Visualization of cardiac ventricular myosin heavy chain homodimers and heterodimers by monoclonal antibody epitope mapping.

Two mAbs, one specific for cardiac alpha-myosin heavy chains (MHC) and the other specific for cardiac beta-MHC, were used to investigate the heavy-chain dimeric organization of rat cardiac ventricular myosin. Epitopes of the two mAbs were mapped on the myosin molecule by electron microscopy of rotary shadowed mAb-myosin complexes. mAbs were clearly identifiable by the different locations of their binding sites on the myosin rod. Thus, myosin molecules could be directly discriminated according to their alpha-or beta-MHC content. alpha alpha-MHC and beta beta-MHC homodimers were visualized in complexes consisting of two molecules of the same mAb bound to one myosin molecule. By simultaneously using the alpha-MHC-specific mAb and the beta-MHC-specific mAb, alpha beta-MHC heterodimers were visualized in complexes formed by one molecule of each of the two mAbs bound to one myosin molecule. Proportions of alpha alpha-and beta beta-MHC homodimers and alpha beta-MHC heterodimers were estimated from quantifications of mAb-myosin complexes and compared with the proportions given by electrophoreses under nondenaturing conditions. This visualization of cardiac myosin molecules clearly demonstrates the arrangement of alpha- and beta-MHC in alpha alpha-MHC homodimers, beta beta-MHC homodimers, and alpha beta-MHC heterodimers, as initially proposed by Hoh, J. F. Y., G. P. S. Yeoh, M. A. W. Thomas, and L. Higginbottom (1979).

IL-1ß- and IL-4-polarized macrophages have opposite effects on adipogenesis of intramuscular fibro-adipogenic progenitors in humans.

Intramuscular fat deposition represents a negative prognostic factor for several myopathies, metabolic diseases and aging. Fibro-adipogenic progenitors (FAPs) are considered as the main source of intramuscular adipocytes, but the mechanisms controlling their adipogenic potential are still not elucidated in humans. The aim of this study was to explore the regulation of human FAP adipogenesis by macrophages. We found that CD140a-expressing FAPs were located close to CD68 positive macrophages in muscles from patients with Duchenne muscular dystrophy (DMD). This strongly suggests a potential interaction between FAPs and macrophages in vivo. Isolated human primary FAPs were then differentiated in the presence of conditioned media obtained from primary blood monocyte-polarized macrophages. Molecules released by IL-1ß-polarized macrophages (M(IL-1ß)) drastically reduced FAP adipogenic potential as assessed by decreased cellular lipid accumulation and reduced gene expression of adipogenic markers. This was associated with an increased gene expression of pro-inflammatory cytokines in FAPs. Conversely, factors secreted by IL-4-polarized macrophages (M(IL-4)) enhanced FAP adipogenesis. Finally, the inhibition of FAP adipocyte differentiation by M(IL-1ß) macrophages requires the stimulation of Smad2 phosphorylation of FAPs. Our findings identify a novel potential crosstalk between FAPs and M(IL-1ß) and M(IL-4) macrophages in the development of adipocyte accumulation in human skeletal muscles.

E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway.

Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

The primary cilium is necessary for the differentiation and the maintenance of human adipose progenitors into myofibroblasts.

The primary cilium is an organelle, present at the cell surface, with various biological functions. We, and others, have shown that it plays a role in the differentiation of adipose progenitors (APs) into adipocytes. APs can also differentiate into myofibroblasts when treated with TGF-ß1. Several components of the TGF-ß1 pathway are located within the cilium suggesting a function for this organelle in AP myofibrogenesis. We studied differentiation of APs into myofibroblasts in two human models: APs of the adipose tissue (aAPs) and APs resident in the skeletal muscles (mAPs). We showed that, in vivo, myofibroblasts within muscles of patients with Duchenne Muscular Dystrophy were ciliated. In vitro, myofibroblasts derived from APs maintained a functional primary cilium. Using HPI4, a small molecule that inhibits ciliogenesis, and siRNA against Kif-3A, we provide evidence that the primary cilium is necessary both for the differentiation of APs into myofibroblasts and the maintenance of the phenotype. Disruption of the primary cilium inhibited TGF-ß1-signalisation providing a molecular mechanism by which the cilium controls myofibroblast differentiation. These data suggest that myofibroblasts from various origins are controlled differently by their primary cilium.

