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Results for “Phenotypic modulation”

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PubMed2006

Differences in retinol metabolism and proliferative response between neointimal and medial smooth muscle cells.

Vascular disease is multifactorial and smooth muscle cells (SMCs) play a key role. Retinoids have been shown to influence many disease-promoting processes including proliferation and differentiation in the vessel wall. Phenotypic heterogeneity of vascular SMCs is a well-known phenomenon and phenotypic modulation of SMCs precedes intimal hyperplasia. The SMCs that constitute the intimal hyperplasia demonstrate a distinct phenotype and differ in gene expression compared to medial SMCs. Cellular retinol-binding protein-1 (CRBP-I), involved in retinoid metabolism, is highly expressed in intimal SMCs, indicating altered retinoid metabolism in this subset of cells. The aim of this study was to evaluate the metabolism of all-trans ROH (atROH), the circulating prohormone to active retinoids, in vascular SMCs of different phenotypes. The results show an increased uptake of atROH in intimal SMCs compared to medial SMCs as well as increased expression of the retinoid-metabolizing enzymes retinol dehydrogenase-5 and retinal dehydrogenase-1 and, in conjunction with this gene expression, increased production of all-trans retinoic acid (atRA). Furthermore, the retinoic acid-catabolizing enzyme CYP26A1 is expressed at higher levels in medial SMCs compared to intimal SMCs. Thus, both retinoid activation and deactivation processes are in operation. To analyze if the difference in ROH metabolism was also correlated to differences in the biological response to retinol, the effects of ROH on proliferation of SMCs with this phenotypic heterogeneity were studied. We found that intimal SMCs showed a dose- and time-dependent growth inhibition when treated with atROH in contrast to medial SMCs, in which atROH had a mitogenic effect. This study shows, for the first time, that (1) vascular SMCs are able to synthesize biologically active atRA from the prohormone atROH, (2) intimal SMCs have a higher capacity to internalize atROH and metabolize atROH into atRA compared to medial SMCs and (3) atROH inhibits growth of intimal SMCs, but induces medial SMC growth.

Alcohol Oxidoreductases↗

PubMed1996

Role of plasminogen activator inhibitor in the reciprocal regulation of bovine aortic endothelial and smooth muscle cell migration by TGF-beta 1.

Vascular endothelial and smooth muscle cells exhibit reciprocal migratory responses after transforming growth factor (TGF)-beta 1 treatment. Endothelial cells exhibit a decreased migratory rate and smooth muscle cells exhibit an increased migratory rate. Previous studies have demonstrated increases in extracellular matrix and integrin synthesis and expression in response to TGF-beta 1. In this report, we illustrate the roles of plasminogen activator inhibitor in modulating the migratory rates in these two cell types. Endothelial cells appear to require a proteolytic phenotype for rapid migration, whereas vascular smooth muscle cells appear to require an anti-proteolytic phenotype. Modulation of proteinase/anti-proteinase activity ratios was accomplished via TGF-beta 1 induction, addition of exogenous plasminogen activator inhibitor, addition of anti-catalytic antibodies directed against urokinase plasminogen activator, overexpression of plasminogen activator inhibitor utilizing stable transfectants, and the use of vitronectin as a substratum. The reciprocal migratory behaviors exhibited by these two vascular cell types in response to TGF-beta 1 is discussed in the context that these two vascular cell types utilize distinct adhesive and signaling pathways in their interactions with extracellular matrix components and responsiveness to proteolytic activity.

Animals↗

PubMed1999

EVEC, a novel epidermal growth factor-like repeat-containing protein upregulated in embryonic and diseased adult vasculature.

A hallmark of vascular lesions is the phenotypic modulation of vascular smooth muscle cells (VSMCs) from a quiescent, contractile state to a more primitive, proliferative phenotype with a more fetal pattern of gene expression. Using subtraction hybridization to identify genes that may regulate this transition, we cloned a novel gene named EVEC, an acronym for its expression in the embryonic vasculature and the presence of Ca2+ binding epidermal growth factor-like repeats contained in the predicted protein structure. Although these repeats are characteristic of the extracellular matrix proteins, fibrillin, fibulin, and the latent transforming growth factor-beta binding proteins, EVEC most closely resembles the H411 and T16/S1-5 gene products, the latter of which are believed to regulate DNA synthesis in quiescent fibroblasts. Using in situ hybridization, we demonstrated that EVEC is expressed predominantly in the VSMCs of developing arteries in E11.5 through E16.5 mouse embryos. Lower levels of expression are also observed in endothelial cells, perichondrium, intestine, and mesenchyme of the face and kidney. EVEC mRNA expression is dramatically downregulated in adult arteries, except in the uterus, where cyclic angiogenesis continues; however, EVEC expression is reactivated in 2 independent rodent models of vascular injury. EVEC mRNA is observed in cellular elements of atherosclerotic plaques of LDL receptor-deficient, human apolipoprotein B transgenic mice and in VSMCs of the media and neointima of balloon-injured rat carotid arteries. These data suggest that EVEC may play an important role in the regulation of vascular growth and maturation during development and in lesions of injured vessels.

