Expression Of Sarcomeric Proteins Research Articles

Effects of chronic exposure to simulated microgravity on skeletal muscle cell proliferation and differentiation.

Cell culture models that mimic long-term exposure to microgravity provide important insights into the cellular biological adaptations of human skeletal muscle to long-term residence in space. We developed insert scaffolding for the NASA-designed rotating cell culture system (RCCS) in order to study the effects of time-averaged microgravity on the proliferation and differentiation of anchorage-dependent skeletal muscle myocytes. We hypothesized that prolonged microgravity exposure would result in the retardation of myocyte differentiation. Microgravity exposure in the RCCS resulted in increased cellular proliferation. Despite shifting to media conditions promoting cellular differentiation, 5 d later, there was an increase in cell number of approximately 62%, increases in total cellular protein (52%), and cellular proliferating cell nuclear antigen (PCNA) content (2.7 times control), and only a modest (insignificant) decrease (10%) in sarcomeric myosin protein expression. We grew cells in an inverted orientation on membrane inserts. Changes in cell number and PCNA content were the converse to those observed for cells in the RCCS. We also grew cells on inserts at unit gravity with constant mixing. Mixing accounted for part, but not all, of the effects of microgravity exposure on skeletal muscle cell cultures (53% of the RCCS effect on PCNA at 4-6 d). In summary, the mechanical effects of simulated microgravity exposure in the RCCS resulted in the maintenance of cellular proliferation, manifested as increases in cell number and expression of PCNA relative to control conditions, with only a modest reciprocal inhibition of cellular differentiation. Therefore, this model provides conditions wherein cellular differentiation and proliferation appear to be uncoupled.

Direct involvement of N-cadherin-mediated signaling in muscle differentiation.

Cell-cell interactions, mediated by members of the cadherin family of Ca2+-dependent adhesion molecules, play key roles in morphogenetic processes as well as in the transduction of long-range growth and differentiation signals. In muscle differentiation cell adhesion is involved in both early stages of myogenic induction and in later stages of myoblast interaction and fusion. In this study we have explored the involvement of a specific cadherin, namely N-cadherin, in myogenic differentiation. For that purpose we have treated different established lines of cultured myoblasts with beads coated with N-cadherin-specific ligands, including a recombinant N-cadherin extracellular domain, and anti-N-cadherin antibodies. Immunofluorescent labeling for cadherins and catenins indicated that treatment with the cadherin-reactive beads for several hours enhances the assembly of cell-cell adherens-type junctions. Moreover, immunofluorescence and immunoblotting analyses indicated that treatment with the beads for 12-24 h induces myogenin expression and growth arrest, which are largely independent of cell plating density. Upon longer incubation with the beads (2-3 d) a major facilitation in the expression of several muscle-specific sarcomeric proteins and in cell fusion into myotubes was observed. These results suggest that surface clustering or immobilization of N-cadherin can directly trigger signaling events, which promote the activation of a myogenic differentiation program.

Influence of calcium on proliferation and phenotype alteration of cardiomyocyte in vitro.

An accelerated weight gain is noted in the heart of Ca-deficient, hypertensive chick embryos maintained in a shell-less culture in vitro. We previously observed that the Ca handling property of cardiomyocytes isolated from the shell-less embryo is altered, i.e., faster Ca uptake, suggesting a requirement for adequate Ca supply and/or proper Ca handling in embryonic cardiac development. In this study, we have examined the function of Ca on cardiomyocytes by analyzing the effects of 1) various Ca concentration in the culture medium (NCa, 1.8 mmol/ L; HCa, 2.8 mmol/L; LCa, 0.9 mmol/L), and 2) various modulators of Ca handling on cell proliferation and phenotype regulation in chick embryonic cardiomyocytes. The analytical parameters included cell number, DNA content, expression of cell cycle-specific and cardiomyocyte-specific proteins, and creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) enzyme activities. Cell number and total DNA were significantly larger (P < 0.01) in LCa cultures compared with those in NCa. The level of LDH was elevated (P < 0.01), but that of CPK was lowered in LCa. Expression of the G1-S-specific protein PCNA was raised, but that of the contractile proteins myosin and tropomyosin was substantially suppressed in LCa; in HCa, the cells did not proliferate as well, whereas the level of contractile proteins was higher. Thapsigargin, a sarcoplasmic reticulum (SR)-specific, Ca-ATPase inhibitor, simulated the effects of LCa by enhancing cell proliferation and lowering the expression of tropomyosin. These results suggest that culturing in low Ca concentration and inhibition of SR Ca pumping enhance myocardial cell proliferation and suppress sarcomeric protein expression, perhaps by inducing cellular de-differentiation. The in vitro effects of medium Ca concentration and Ca handling modulators on cardiomyocytes also suggest that the in vivo cardiomegaly of the SL embryos is a direct result of Ca-deficiency, and that Ca is important in the phenotype regulation of cardiomyocytes.

Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms.

Cardiac hypertrophy is an adaptive response that normalizes wall stress and compensates for increased workload. It is accompanied by distinct qualitative and quantitative changes in the expression of protein isoforms concerning contractility, intracellular Ca(2+)-homeostasis and metabolism. Changes in the myosin subunit isoform expression improves contractility by an increase in force generation at a given Ca(2+)-concentration (increased Ca(2+)-sensitivity) and by improving the economy of the chemo-mechanical transduction process per amount of utilised ATP (increased duty ratio). In the human atrium this is achieved by partial replacement of the endogenous fast myosin by the ventricular slow-type heavy and light chains. In the hypertrophic human ventricle the slow-type beta-myosin heavy chains remain unchanged, but the ectopic expression of the atrial myosin essential light chain (ALC1) partially replaces the endogenous ventricular isoform (VLC1). The ventricular contractile apparatus with myosin containing ALC1 is characterised by faster cross-bridge kinetics, a higher Ca(2+)-sensitivity of force generation and an increased duty ratio. The mechanism for cross-bridge modulation relies on the extended Ala-Pro-rich N-terminus of the essential light chains of which the first eleven residues interact with the C-terminus of actin. A change in charge in this region between ALC1 and VLC1 explains their functional difference. The intracellular Ca(2+)-handling may be impaired in heart failure, resulting in either higher or lower cytosolic Ca(2+)-levels. Thus the state of the cardiomyocyte determines whether this hypertrophic adaptation remains beneficial or becomes detrimental during failure. Also discussed are the effects on contractility of long-term changes in isoform expression of other sarcomeric proteins. Positive and negative modulation of contractility by short-term phosphorylation reactions at multiple sites in the myosin regulatory light chain, troponin-I, troponin-T, alpha-tropomyosin and myosin binding protein-C are considered in detail.

Expression of sarcomeric proteins and assembly of myofibrils in the putative myofibroblast cell line BHK-21/C13.

The expression and organization patterns of several myofibrillar proteins were analysed in the putative myofibroblast cell line BHK-21/C13. Although this cell line originates from renal tissue, the majority of the cells express titin. In these cells, titin is, under standard culture conditions, detected in myofibril-like structures (MLSs), where it alternates with non-muscle myosin (NMM). Expression of sarcomeric myosin heavy chain (sMyHC) is observed in a small minority of cells, while other sarcomeric proteins, such as nebulin, myosin binding protein C (MyBP-C), myomesin and M-protein are not expressed at all. By changing the culture conditions in a way equal to conditions that induce differentiation of skeletal muscle cells, a process reminiscent of sarcomerogenesis in vitro is induced. Within one day after the switch to a low-nutrition medium, myofibrillar proteins can be detected in a subset of cells, and after two to five days, all myofibrillar proteins examined are organized in typical sarcomeric patterns. Frequently, cross-striations are visible with phase contrast optics. Transfection of these cells with truncated myomesin fragments showed that a specific part of the myomesin molecule, known to contain a titin-binding site, binds to MLSs, whereas other parts do not. These results demonstrate that this cell line could serve as a powerful model to study the assembly of myofibrils. At the same time, its transfectability offers an invaluable tool for in vivo studies concerning binding properties of sarcomeric proteins.

Major risk factors for atherosclerosis are manifested in experimental Ca-deficiency.