Contribution of the plasmalemma to Ca2+ homeostasis in hair cells.

Calcium influx through transduction channels and efflux via plasmalemmal Ca(2+)-ATPases (PMCAs) are known to contribute to calcium homeostasis and modulate sensory transduction in vertebrate hair cells. To examine the relative contributions of apical and basolateral pathways, we analyzed the calcium dynamics in solitary ciliated and deciliated guinea pig type I and type II vestibular hair cells. Whole-cell patch-clamp recordings demonstrated that these cells had resting potentials near -70 mV and could be depolarized by 10-20 mV by superfusion with high potassium. Fura-2 measurements indicated that ciliated type II cells and deciliated cells of either type had low basal [Ca(2+)](i), near approximately 90 nm, and superfusion with high potassium led to transient calcium increases that were diminished in the presence of Ca(2+) channel blockers. In contrast, measurements of type I ciliated cells, hair cells with large calyceal afferents, were associated with a higher basal [Ca(2+)](i) of approximately 170 nm. High-potassium superfusion of these cells induced a paradoxical decrease in [Ca(2+)](i) that was augmented in the presence of Ca(2+) channel blockers. Optical localization of dihydropyridine binding to the kinocilium suggests that they contain L-type calcium channels, and as a result apical calcium influx includes a contribution from voltage-dependent ion channels in addition to entry via transduction channels localized to the stereocilia. Eosin block of PMCA significantly altered both [Ca(2+)](i) baseline and transient responses only in ciliated cells suggesting that, in agreement with immunohistochemical studies, PMCA is primarily localized to the bundles.

Large-scale analysis of differential gene expression in the hindlimb muscles and diaphragm of mdx mouse.

The mdx mouse is an animal model for Duchenne muscular dystrophy (DMD), which is caused by the absence of dystrophin. Mdx limb muscles substantially compensate for the lack of dystrophin while the diaphragm is affected like DMD skeletal muscles. To understand better the complex cascade of molecular events leading to muscle degeneration and compensatory processes in mdx muscles, we analyzed alterations of gene expression in mdx hindlimb and diaphragm muscles as compared to their normal counterparts. The strategy was based on suppression subtractive hybridization followed by reverse Northern quantitative hybridization. Four subtracted/normalized libraries, containing cDNA clones up- or downregulated in mdx hindlimb muscles or diaphragm, were constructed and a total of 1536 cDNA clones were analyzed. Ninety-three cDNAs were found to be differentially expressed in mdx hindlimb muscles and/or diaphragm. They corresponded to 54 known genes and 39 novel cDNAs. The potential role of the known genes is discussed in the context of the mdx phenotype.

The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation.

DNA topoisomerase I (Topo1) contributes to vital biological functions, but its regulation is not clearly understood. The BTBD1 protein was recently cloned on the basis of its interaction with the core domain of Topo1 and is expressed particularly in skeletal muscle. To determine BTBD1 functions in this tissue, the in vitro model used was the C2C12 mouse muscle cell line, which expresses BTBD1 mainly after myotube differentiation. We studied the effects of a stably overexpressed BTBD1 protein truncated of the 108 N-terminal amino-acid residues and harbouring a C-terminal FLAG tag (Delta-BTBD1). The proliferation speed of Delta-BTBD1 C2C12 cells was significantly decreased and no myogenic differentiation was observed, although these cells maintained their capacity to enter adipocyte differentiation. These alterations could be related to Topo1 deregulation. This hypothesis is further supported by the decrease in nuclear Topo1 content in Delta-BTBTD1 proliferative C2C12 cells and the switch from the main peripheral nuclear localization of Topo1 to a mainly nuclear diffuse localization in Delta-BTBTD1 C2C12 cells. Finally, this study demonstrated that BTBD1 is essential for myogenic differentiation.

Characterization of adipocytes derived from fibro/adipogenic progenitors resident in human skeletal muscle.