Age Factors↗

PubMedDate not supplied

Cyclic GMP-dependent protein kinase regulates vascular smooth muscle cell phenotype.

Nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) have been reported to prevent vascular smooth muscle cell (VSMC) proliferation and have beneficial effects to reduce intimal thickening in response to arterial injury. The purpose of this study was to determine whether the downstream effector molecule of NO-cGMP signaling, cyclic GMP-dependent protein kinase (PKG), regulates phenotypic modulation and proliferation in cultured rat aortic VSMC. PKG-expressing VSMC lines were created by transfection of PKG-deficient cell lines and characterized. All forms of PKG, i.e. PKG-I alpha and PKG-I beta, as well as the constitutively active catalytic domain of PKG-I, transformed dedifferentiated 'synthetic' VSMC to a more contractile-like morphology. PKG expression resulted in an increased production of the contractile phenotype marker proteins, smooth muscle myosin heavy chain-2, calponin and alpha-actin and restored the capacity of cAMP and cGMP analogues to inhibit platelet-derived growth factor (PDGF)-induced cell migration. On the other hand, PKG expression had no significant effects on PDGF-induced cell proliferation. These results suggest that PKG expression contributes to the regulation of a contractile-like phenotypic expression in cultured VSMC, and the suppression of PKG expression during cultured growth in vitro may permit the modulation of cells to a more synthetic, dedifferentiated phenotype.

Animals↗

PubMed2005

Neurogenic potential of human mesenchymal stem cells revisited: analysis by immunostaining, time-lapse video and microarray.

The possibility of generating neural cells from human bone-marrow-derived mesenchymal stem cells (hMSCs) by simple in vitro treatments is appealing both conceptually and practically. However, whether phenotypic modulations observed after chemical manipulation of such stem cells truly represent a genuine trans-lineage differentiation remains to be established. We have re-evaluated the effects of a frequently reported biochemical approach, based on treatment with butylated hydroxyanisole and dimethylsulphoxide, to bring about such phenotypic conversion by monitoring the morphological changes induced by the treatment in real time, by analysing the expression of phenotype-specific protein markers and by assessing the modulation of transcriptome. Video time-lapse microscopy showed that conversion of mesenchymal stem cells to a neuron-like morphology could be reproduced in normal primary fibroblasts as well as mimicked by addition of drugs eliciting cytoskeletal collapse and disruption of focal adhesion contacts. Analysis of markers revealed that mesenchymal stem cells constitutively expressed multi-lineage traits, including several pertaining to the neural one. However, the applied ;neural induction' protocol neither significantly modulated the expression of such markers, nor induced de novo translation of other neural-specific proteins. Similarly, global expression profiling of over 21,000 genes demonstrated that gene transcription was poorly affected. Most strikingly, we found that the set of genes whose expression was altered by the inductive treatment did not match those sets of genes differentially expressed when comparing untreated mesenchymal stem cells and immature neural tissues. Conversely, by comparing these gene expression profiles with that obtained from comparisons between the same cells and an unrelated non-neural organ, such as liver, we found that the adopted neural induction protocol was no more effective in redirecting human mesenchymal stem cells toward a neural phenotype than toward an endodermal hepatic pathway.

Adult↗

PubMed2005

Modulation of smooth muscle cell proliferation and migration: role of smooth muscle cell heterogeneity.

Proliferation and migration of smooth muscle cells (SMCs) from the media towards the intima are key events in atherosclerosis and restenosis. During these processes, SMC undergo phenotypic modulations leading to SMC dedifferentiation. The identification and characterization of factors controlling these phenotypic changes are crucial in order to prevent the formation of intimal thickening. One of the questions which presently remains open, is to know whether any SMCs of the media are capable of accumulating into the intima or whether only a predisposed medial SMC subpopulation is involved in this process. The latter hypothesis implies that arterial SMCs are phenotypically heterogenous. In this chapter, we will describe the distinct SMC phenotypes identified in arteries of various species, including humans. Their role in the formation of intimal thickening will be discussed.

