Ultrastructural analysis of the smooth-to-striated transition zone in the developing mouse esophagus: emphasis on apoptosis of smooth and origin and differentiation of striated muscle cells.

The exact mechanism of smooth-to-striated muscle conversion in the mouse esophagus is controversial. Smooth-to-striated muscle cell transdifferentiation vs. distinct differentiation pathways for both muscle types were proposed. Main arguments for transdifferentiation were the failure to detect apoptotic smooth and the unknown origin of striated muscle cells during esophageal myogenesis. To reinvestigate this issue, we analyzed esophagi of 4-day-old mice by electron microscopy and a fine-grained sampling strategy considering that, in perinatal esophagus, the replacement of smooth by striated muscle progresses craniocaudally, while striated myogenesis advances caudocranially. We found numerous (1) apoptotic smooth muscle cells located mainly in a transition zone, where smooth intermingled with developing striated muscle cells, and (2) mesenchymal cells in the smooth muscle portion below the transition zone, which appeared to give rise to striated muscle fibers. Taken together, these results provide further evidence for distinct differentiation pathways of both muscle types during esophagus development.     

Smooth muscle persists in the muscularis externa of developing and adult mouse esophagus

Following initial patterning as differentiated smooth muscle (SM) cells, the muscularis externa of the murine esophagus is replaced by skeletal muscle, but the mechanism underlying this process is controversial. The hypothesis that committed SM cells transdifferentiate into striated muscle is not consistent with fate mapping studies. Similarly, apoptosis does not fully explain the process. Using immunohistochemical techniques and transgenic mice that express eGFP and Cre-recombinase exclusively in SM, we have identified a population of remnant SM cells that persist throughout the developing and mature murine esophagus. These cells display an atypical phenotype, are not associated with microvasculature, but are often apposed to cKit positive, interstitial cells of Cajal. The absolute length of the SM component of the developing esophagus remains constant during a period when total esophageal length increases 4-fold, resulting in a small maintained distal segment of smooth muscle. Esophageal SM cells fail to express myogenin during development, and striated muscle cell precursors expressing myogenin fail to express specific SM cell markers, indicating that they did not transdifferentiate from SM cells. Moreover, smooth muscle-specific myogenin inactivation has no effect on esophageal skeletal myogenesis. Taken together, our results provide an alternative hypothesis regarding the fate of SM cells in the developing murine esophagus, which does not invoke apoptosis or transdifferentiation.     

Deletion of Pax7 changes the tunica muscularis of the mouse esophagus from an entirely striated into a mixed phenotype

The mechanisms responsible for the different amounts of striated muscle in mammalian esophagi are still enigmatic. A recent ultrastructural analysis in mouse esophagus pointed to a particular role of satellite cells during postnatal growth of striated muscle. The aim of this study was to investigate satellite cell development and the influence of Pax7 on this process. Developing and adult esophagi of wild‐type and mice carrying a targeted mutation in Pax7 were analyzed by electron microscopy. We found a gene dose‐dependent delayed development of striated muscle and a severe loss of satellite cells in Pax7^+/? and Pax7^?/? esophagi. In contrast to the entirely striated wild‐type esophagus, Pax7^?/? mutants developed a mixed phenotype with predominantly smooth muscle caudally. We conclude that Pax7‐dependent myogenic progenitor cells are of prime importance for striated muscle formation and the degree of smooth‐to‐striated muscle conversion during esophageal ontogeny. Developmental Dynamics 238:864–874, 2009. © 2009 Wiley‐Liss, Inc.     

The neural regulation of the mammalian esophageal motility and its implication for esophageal diseases

In contrast to the tunica muscularis of the stomach, small intestine and large intestine, the external muscle layer of the mammalian esophagus contains not only smooth muscle but also striated muscle fibers. Although the swallowing pattern generator initiates the peristaltic movement via vagal preganglionic neurons that project to the myenteric ganglia in the smooth muscle esophagus, the progressing front of contraction is organized by a local reflex circuit composed by intrinsic neurons similarly to other gastrointestinal tracts. On the other hand, the peristalsis of the striated muscle esophagus is both initiated and organized by the swallowing pattern generator via vagal motor neurons that directly innervate the muscle fibers. The presence of a distinct ganglionated myenteric plexus in the striated muscle portion of the esophagus had been enigmatic and neglected in terms of peristaltic control for a long time. Recently, the regulatory roles of intrinsic neurons in the esophageal striated muscle have been clarified. It was reported that esophageal striated muscle receives dual innervation from both vagal motor fibers originating in the brainstem and varicose intrinsic nerve fibers originating in the myenteric plexus, which is called ‘enteric co-innervation’ of esophageal motor endplates. Moreover, a putative local neural reflex pathway that can control the motility of the striated muscle was identified in the rodent esophagus. This reflex circuit consists of primary afferent neurons and myenteric neurons, which can modulate the release of neurotransmitters from vagal motor neurons in the striated muscle esophagus. The pathogenesis of some esophageal disorders such as achalasia and gastroesophageal reflux disease might be involved in dysfunction of the neural networks including alterations of the myenteric neurons. These evidences indicate the physiological and pathological significance of intrinsic nervous system in the regulation of the esophageal motility. In addition, it is assumed that the components of intrinsic neurons might be therapeutic targets for several esophageal diseases.     