A population of fibro/adipogenic but non-myogenic progenitors located between skeletal muscle fibers was recently discovered. The aim of this study was to determine the extent to which these progenitors differentiate into fully functional adipocytes. The characterization of muscle progenitor-derived adipocytes is a central issue in understanding muscle homeostasis. They are considered as being the cellular origin of intermuscular adipose tissue that develops in several pathophysiological situations. Here fibro/adipogenic progenitors were isolated from a panel of 15 human muscle biopsies on the basis of the specific cell-surface immunophenotype CD15+/PDGFRα+CD56-. This allowed investigations of their differentiation into adipocytes and the cellular functions of terminally differentiated adipocytes. Adipogenic differentiation was found to be regulated by the same effectors as those regulating differentiation of progenitors derived from white subcutaneous adipose tissue. Similarly, basic adipocyte functions, such as triglyceride synthesis and lipolysis occurred at levels similar to those observed with subcutaneous adipose tissue progenitor-derived adipocytes. However, muscle progenitor-derived adipocytes were found to be insensitive to insulin-induced glucose uptake, in association with the impairment of phosphorylation of key insulin-signaling effectors. Our findings indicate that muscle adipogenic progenitors give rise to bona fide white adipocytes that have the unexpected feature of being insulin-resistant.

Antibodies to a C-terminal peptide of the rat brain glutamate receptor subunit, GluR-A, recognize a subpopulation of AMPA binding sites but not kainate sites.

Antibodies were made to a thirteen amino acid synthetic peptide corresponding to the C-terminal portion of the glutamate (glu) receptor, GluR-A. The immunoprecipitation of kainic acid (KA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites by the anti-peptide antibodies was studied using a detergent-solubilized preparation of rat brain membranes. Under these conditions a subpopulation of AMPA binding sites was recognized by the antibodies, but no KA binding sites were recognized. Scatchard analysis of this subpopulation of AMPA binding sites yields a curvilinear plot which fits a two-site model with dissociation constants of 4.6 and 323 nM. These studies show that the glu receptor complex, GluR-A, binds AMPA but not KA and suggest that (i) the binding sites for these two ligands reside on different proteins, and (ii) the KA receptor identified physiologically is not equivalent to the KA binding sites identified with 3H-labelled KA.

Down-regulation of mitochondrial mRNAs in the mdx mouse model for Duchenne muscular dystrophy.

In our search for genes up- or down-regulated genes in the mdx mouse model for Duchenne muscular dystrophy, we isolated a down-regulated mitochondrial DNA clone. In addition to this clone, all protein-coding mitochondrial genes tested had tissue-specific and age independent down-regulated expression. This implied mechanisms at the RNA level since no change in the mitochondrial DNA contents were detected. Cytochrome c oxidase activity showed the same range of down-regulated expression. These data provide a molecular basis for energetic metabolism modifications in mdx mice.

Development of calretinin immunoreactivity in the mouse inner ear.

Calretinin is a calcium-binding protein of the EF-hand family. It has been previously identified in particular cell types of adult guinea pig, rat, and chinchilla inner ear. Development of calretinin immunoreactivity in the mouse inner ear was investigated from embryonic day 13 (E13) to the adult stage. In the adult mouse vestibule, calretinin immunoreactivity was present in the same structures as described for the rat and guinea pig: the population of afferent fibers forming calyx units and a small number of ganglion neurons. The earliest immunoreactivity was found at E17 in vestibular hair cells (VHCs), then, at E19, in afferent fibers entering the sensory epithelia and in rare ganglion neurons. At postnatal day 4 (P4), a few vestibular nerve fibers and ganglion neurons were reactive. From this stage until P14, immunoreactivity developed in the calyx units and disappeared from VHCs. At P14, immunostaining was adult-like. In the adult mouse cochlea, immunoreactivity was present in the same cell populations as described in the rat: the inner hair cells (IHCs) and most of Corti's ganglion neurons. Calretinin immunoreactivity appeared at E19-P0 in IHCs and ganglion neurons of the basal turn. At P1, outer hair cells (OHCs) of the basal turn were positive. Calretinin immunoreactivity then appeared in IHCs, OHCs, and ganglion neurons of the medial turn, then of the apical turn. At P4, all IHCs and OHCs and most of the ganglion neurons were immunostained. Immunoreactivity gradually disappeared from the OHCs starting at P10 and, at P22, only IHCs and ganglion neurons were positive. The sequences of appearance of calretinin were specific to each cell type of the inner ear and paralleled their respective maturation. Calretinin was transiently expressed in VHCs and OHCs.

Structural differences between atrial and ventricular myosins from normal human hearts.