Animals↗

PubMed2004

Comparison of phenotypic characterization between "alginate bead" and "pellet" culture systems as chondrogenic differentiation models for human mesenchymal stem cells.

Chondrogenesis involves the recruitment of mesenchymal cells to differentiate into chondroblasts, and also the cells must synthesize a cartilage-specific extracellular matrix. There were two representative culture systems that promoted the chondrogenic differentiation of human mesenchymal stem cells. These systems were adaptations of the "pellet" culture system, which was originally described as a method for preventing the phenotypic modulation of chondrocytes, and the "alginate bead" culture system, which was used to maintain encapsulated cells at their differentiated phenotype over time, and also it was used to maintain the cells' proteoglycan synthesis at a rate similar to that of primary chondrocytes. We performed test on the differences of phenotypic characterization with the two methods of differentiating human mesenchymal stem cells into chondrocytes. The typical gene for articular cartilage, collagen type II, was more strongly expressed in the "alginate bead" system than in the "pellet" culture system, in addition, specific gene for hypertrophic cartilage, collagen type X, was more rapidly expressed in the "pellet" system than in "alginate bead" culture system. Therefore, the "alginate bead" culture system is a more phenotypical, practical and appropriate system to differentiate human mesenchymal stem cells into articular chondrocytes than the "pellet" culture system.

Adult↗

PubMed2001

Perturbation of rat renal tubule transport of the organic cation amantadine in recent onset streptozotocin-induced diabetes and in uninephrectomy.

The effects of early-stage diabetes mellitus and uninephrectomy on the renal tubule transport of amantadine were investigated. Kidney tubules were isolated from streptozotocin-induced diabetic (+/- insulin treatment) uninephrectomized, and control male Sprague-Dawley rats. There were no differences in the Km of amantadine uptake in renal proximal and distal tubules for the imposed treatments compared with control values. Vmax for amantadine uptake in the proximal tubules of diabetic and uninephrectomized rats was higher than the respective control (P < 0.05). Vmax for insulin-treated diabetic rats was similar to control values but was lower than that for untreated diabetic rats (P < 0.05). Vmax for distal tubule uptake was not altered by any treatment. Structure-activity studies demonstrated that bicarbonate-dependent amantadine uptake was inhibited by glycolate and lactate, but not by propionate or alpha-, beta-, or gamma-hydroxybutyrate. Early stage streptozotocin-induced diabetes mellitus and uninephrectomy induced changes in the kidney that resulted in a similar selective increase in proximal tubule amantadine uptake. These data represent the first description that experimentally induced diabetes mellitus and uninephrectomy modulate the function of the renal tubule organic cation (amantadine) transport system. Both interventions represent potential models in which phenotypic modulation of the renal elimination of organic cationic drugs may be achieved and studied.

Amantadine↗

PubMed2003

Modulation of cultured corneal keratocyte phenotype by growth factors/cytokines control in vitro contractility and extracellular matrix contraction.

The purpose of this study was to evaluate specific keratocyte phenotypes (keratocyte, fibroblast, myofibroblast) for cell contractility and ability to contract extracellular matrix. Rabbit keratocyte phenotype was modulated by exposure to optimal proliferative doses of IGF-I, IL-1alpha, FGF2, PDGF-AB, and TGFbeta(1). Cells were then evaluated by immunocytochemistry, western blot, collagen gel contraction and LPA stimulation to measure: (1) focal adhesion (FA), fibronectin (FN) and f-actin assembly; (2) expression of alpha-smooth muscle actin (alpha-SMA); (3) ability to contract extracellular matrix and (4) determine contractile ability, respectively. Untreated keratocytes showed no ability to contract collagen matrix. IGF-I and IL-1alpha increased cell proliferation (70.2 and 74.3%, respectively) but did not alter keratocyte phenotype or ability to contract matrix. FGF2 and PDGF induced fibroblast differentiation with FA and FN assembly and significant (p<0.05) extracellular matrix contraction. TGFbeta(1) induced myofibroblast differentiation with prominent FA and FN assembly, expression of alpha-SMA and significantly greater (p<0.05) matrix contraction. Addition of LPA induced actin filament assembly in growth factor starved fibroblasts and myofibroblasts but had no effect on the cultured keratocyte phenotype. We report for the first time that the keratocyte phenotype is non-contractile and that cell quiescence is not a defining characteristic. We further establish that changes in environmental conditions modulate the keratocyte phenotype resulting in physiologically functional differences regarding cell contractility and capacity to contract extracellular matrix.

