzard smooth muscle was studied in chicken embryos. Nervous system and smooth-muscle-specific antibodies were employed in immunofluorescence stainings on tissue sections to investigate the temporal and spatial frame of neural and muscular differentiation in relation to each other. Subserosal clusters of neural cells were clearly demonstrable at embryonic day 5 (ED5), the earliest stage analysed, with the monoclonal antibody El (SGIII-1). Fine nerve fibres (ED6) and, later, large axon bundles projecting from subserosal neuron clusters towards the lumen were followed and found to reach the luminal border by ED11. Already in early development the area of the future laminar tendons on the ventral and dorsal surface of the gizzard was devoid of neuroblasts, and nerve fibres were not extending to the muscle-tendon borderline until ED16. Double stainings with antibodies to smooth muscle myosin (SMM) and El revealed that SMM expression, taken as an indicator for muscle differentiation, followed neural growth. It was first detectable in close apposition to the differentiating neuroblasts in the caudal and cranial portion of the gizzard at ED6. With further development, myosin expression proceeded inward towards the lumen in a wave which followed the ingrowth of E1-positive nerve fibres from the prospective Auerbach plexus. Neuromuscular differentiation deviated from this pattern in the lateral tendon area where nerve growth was delayed and myosin expression preceeded the arrival of E1-positive nerve fibres. The findings suggest that the gizzard could serve as a model system for the analysis of potential early nervous system imprints on smooth premuscle mesenchyme differentiation.

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, alpha-actin-in and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryo-specific isoform with a different molecular mass was detected.

Expression of smooth muscle myosin in relation to growth kinetics of cultured aortic smooth muscle cells

The goal of this study is to quantify smooth muscle myosin (SMM) expression at the level of the individual cell and to ascertain whether SMM expression in cultured aortic smooth muscle cells is related to definite growth phases, and whether the initial seeding density affects growth or SMM staining. Rabbit aortic smooth muscle cells (SMCs) were harvested by enzyme digestion of aortic tissue and plated at low (100 cells cm −2), medium (1000 cells cm −2), and high (10,000 cells cm −2) densities. Independent of seeding density, the lag phase lasted 2 to 3 days and, at all three densities, the growth rate during the logarithmic growth phase was almost the same. However, the time, the number of population doubling needed to reach the plateau phase and the cell number in the plateau were influenced by the initial seeding density. Immunofluorescence staining with anti-smooth muscle myosin (ASMM) revealed intensive staining of striated and filamentous patterns in all cells during the lag and early logarithmic growth phases. During the late logarithmic growth phase, two subpopulations of cells appeared, one showing a positive and the other no reaction with SMM antiserum. The lowest relative number of cells which showed positive reactions with SMM antiserum was observed toward the end of the logarithmic growth phase. During the plateau phase, the SMM-positive subpopulation increased, amounting to about 60 % of the total number of cells, independent of the seeding density. In terms of absolute numbers, the number of SMM-positive cells increased over the course of 21 days by factors of 13, 72, and 342 for high, medium, and low seeded cultures, respectively. We conclude that a SMC subpopulation can divide without loss of SMM and that some, but not all, cells which lose their SMM may possibly regain it in the postconfluent state.