cis-4-Hydroxy-L-proline and ethyl-3,4-dihydroxybenzoate prevent myogenesis of C2C12 muscle cells and block MyoD1 and myogenin expression.

cis-4-Hydroxy-L-proline (cis-OH-Pro) and ethyl-3,4-dihydroxybenzoate (EDHB), two distinct inhibitors of collagen synthesis, prevented myogenesis in C2C12 mouse skeletal muscle cells. Both inhibitors blocked myotube formation and the expression of sarcomeric myosin heavy chain. Northern blot analysis showed that cis-OH-Pro- and EDHB-treated C2C12 muscle cells did not express the myogenic regulatory genes, MyoD1 and myogenin, but continued to express non-muscle isoforms of actin (beta and gamma) and alpha-tropomyosin. 10TFL2-3B cells, a C3H10T1/2 cell line permanently transfected with myogenin cDNA, constitutively expressed exogenous myogenin in the presence of cis-OH-Pro but failed to activate endogenous myogenin and to undergo myogenesis. These results demonstrate that commitment to terminal differentiation and activation of myogenic regulatory genes requires active synthesis of the extracellular matrix component collagen.     

Smoothelin-positive cells in human and porcine semilunar valves

Our aim was to further characterize the interstitial cell phenotypes of normal porcine and human semilunar valves, information necessary for the design of bioengineered valves and for the understanding of valve disease processes such as aortic valve sclerosis. Existence of fibroblasts, myofibroblasts, and smooth muscle-like cells within semilunar heart valves has been established. However, the nature of the smooth muscle cell population has been controversial. We used immunochemical and western blotting methods to determine the status of smoothelin and smooth muscle -actin in the valve. Our examination of valve interstitial cells confirmed the presence of terminally differentiated, contractile smooth muscle cells in situ. They were arranged in small bundles of 5–35 cells within the ventricularis or as individual cells scattered throughout the valvular layers in vivo, and were present in cells explanted from the valves in vitro. Colocalization of these proteins in semilunar heart valves was achieved with double-labeling experiments. Protein extraction, followed by coimmunoprecipitation, electrophoresis, and western blotting confirmed the immunochemical analysis and suggested that smooth muscle -actin and smoothelin interact, as has been previously postulated. The presence of contractile smooth muscle within the valve may be an important factor in understanding valve pathology and in the design of tissue engineering efforts.     

Sarcomeric Gene Expression and Contractility in Myofibroblasts

Myofibroblasts are unusual cells that share morphological and functional features of muscle and nonmuscle cells. Such cells are thought to control liver blood flow and kidney glomerular filtration rate by having unique contractile properties. To determine how these cells achieve their contractile properties and their resemblance to muscle cells, we have characterized two myofibroblast cell lines. Here, we demonstrate that myofibroblast cell lines from kidney mesangial cells (BHK) and liver stellate cells activate extensive programs of muscle gene expression including a wide variety of muscle structural proteins. In BHK cells, six different striated myosin heavy chain isoforms and many thin filament proteins, including troponin T and tropomyosin are expressed. Liver stellate cells express a limited subset of the muscle thick filament proteins expressed in BHK cells. Although these cells are mitotically active and do not morphologically differentiate into myotubes, we show that MyoD and myogenin are expressed and functional in both cell types. Finally, these cells contract in response to endothelin-1 (ET-1); and we show that ET-1 treatment increases the expression of sarcomeric myosin.     

Localization of N-terminal sequences in human AMP deaminase isoforms that influence contractile protein binding.

The reversible association of AMP deaminase (AMPD, EC 3.5.4.6) with elements of the contractile apparatus is an identified mechanism of enzyme regulation in mammalian skeletal muscle. All three members of the human AMPD multigene family contain coding information for polypeptides with divergent N-terminal and conserved C-terminal domains. In this study, serial N-terminal deletion mutants of up to 111 (AMPD1), 214 (AMPD2), and 126 (AMPD3) residues have been constructed without significant alteration of catalytic function or protein solubility. The entire sets of active enzymes are used to extend our understanding of the contractile protein binding of AMPD. Analysis of the most truncated active enzymes demonstrates that all three isoforms can associate with skeletal muscle actomyosin and suggests that a primary binding domain is located within the C-terminal 635-640 residues of each polypeptide. However, discrete stretches of N-terminal sequence alter this behavior. Residues 54-83 in the AMPD1 polypeptide contribute to a high actomyosin binding capacity of both isoform M spliceoforms, although the exon 2- enzyme exhibits significantly greater association compared to its exon 2+ counterpart. Conversely, residues 129-183 in the AMPD2 polypeptide reduce actomyosin binding of isoform L. In addition, residues 1-48 in the AMPD3 polypeptide dramatically suppress contractile protein binding of isoform E, thus allowing this enzyme to participate in other intracellular interactions.     