Animals↗

PubMed1996

Differentiated vascular myocytes: are they involved in neointimal formation?

The role of differentiated vascular myocytes are neointimal formation in canine carotid artery was investigated. Using antibodies and cDNA probes, cells were characterized in situ and after isolation. In situ characterization indicated the majority of medial cells expressed both smooth muscle myosin and alpha actin but many cells were negative to these markers. All adventitial cells were negative for these proteins. The muscle protein-positive cells were designated differentiated, vascular myocytes (VSMC). The others were designated type 2 cells. Sequential enzyme digestion from lumenal surface yielded VSMC ( > 90%) while digestions from the adventitial surface yielded type 2 cells ( > 90%). VSMC were viable in culture but did not spread, proliferate, or alter expression of muscle proteins. Type 2 cells proliferated and increased their expression of muscle actin but did not express muscle myosin. Characterization of neointimal cells from injured carotid arteries indicated they were morphologically and immunologically identical to cultured type 2 cells. We concluded that: (a) canine carotid artery media consists of a heterogeneous cell population: (b) serum does not stimulate isolated VSMC to undergo phenotypic modulation or proliferate: and (c) type 2 cells may be responsible for neointimal formation because they proliferate and acquire a phenotype identical to in situ neointimal cells.

Actins↗

PubMed1998

Differentiated phenotype of smooth muscle cells depends on signaling pathways through insulin-like growth factors and phosphatidylinositol 3-kinase.

Under conventional culture conditions, smooth muscle cells display their phenotypic modulation from a differentiated to a dedifferentiated state. Here, we established a primary culture system of smooth muscle cells maintaining a differentiated phenotype, as characterized by expression of smooth muscle-specific marker genes such as h-caldesmon and calponin, cell morphology, and ligand-induced contractility. Laminin retarded the progression of dedifferentiation of smooth muscle cells. Insulin-like growth factors (IGF-I and IGF-II) and insulin markedly prolonged the differentiated phenotype, with IGF-I being the more potent. In contrast, serum, epidermal growth factor, transforming growth factors, and platelet-derived growth factors potently induced dedifferentiation compared with angiotensin II, arginine-vasopressin, and basic fibroblast growth factor. Using the present culture system, we investigated signaling pathways regulating a phenotype of smooth muscle cells. In cultured cells, IGF-I specifically activated phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, protein kinase B, but not mitogen-activated protein kinases. Specific inhibitors of PI3-kinase (wortmannin and LY294002) induced dedifferentiation of smooth muscle cells even when they were cultured on laminin under IGF-I-stimulated conditions. The sole effect of laminin to retard the dedifferentiation was completely blocked by anti-IGF-I antibody, and laminin promoted the endogenous expression of IGF-I in cultured cells. The reduced promoter activity of the caldesmon gene induced by platelet-derived growth factor BB was overcome by the forced expression of the constitutive active form of PI3-kinase p110alpha catalytic subunit. These findings suggest that an IGF-I signaling pathway through PI3-kinase plays a critical role in maintaining a differentiated phenotype of smooth muscle cells.

Animals↗

PubMed1996

Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening.

It is well accepted that smooth muscle cells (SMCs) cultured from normal rat arterial media have different morphological and biological features compared with SMCs cultured from experimental intimal thickening (IT) 15 days after endothelial injury. It is not known, however, whether the phenotypic modulation producing IT cells occurs in any medial SMCs or only in a particular SMC subpopulation. To distinguish among these possibilities, the phenotypic features of SMC clones derived from normal adult media and the IT 15 days after endothelial lesion were analyzed according to morphological appearance, replicative activity in the presence and absence of fetal calf serum, and [3H]thymidine incorporation and motile activity; these features were compared with those of the respective SMC parental populations. Two categories of SMC clones predominated: spindle clones, with morphological features similar to those of the parental population from the normal media, and epithelioid clones, with morphological features similar to those of the IT parental population. Both categories were present among clones produced from normal media and IT; however, spindle was more common among normal media clones, and epithelioid, among IT clones. The behavior in vitro was distinct for each category of clones and did not depend on their origin. Our results are compatible with the possibility that the SMC population of IT in vivo derives mainly from SMCs belonging to the category exhibiting epithelioid features in vitro.