The eggshell is the major source of Ca required during growth of chick embryos. Therefore, chick embryos placed ex ovo for long-term (SL) are rendered severe systemic calcium deficiency. We report here that SL chick embryos express Ca-deficiency related atherogenic disorders, and that in vitro Ca-deficiency induces dedifferentiation, i.e. loss of cell-type specific features and accelerated proliferative activities, in the various types of cultured cells. Systemic blood pressure is significantly higher and an accelerated weight gain of the heart is noted in SL compared to normal embryos (NL) at the incubation Day-14. Plasma cholesterol was lower, while triglyceride and glucose were higher in SL. Varying Ca in the culture medium (FCa, 1.8 mM; HCa, 2.8 mM; Ca/2, 0.9 mM) clearly affected the phenotype of the cultured cardiomyocytes and vascular cells isolated from the chick embryos. The cell number and total DNA were significantly larger and the level of LDH and proliferating cell nuclear antigen (PCNA) was elevated in Ca/2 compared to FCa. On the contrary, the level of CPK and contractile proteins were lowered in Ca/2. Thus, it is indicated that Ca-deficiency induces atherogenic disorders in vivo, and accelerates cell proliferation and decelerates sarcomeric protein expression in vitro. Taken together, it is suggested that the atherogenic, developmental disorders in SL may be the integrated result of the phenotype alteration in the various cell types directly induced by Ca-deficiency.

Initiation of cardiac differentiation occurs in the absence of anterior endoderm.

Anterior endoderm has been proposed to be a specific inducer of cardiac differentiation in vertebrates (reviewed in Jacobson and Sater, Development 104, 341-359, 1988). The ability of cardiogenic mesoderm to differentiate in a minimal culture system was examined using cardiac-specific gene expression as an assay. Anterior lateral plate mesoderm was explanted from chick embryos with and without associated endoderm at developmental stages from just after gastrulation (stage 4; Hamburger and Hamilton, J. Morph. 88, 49-67, 1951) to just prior to contraction (stage 9). At all stages examined, cardiogenic mesoderm expressed a profile of cardiac-specific mRNAs after two days in minimal medium independent of the presence of endoderm. Our studies indicate that endoderm is necessary for the generation of stable sarcomeric protein expression, organized myofibrils and beating tissue from stage 4-6. Subsequent to this stage, an interaction with anterior endoderm is no longer required. Examination of cardia bifida embryos from which anterior endoderm had been unilaterally removed also showed a stage-dependent effect of endoderm on beating, while cardiac gene expression and heart morphogenesis were unaffected. These results demonstrate that anterior endoderm does not induce or maintain cardiac gene expression, nor is it required for terminal differentiation. Endoderm does appear to be necessary for a short period of time between initiation of cardiac gene expression and the onset of contraction.

Apolipoprotein A-1 expression is resistant to dimethyl sulfoxide inhibition of myogenic differentiation

Primary cultures of chick embryonic muscle (CEM) were analyzed for the differential expression of a 26-kDa protein during myogenesis. We have identified this 26-kDa protein as apolipoprotein A-1 (Apo A-1), the major protein of serum high density lipoprotein particles. Apo A-1 was expressed in a pattern temporally similar to those of muscle-specific proteins, by myoblasts at very low levels, and by myotubes at high levels. The half-life of Apo A-1 in CEM cell homogenates was 23 min. This fast turnover rate appeared to be due to the secretion of Apo A-1 into the culture medium. To further characterize the relationship of Apo A-1 expression and myogenic differentiation, CEM cultures were treated with dimethyl sulfoxide (DMSO). In the presence of 2% DMSO, myotubes exhibited an atrophied morphology and an inhibition of the synthesis and accumulation of muscle-specific sarcomeric proteins. During recovery from DMSO treatment, the expression and accumulation of muscle-specific proteins returned to high levels. In contrast, the rates of synthesis and secretion of Apo A-1 in control, DMSO-treated, and DMSO-recovered CEM cells were nearly equivalent. These results indicate that the expression of Apo A-1 is not strictly linked to the expression of muscle-specific sarcomeric proteins in skeletal muscle and suggest that independent, or additional regulatory mechanisms exist which modulate Apo A-1 expression during myogenesis.

A molecular view of cardiogenesis

Cardiac development involves a complex integration of subcellular processes into multicellular and, finally, whole organ effects. Until recently it has been difficult to investigate the genetic control of this organ level differentiation of the heart. The proliferation of molecular biology methodologies has provided mechanisms to directly investigate the control of these processes. This article focuses on molecular lines of research on two key areas in cardiac development: the regulation of expression of sarcomeric contractile and regulatory proteins, and atrial natriuretic factor. Molecular approaches are described which have allowed investigators to begin to determine the tissue and stage-specific expression of genes, to locate those genes in the genome, determine their sequences, and to directly investigate the mechanisms controlling their expression.