Comparisons were made between myosins isolated from the right and left ventricles and the atria of normal human hearts. Parameters examined included electrophoretic mobilities of native molecules, K+ and Ca2+ dependent enzymatic activities, light chain composition, peptide patterns from partial proteolytic digests of entire heavy chains or rods, and maps of complete digests of specific 21 and 25 kilodalton heavy chain fragments. Human ventricular and atrial myosins differ in all parameters except in the charge of molecules. Structural differences between cardiac myosins derived from the two sources were apparent in both the head and tail portions of the heavy chains. With respect to the above parameters no differences were found between myosins from left and right human ventricles.

Identification of altered gene expression in skeletal muscles from Duchenne muscular dystrophy patients.

Mutations in the dystrophin gene lead to dystrophin deficiency, which is the cause of Duchenne muscular dystrophy (DMD). This important discovery more than 10 years ago opened a new field for very productive investigations. However, the exact functions of dystrophin are still not fully understood and the complex process leading to subsequent muscle fiber necrosis has not been clearly described; hence there has not yet been any marked improvement in patient treatment. To decipher the molecular mechanisms induced by a lack of dystrophin, we started identifying genes whose expression is altered in DMD skeletal muscles. The approach was based on differential screening of a human muscle cDNA array. Nine genes were found to be up- or downregulated. Our results indicate expression alterations in mitochondrial genes, titin, a muscle transcription factor and three novel genes. First characterizations of these novel genes indicated that two of them have striated muscle tissue specificity.

Exclusion of muscle specific actinin-associated LIM protein (ALP) gene from 4q35 facioscapulohumeral muscular dystrophy (FSHD) candidate genes.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder for which no candidate gene has yet been identified. The gene corresponding to one of the novel human cDNAs that we cloned on the basis of a muscle restricted expression pattern [Piétu G, Alibert O, Guichard B, et al. Genome Res 1996;6:492-503] was mapped in the region of the FSHD1A genetic locus, i.e. one of the loci involved in this muscular dystrophy. The corresponding encoded protein contains a PDZ and a LIM domain, two protein-protein interaction domains, and was very recently shown to bind alpha-actinin-2 and was named ALP (actinin-associated LIM protein) [Xia H, Winokur S, Kuo W, Altherr M, Bredt D. J Cell Biol 1997;139:507-515]. We raised a specific polyclonal anti-ALP serum against an ALP recombinant polypeptide to evaluate the size, level of expression and subcellular localization of ALP in three patients, clearly diagnosed with FSHD disease. Quantitative or qualitative alterations of ALP expression have not been detected in any of them, thus prompting us to exclude ALP as a FSHD gene candidate.

The efferent vestibular system in the cat: a horseradish peroxidase and fluorescent retrograde tracers study.

Neurons of the efferent vestibular system were investigated in the cat using retrograde axonal transport of horseradish peroxidase and fluorescent retrograde double labelling techniques. The number of efferent neurons was clearly higher than previously reported. A three dimensional reconstruction of the location of these neurons showed that they constitute a single group and did not give evidence of an eventual specialization based on neuron subpopulations. However, a study of cross-sectional areas of the horseradish peroxidase-labelled efferent neurons detected that the ipsilateral population contained a larger number of small neurons than the contralateral one. Double labelling by means of either 4',6-diamidino-2- phenylindol 2HCl in combination with horseradish peroxidase or Fast Blue in combination with Nuclear Yellow showed that 20% of efferent neurons project to both labyrinths. Such a high percentage raises the question of the role of these double-projecting cells and the specificity of their branching on vestibular receptors. This study expands previous work in the cat demonstrating that a much greater number of efferent neurons exists than had hitherto been assumed, among them 20% have both crossed and uncrossed projections.

Isolation, localization, and cloning of a kainic acid binding protein from frog brain.

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.

Calbindin (CaBP 28 kDa) appearance and distribution during development of the mouse inner ear.