Animals↗

PubMed1998

Myofibroblastic tumours: an update.

The concept of the myofibroblast is relatively new, since this cell type was defined less than 30 years ago, and there is as yet no firm consensus as to how a myofibroblast should be defined either morphologically or phenotypically. Because its attributes place it midway between a fibroblast and a smooth muscle cell and because it appears capable of functional and phenotypic modulation, some authorities argue that there is no such thing as a truly myofibroblastic tumour. However, since most diagnostic tumour pathology is based on phenotypic features of the excised lesion, it is an unavoidable fact that a wide range of soft tissue tumours show convincing myofibroblastic features. Lesions in this category fall into four main groups--the family of reactive fasciitis-like lesions, a group of benign lesions most of which have been fairly recently recognized (e.g., mammary myofibrolastoma, intranodal myofibroblastoma, angiomyo-fibroblastoma and dermatomyofibroma), the locally aggressive fibromatoses (either superficial or deep) which share features of fibroblasts and myofibroblasts in varying degree and, finally, sarcomas showing myofibroblastic differentiation. These latter include low grade lesions such as so-called infantile fibrosarcoma, inflammatory myofibroblastic tumour and a distinctive form of low grade myofibroblastic sarcoma, as well as some of the high grade lesions formerly known as MFH. Based on the advances made in the past 20 years, it makes no sense to deny the existence of myofibroblastic tumours (especially in an era when histogenetic concepts have crumbled)--instead we need to work towards a reproducible and agreed definition of the myofibroblast.

Child↗

PubMed1988

Differential susceptibility to recombinant interferon-gamma-induced HLA-DQ antigen modulation among clones from a human metastatic melanoma.

Twenty-one clones from an early culture of a histocompatibility leukocyte antigen (HLA) class II negative human metastatic melanoma (Me 9229) were screened for susceptibility to phenotypic modulation induced by recombinant interferon-gamma (rIFN-gamma) by using SPV-L3, a monoclonal antibody to HLA-DQ antigens, in indirect immunofluorescence followed by fluorescence-activated cell sorter analysis. After treatment with 500 U/ml of rIFN-gamma for 3 days one of the clones (9229/18) expressed high levels of DQ antigens, in terms of percentage of positive cells, whereas many other clones were much less susceptible or remained DQ negative. Scatchard analysis of the data of specific binding of 125-I-labeled rIFN-gamma revealed that one clone susceptible (9229/18) and one clone resistant (9229/5) to HLA-DQ modulation expressed similar numbers of interferon-gamma binding sites per cell; dose-response experiments showed that all clones could be induced to express HLA-DR and -DP antigens after exposure to rIFN-gamma. However, the DQ-negative profile of clone 9229/5 was not modified even after incubation with up to 1 X 10(4) U/ml of rIFN-gamma or by extending the culture time in the presence of this lymphokine up to 120 hr. Furthermore, Northern blot analysis indicated a direct correlation between changes in the levels of HLA-DR and -DQ-specific mRNA after rIFN-gamma treatment, and the lack or expression of HLA class II antigens at the cell surface of the two different clones. Karyotype studies did not reveal differences between clones 9229/5 and 9229/18 and Southern blot analysis indicated that both clones had similar EcoRI and HindIII restriction patterns for DR and DQ gene sequences. Finally, strong DQ-specific mRNA signal and antigen expression at the cell surface could be induced even on clone 9229/5 by treating the cells with supernatants from mixed lymphocyte cultures, recently shown to contain a class II-inducing factor different from interferon-gamma. Taken together these results indicate that DQ antigens can be modulated even in clones resistant to rIFN-gamma induction and suggest that the differential susceptibility observed in response to this lymphokine could play a role in the genesis of the phenomenon of intratumor heterogeneity.

DNA, Neoplasm↗

PubMed1997

Age, gender, and non-modulation. A sexual dimorphism in essential hypertension.