Molecular and functional characteristics of heart-valve interstitial cells

The cells that reside within valve cusps play an integral role in the durability and function of heart valves. There are principally two types of cells found in cusp tissue: the endothelial cells that cover the surface of the cusps and the interstitial cells (ICs) that form a network within the extracellular matrix (ECM) within the body of the cusp. Both cell types exhibit unique functions that are unlike those of other endothelial and ICs found throughout the body. The valve ICs express a complex pattern of cell-surface, cytoskeletal and muscle proteins. They are able to bind to, and communicate with, each other and the ECM. The endothelial cells on the outflow and inflow surfaces of the valve differ from one another. Their individual characteristics and functions reflect the fact that they are exposed to separate patterns of flow and pressure. In addition to providing a structural role in the valve, it is now known that the biological function of valve cells is important in maintaining the integrity of the cusps and the optimum function of the valve. In response to inappropriate stimuli, valve interstitial and endothelial cells may also participate in processes that lead to valve degeneration and calcification. Understanding the complex biology of valve interstitial and endothelial cells is an important requirement in elucidating the mechanisms that regulate valve function in health and disease, as well as setting a benchmark for the function of cells that may be used to tissue engineer a heart valve.     

The cardiac valve interstitial cell

Cardiac valve interstitial cells (ICs) are a heterogeneous and dynamic population of specific cell types that have many unique characteristics. They are responsible for maintaining the extracellular scaffold that provides the mechanical characteristics vital for sustaining the unique dynamic behaviour of the valve. A number of cellular phenotypes can be distinguished: some are sparsely arranged throughout the valve leaflets, whilst others are arranged in thin bundles. These cells express molecular markers similar to those of skeletal, cardiac and smooth muscle cells (SMCs) and in particular, many ICs express smooth muscle (SM) alpha-actin, a marker of myofibroblasts. In this respect, these cells exhibit a profile unlike skin fibroblasts, which may allude to their role in valve function.     

Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.     

The expression of sarcomeric muscle-specific contractile protein genes in BC3H1 cells: BC3H1 cells resemble skeletal myoblasts that are defective for commitment to terminal differentiation

The BC3H1 cell line has been used widely as a model for studying regulation of muscle-related proteins, such as the acetylcholine receptor, myokinase, creatine kinase, and actin. These cells, derived from a nitrosourea-induced mouse brain neoplasm, have some of the morphological characteristics of smooth muscle and have been shown to express the vascular smooth muscle isoform of alpha-actin. To provide further information about the contractile protein phenotype of BC3H1 and to gain additional insights into the possible tissue of origin of these cells, we have examined the expression of a battery of contractile protein genes. During rapid growth, subconfluent BC3H1 cells express the nonmuscle isoform of alpha-tropomyosin (alpha-Tm) and the nonsarcomeric isoforms of myosin heavy and light chains (MHCs and MLCs, respectively), but do not express troponin T(TnT). However, when BC3H1 cells differentiate in response to incubation in serum-deprived medium or upon approaching confluence, they express TnT as well as sarcomeric muscle isoforms of MHC, MLC 2 and 3, alpha-Tm, and alpha-actin. These results suggest that BC3H1 is a skeletal muscle cell line of ectodermal origin that is defective for commitment to terminal differentiation.     