Previous reports of the distribution of calbindin, a 28 kDa vitamin D-induced calcium-binding protein, in the mammalian peripheral vestibular system postulated that this protein was involved in the calcium-dependent mechanisms occurring in the hair cells and ganglion cells. In this study, we examined the possibility of a relationship between the presence of calbindin and neurotransmission by comparing calbindin appearance in the inner ear to the sequence of synaptogenesis. Calbindin distribution was studied by immunocytochemistry, in the developing mouse inner ear from gestational day 12 to postnatal day 40. During the early development, calbindin was localized in non-neuronal structures: Kolliker's organ, spiral limbus and crista supporting cells; and in cochlear and vestibular ganglion neurons and sensory cells. At later stages and in the adult, no reactivity was observed in the non-neuronal cell populations and only certain sensory and nerve cells remained stained: inner hair cells, outer hair cells, vestibular hair cells of the apex of the cristae and of the striola in the maculae, all Corti's ganglion neurons and some vestibular ganglion neurons. The sequence of appearance of calbindin immunoreactivity in the sensory and nerve cells was not completely parallel to the maturation sequence of the inner ear, especially synaptogenesis. The dual distribution of calbindin during development and its expression in specific sensory and nerve cells opens new perspectives on its role in the inner ear.

Appearance and development of neuron-specific enolase immunoreactivity in organotypic cultures of mouse embryo otocysts.

The appearance of neuron-specific enolase (NSE) immunoreactivity was studied in sensory and vestibular ganglion cells during the development of mouse embryo otocysts grown in vitro from the 13th gestation day. NSE appeared sequentially in the ganglion and sensory cell populations of the inner ear with a pattern that paralleled their successive maturation. Comparison with NSE immunoreactivity profile during in vivo development shows that NSE appears earlier during organotypic in vitro maturation.

Characterization of different neuron populations in mouse statoacoustic ganglion cultures.

Statoacoustic ganglion (SAG) cells were grown in primary culture and the appearance of different neuronal phenotypes was investigated. Analysis criteria were shape, size and staining for the immunocytochemical markers: neurofilament proteins (NF-200 kDa), neuron-specific enolase (NSE), calretinin, a calcium-binding protein and substance P, a neurotransmitter. Cultures were prepared from dissociated SAG cells of 13 gestation-day-old mouse embryos. Neurons were identified and counted after 7 days in vitro. At this stage, neurons were organized in small clusters forming an extensive network of neurites grown on a layer of fibroblasts and glia. Most neurons identified by NF or NSE immunoreactivity showed a typical adult-like bipolar profile. The diameters of the neurons were between 5.62 and 17.00 microns and displayed a normal distribution (mean: 10.6 microns). Two distinct subpopulations were identified by the expression of calretinin and substance P. Calretinin-immunoreactive neurons were large and very rare and had a mean diameter of 11.3 microns; the distribution of substance P was more extensive than that of calretinin and identified a population of small neurons with a mean diameter of 8.9 microns. The distributions of these two markers in SAG cultures were consistent with in vivo results. In conclusion, dissociated SAG cell cultures appear to be a suitable model for analyzing the development of the immunocytochemical and functional characteristics of the neurons of this inner ear ganglion.

Cellular and subcellular localization of AMPA-selective glutamate receptors in the mammalian peripheral vestibular system.

The cellular and subcellular distribution of AMPA-selective glutamate receptors in the mammalian peripheral vestibular system was examined using antibodies against peptides corresponding to the C-terminal portions of AMPA receptor subunits: GluR1, GluR2/R3 and GluR4. The light and electron microscopic immunocytochemical studies were carried out on Vibratome sections of rat and guinea pig vestibular sensory epithelial and ganglia. In the epithelium, GluR1 subunit immunoreactivity appeared as accumulations of patches outlining the baso-lateral periphery of the type I sensory cells. The GluR1-immunoreactive microareas were postsynaptically distributed on the membranes of calyceal afferent fibers. GluR2/R3 immunoreactivity was present in the sensory cells. GluR4 was not detected. In the vestibular ganglion, the neurons were densely stained with antibodies to GluR2/R3 and GluR4. The fibroblasts and the Schwann cells were also intensely stained with antibodies to GluR2/R3 and GluR4. In the sensory cells, the AMPA receptors, GluR2/R3, may function as (1) autoreceptors controlling afferent neurotransmitter release or (2) 'postsynaptic' receptors activated by the neurotransmitter release of the afferent calyx. The detection of GluR1 at postsynaptic sites in the afferent fibers provides anatomical evidence for the role of glutamate as a neurotransmitter of sensory cells. In the ganglion neurons, GluR2/R3 and GluR4 may represent reserve intracytoplasmic pools of receptor subunits in transit to the postsynaptic sites. In the Schwann cells, GluR2/R3 and GluR4 may be involved in neuronal-glial signalling at the nodes of Ranvier.