The angiotensinogen gene is one of the very few related by linkage analysis to human hypertension, but the linkage has been consistently shown only among males. Moreover, polymorphisms in this gene predict an abnormal renal responsiveness to angiotensin II, a feature of non-modulation, but again, only among males. To pursue these related bridges between genetics and physiology, we evaluated the effects of sex on a second feature of non-modulation, the aldosterone response to infused angiotensin II during low sodium balance. We tested the resultant hypothesis-that non-modulation would be less frequent in women-by conducting identical protocols on 225 hypertensive inpatients (70 women, 155 men). Non-modulation was strikingly less frequent among women (26%; 95% confidence interval, 16% to 37%) than men (49%; 95% confidence interval, 40% to 57% (P = .001). We tested the hypothesis that sex steroids play a role by comparing young, premenopausal women (< 35 years) with women who were perimenopausal (45 to 55 years) and postmenopausal (> 55 years). Among the youngest women, the frequency of non-modulation was only 7%, significantly less than in young men (41%, P = .02). A steady increase in non-modulation frequency accompanied advancing age in women, reaching 47% in those older than 55 years, equal to the fraction of men affected. Age influenced non-modulation frequency in men far less. We conclude that a striking sex difference underlies the non-modulation phenotype and that female sex hormones may confer protection against a genotypic predisposition in women. This "override" of genotype, manifest by a very low frequency of non-modulation in young women, may participate in their known protection against cardiovascular disease.

Adolescent↗

PubMed2003

Regulation of smooth muscle phenotype.

Differentiated smooth muscle cells (SMCs) remain highly plastic, enabling them to alter their phenotype in response to environmental and pathologic stimuli. SMCs in vascular pathologies such as atherosclerosis exhibit phenotypes clearly different from those of the mature cells in normal blood vessels. These phenotypically modulated SMCs play an integral role in the development of vascular diseases. This review addresses recent progress in our understanding of the mechanisms that control SMC phenotype during vascular development and in vascular disease. A particular focus is on the transcriptional control programs of the differentiated state of SMCs.

Gene Expression Regulation↗

PubMed2000

Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma.

Chronic persistent asthma is characterized by poorly reversible airflow obstruction and airways inflammation and remodelling. Histopathological studies of airways removed at post mortem from patients with severe asthma reveal marked inflammatory and architectural changes associated with airway wall thickening. Increased airway smooth muscle content, occurring as a result of hyperplastic and/or hypertrophic growth, is believed to be one of the principal contributors to airway wall thickening. In recent years, significant advances have been made in elucidating the mediators and the intracellular pathways that regulate proliferation of airway smooth muscle. The contribution that smooth muscle makes to persistent airflow obstruction may not, however, be limited simply to its increased bulk within the airway wall. Interest is growing in the possibility that reversible phenotypic modulation and increased heterogeneity of airway smooth muscle function may also be a feature of the asthmatic airway. This review focuses on possible mechanisms controlling smooth muscle phenotype heterogeneity as well as on the mediators and intracellular pathways implicated in its cellular proliferation. Particular attention is paid to mechanisms involving activation of the extracellular signal regulated kinase-, protein kinase C- and phosphoinositide 3-kinase-dependent pathways, since these appear to be the major candidate second messenger pathways for G protein- and tyrosine kinase-coupled receptor-stimulated proliferation.

Animals↗

PubMed1990

Effects of electrically induced contractile activity on cultured embryonic chick breast muscle cells.

Development of chicken breast muscle is characterized by the sequential appearance of six electrophoretically distinct myosin heavy chain (HC) isoforms. Cultured secondary myotubes, derived from 12-day embryonic chick breast muscle, mainly express the early embryonic HC isoform HCemb/e, normally present in 8-day embryonic breast muscle, and the two fast light chain isoforms LC1f and LC2f. Direct low-frequency (2.5 Hz) stimulation of these myotubes via platinum electrodes leads to a shift in myosin HC expression with increases in the late embryonic HC isoform HCemb/l amounting to 35% of total HC in 19-day-stimulated cultures. Measurements of 35S-methionine incorporation and immunohistochemical analyses demonstrate increases in LC3f. This increase is also seen at the mRNA level. These results indicate that induced contractile activity promotes myotube maturation in vitro. The observation that chronic stimulation enhances the expression of the slow isoform LC2s at the RNA, as well as the protein level, suggests an additional effect consisting of a fast-to-slow change in phenotype expression. In view of the fact that muscle maturation and phenotype expression is under neural control during development in vivo, our results on directly stimulated, aneural myotubes indicate that neurally transmitted contractile activity may be an important factor in modulating phenotype expression of secondary myotubes.

Animals↗