Comparative analysis of the isoform expression pattern of Ca(2+)-regulatory membrane proteins in fast-twitch, slow-twitch, cardiac, neonatal and chronic low-frequency stimulated muscle fibers

Although all muscle cells generate contractile forces by means of organized filament systems, isoform expression patterns of contractile and regulatory proteins in heart are not identical compared to developing, conditioned or mature skeletal muscles. In order to determine biochemical parameters that may reflect functional variations in the Ca(2+)-regulatory membrane systems of different muscle types, we performed a comparative immunoblot analysis of key membrane proteins involved in ion homeostasis. Cardiac isoforms of the alpha(1)-dihydropyridine receptor, Ca(2+)-ATPase and calsequestrin are also present in skeletal muscle and are up-regulated in chronic low-frequency stimulated fast muscle. In contrast, the cardiac RyR2 isoform of the Ca(2+)-release channel was not found in slow muscle but was detectable in neonatal skeletal muscle. Up-regulation of RyR2 in conditioned muscle was probably due to degeneration-regeneration processes. Fiber type-specific differences were also detected in the abundance of auxiliary subunits of the dihydropyridine receptor, the ryanodine receptor and the Ca(2+)-ATPase, as well as triad markers and various Ca(2+)-binding and ion-regulatory proteins. Hence, the variation in innervation of different types of muscle appears to have a profound influence on the levels and pattern of isoform expression of Ca(2+)-regulatory membrane proteins reflecting differences in the regulation of Ca(2+)-homeostasis. However, independent of the muscle cell type, key Ca(2+)-regulatory proteins exist as oligomeric complexes under native conditions.     

Wnt signaling in heart valve development and osteogenic gene induction

Wnt signaling mediated by β-catenin has been implicated in early endocardial cushion development, but its roles in later stages of heart valve maturation and homeostasis have not been identified. Multiple Wnt ligands and pathway genes are differentially expressed during heart valve development. At E12.5, Wnt2 is expressed in cushion mesenchyme, whereas Wnt4 and Wnt9b are predominant in overlying endothelial cells. At E17.5, both Wnt3a and Wnt7b are expressed in the remodeling atrioventricular (AV) and semilunar valves. In addition, the TOPGAL Wnt reporter transgene is active throughout the developing AV and semilunar valves at E16.5, with more localized expression in the stratified valve leaflets after birth. In chicken embryo aortic valves, genes characteristic of osteogenic cell lineages including periostin, osteonectin, and Id2 are expressed specifically in the collagen-rich fibrosa layer at E14. Treatment of E14 aortic valve interstitial cells (VICs) in culture with osteogenic media results in increased expression of multiple genes associated with bone formation. Treatment of VIC with Wnt3a leads to nuclear localization of β-catenin and induction of periostin and matrix gla protein but does not induce genes associated with later stages of osteogenesis. Together, these studies provide evidence for Wnt signaling as a regulator of endocardial cushion maturation as well as valve leaflet stratification, homeostasis, and pathogenesis.     

Structural and functional properties of the non-muscle tropomyosins

Summary The non-muscle tropomyosins (TMs), isolated from such tissues as platelets, brain and thyroid, are structurally very similar to the muscle TMs, being composed of two highly -helical subunits wound around each other to form a rod-like molecule. The non-muscle TMs are shorter than the muscle TMs; sequence analysis demonstrates that each subunit of equine platelet TM consists of 247 amino acids, 37 fewer than for skeletal muscle TM. The major differences in sequence between platelet and skeletal muscle TM are found near the amino and carboxyl terminal ends of the proteins. Probably as the result of such alterations, the non-muscle TMs aggregate in a linear end-to-end manner much more weakly than do the muscle TMs. Since end-to-end interactions are responsible for the highly cooperative manner in which TM binds to actin, the non-muscle TMs have a lower affinity for actin filaments than do the muscle TMs. However, the attachment of other proteins to actin (e.g. the Tn-I subunit of skeletal muscle troponin or the S-1 subfragment of skeletal muscle myosin) can increase the affinity of actin filaments for non-muscle TM. The non-muscle TMs interact functionally with the Tn-I component of skeletal muscle troponin to inhibit the ATPase activity of muscle actomyosin and with whole troponin to regulate the muscle actomyosin ATPase in a Ca⁺⁺-dependent manner, even though one of the binding sites for troponin on skeletal TM is missing in non-muscle TM. A novel actomyosin regulatory system can be produced using Tn-I, calmodulin and non-muscle TM; in this case inhibition is released when the non-muscle TM detaches from the actin filament in the presence of Ca⁺⁺. Although it has not yet been demonstrated that the non-muscle TMs participate in a Ca⁺⁺-dependent contractile regulatory system in vivo it does appear that they are associated with actin filaments in vivo.