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Reduced amount of the glucose-regulated protein GRP94 in skeletal myoblasts results in loss of fusion competence
No full textWe previously showed that skeletal myocytes of the adult rabbit do not accumulate the endoplasmic reticulum glucose-regulated protein GRP94, neither constitutively nor inducibly, at variance with skeletal myocytes during perinatal development (5), Here we show that C2C12 cells upregulate GRP94 during differentiation and, similarly to primary cultures of murine skeletal myocytes, specifically display GRP94 immunoreactivity on the cell surface. Stable transfection of C2C12 cells with grp94 antisense cDNA shows lack of myotube formation in clones displaying >40% reduction in GRP94 amount. The same result is obtained after irt vivo injection of grp94-antisense myoblasts, Conversely, GRP94 overexpression is accompanied by accelerated myotube formation. Analyses of BrdU incorporation, p21 nuclear translocation, and muscle-gene expression show that muscle differentiation is not apparently affected in grp94-antisense clones. In contrast, cell-surface GRP94 is greatly reduced in grp94-antisense clones, as shown by immunocytochemistry and precipitation of cell-surface biotinylated proteins. Thus, cell-surface expression of GRP94 is necessary for maintenance of fusion competence. Furthermore, differentiating C2C12 cells grown in the presence of anti-GRP94 antibody show decreased myotube number suggesting that cell-surface GRP94 is directly involved in myoblast fusion process
Neurofilament M mRNA is expressed in conduction system myocytes of the developing and adult rabbit heart
No full textWe previously demonstrated that conduction-system myocytes of the rabbit heart express cytoskeletal proteins immunologically related to neurofilaments. In order to determine more precisely the nature of these proteins, we screened an expression cDNA library, prepared from the sino-atrial node region of the rabbit heart, using a monoclonal antibody which reacts with the M subunit of neurofilaments, Sequence analysis of the isolated cDNA clones shows high homology with rat and human neurofilament M mRNAs. Northern blot analysis demonstrates hybridization with a transcript expressed in brain, with the size expected for neurofilament M mRNA. An mRNA species of the same size is also detectable in the Northern blot of the sino-atrial node region RNA. bl situ hybridization documents that in the adult rabbit the transcript accumulates in neurons and is localized in myocytes of the sino-atrial and atrio-ventricular nodes and of the atrio-ventricular bundle and bundle branches, but not in working atrial and ventricular myocytes, Developmental analysis was undertaken in order to determine the distribution of the neurofilament M mRNA in the rabbit embryonic heart, In situ hybridization shows that neurofilament M mRNA is detectable in a few ventricular myocytes in proximity to the atrioventricular groove after 9.5 days of embryonic development and it is accompanied by the presence of the protein, At subsequent stages of development neurofilament M mRNA is detectable in a number of cardiac myocytes, which are mainly localized at the atrio-ventricular junction and in the ventricular subendocardium and presumably correspond to myocytes of the heart conduction system
Expression of the glucose regulated protein GRP94 at the cell surface is required for myotube formation
No full textDistribution of conduction system fibers in the developing and adult rabbit heart revealed by an anti-neurofilament antibody.
No full textUsing an immunological approach, we demonstrated previously that a
neurofilament-like protein is expressed in rabbit heart conduction tissue
myocytes, and we proposed that these specialized cardiac muscle cells are of
neuroectodermal origin. In the present study, we used the expression of the
neurofilament-like protein as a marker for identifying conduction tissue cells
and studying their distribution in the developing heart. In 11-day-old rabbit
embryos, myocytes expressing the neurofilament-like protein were localized at the
atrioventricular and the sinoatrial junctions and had a ring-like distribution.
At embryonic day 12, reactive myocytes were found also in the subendocardial
layer of the dorsal ventricular wall, in continuity with labeled myocytes at the
atrioventricular junction. Examination of older embryos and of neonatal and adult
hearts revealed that the expression of the neurofilament-like protein was not
restricted to myocytes of conduction tissue regions, but it was also detectable
in myocytes of the sinoatrial ring bundle, in scattered myocytes localized in the
left sinal horn wall, and in the right atrium in proximity to atrioventricular
sulcus tissue. Thus, using an intracellular marker, we show that precursors of
adult atrial conduction tissue are distributed at the sinoatrial and
atrioventricular junctions; at variance, ventricular conduction tissue precursors
do not have a ring-like distribution but are localized in the subendocardial
layer, in continuity with the atrioventricular junctional myocytes
Neurofilament proteins are co-expressed with desmin in heart conduction sytem myocytes.
No full textWe have recently shown that specialized myocytes of the rabbit heart express a
cytoskeletal protein similar to the M subunit of neurofilaments (NF). Since this
result was obtained using a single anti-NF-M monoclonal antibody, we tested on
conduction myocytes a panel of five anti-NF antibodies, specific for each of the
three NF subunits and for phosphorylated and non-phosphorylated epitopes. Two
antibodies, one specific for the L subunit and one for phosphorylated M subunit
of NF, reacted with specialized myocytes in immunohistochemistry. In immunoblots
on conduction tissue homogenates the two antibodies recognized two polypeptides
with electrophoretic mobility and solubility properties identical to those of
NF-L and NF-M in the sciatic nerve. The subcellular distribution of NF
immunoreactivity in specialized myocytes was very similar to desmin localization;
namely, it was distributed on large filamentous bundles and on fine filaments
localized transversely at the level of the Z line. At the ultrastructural level,
immunoreactive filaments were localized in the intermyofibrillar space and
connected myofibrils with mitochondria. Co-expression of NF proteins and desmin
was also observed in vitro in a minor population of cardiac myocytes cultured
from embryonic rabbit heart. In most cases NF immunoreactivity co-localized with
desmin, especially where filaments were well organized, but in some cells anti-NF
and anti-desmin antibodies labelled different filamentous structures. These
results indicate that NF proteins are structural components of the cytoskeleton
of specialized myocytes and show a subcellular distribution very similar to
desmin. Such a composition of intermediate filaments indicates that in these
cardiac cells muscle differentiation is compatible with the expression of
neuronal proteins
Heart conduction system: a neural crest derivative?
No full textUsing the anti-neurofilament monoclonal antibody iC8 we report here that muscle
fibers of the conduction system of the adult and developing rabbit heart express
a cytoskeletal protein antigenically and electrophoretically similar to the
middle subunit of neurofilaments (NF-M). In the 11-day embryo a number of cardiac
muscle cells also express a neural crest surface marker recognized by the
monoclonal antibody HNK-1. Both markers are found in many cells of the 3rd and
4th branchial arches, which are populated by cells of neural crest origin. In the
11-day embryo cells of the 4th branchial arch are in close proximity to and
intermingled with the atrial myocardium: cells co-expressing sarcomeric myosin
heavy chain with iC8 and HNK-1 immunoreactivity are seen at these sites. The
findings suggest that conduction tissue cells of the rabbit heart originate from
a population of neural crest-derived cells migrating from the branchial arches
into the developing heart
Transfection of skeletal myoblasts with antisense cDNA for GRP94 results in loss of fusion competence.
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