Structure of the esophagus in the adult opossum, Didelphis virginiana.

The structure of the esophagus has been studied in the adult opossum, Didelphis virginiana. A thickening of both layers of the muscularis externa occurs at the origin of the esophagus and may represent the upper esophageal sphincter; a massive expansion of the muscularis mucosae occurs in the region of the lower esophageal sphincter. The distribution of striated, mixed and smooth muscle in the muscularis externa differs in the inner and outer layers and elements of the myenteric plexus are found to occur even in the region of striated muscle; however, the ganglia of this plexus become much more prominent as smooth muscle makes its appearance. Esophageal glands are found in the lamina propria where they are confined to the 2 ends. They are especially prominent at the distal end where they are responsible for the formation of permanent transverse folds. Similar glands are found in the submucosa, scattered throughout the length of the esophagus but distally, in the region of the transverse folds, the submucous glands disappear. In both of these layers, the glands contain mucous, serous and myoepithelial cells.     

A light microscope study of the distribution of muscle in the frog esophagus and stomach.

The present study reports light microscopical observations of the distribution of muscle in the esophagus and stomach of both the bull frog (Rana catesbeiana) and the African clawed frog (Xenopus laevis). The external muscle coat of the upper half of the esophagus in both species had several collagen coated bundles of striated muscle fibres around the circumference. These striated muscle bundles ran longitudinally from the pharynx to around the vicinity of the center of the esophagus. Beneath these striated muscle bundles was an inner circular layer of smooth muscle. In both species, the inner circular layer of smooth muscle was particularly thick in the region close to the pharynx. In the bull frog, the lower half of the esophagus lacked striated muscle. However, the circular smooth muscle layer, extending from the upper half of the esophagus, was also observed throughout the lower half of the esophagus. An outer longitudinal layer of smooth muscle developed towards the terminal portion of the esophagus such that in this region, both outer longitudinal and inner circular layers of smooth muscle were observed. Similarly in the African clawed frog, the inner circular layer of smooth muscle was continuous along the full length of the esophagus. Again, no striated muscle bundles were observed in the lower half of the esophagus. However, the outer longitudinal layer of smooth muscle was seen to develop in the middle region of the esophagus. Its muscle layer extended to the terminal portion of the esophagus. Thus, both outer longitudinal and inner circular layers of smooth muscle were observed throughout the lower half of the esophagus. In both frogs, the thickness of the outer longitudinal and inner circular layers of smooth muscle changed before and after the esophago-gastric junction. In both frogs, no muscularis mucosa was observed in the esophageal wall. However, in the lower half of the esophagus of the African clawed frog, small bundles of smooth muscle were observed here and there in the submucosa. A fully developed muscularis mucosa with both outer longitudinal and inner circular layers was observed in the upper stomach of both frogs.     

The expression of nestin delineates skeletal muscle differentiation in the developing rat esophagus

The muscularis externa of the developing rodent esophagus is initially composed of smooth muscle, and later replaced by skeletal muscle in a craniocaudal progression. There is growing evidence of distinct developmental origins for esophageal smooth and skeletal muscles. However, the identification of skeletal muscle progenitor cells is controversial, and the detailed cell lineage of their descendants remains elusive. In the current study, we carried out multiple labeling immunofluorescence microscopy of nestin and muscle type-specific markers to characterize the dynamic process of rat esophageal myogenesis. The results showed that nestin was transiently expressed in immature esophageal smooth muscle cells in early developing stages. After nestin was downregulated in smooth muscle cells, a distinct population of nestin-positive cells emerged as skeletal muscle precursors. They were mitotically active, and subsequently co-expressed MyoD, followed by the embryonic and later the fast type of skeletal muscle myosin heavy chain. Thus, the cell lineage of esophageal skeletal muscle differentiation was established by an immunotyping approach, which revealed that skeletal myocytes arise from a distinct lineage rather than through transdifferentiation of smooth muscle cells during rat esophageal myogenesis.     

Histological analysis of esophageal muscular layers from 27 autopsy cases with mixed connective tissue disease (MCTD)

Esophageal symptoms in mixed connective tissue disease (MCTD) have been investigated radiologically. We investigated the esophageal lesions in MCTD histopathologically, and analyzed relationships between these lesions and autoantibodies extracted from the serum of MCTD patients. Esophageal tissues from 27 MCTD patients submitted to autopsy were examined. We compared histopathological features of the esophagus in different wall layers from the mucosa, submucosa, and muscular layer to the adventitia, and in the upper, middle, and lower portions of esophagus. The most striking change observed was severe atrophy and occasional loss of smooth muscle cells in the muscular layer, followed by fibrosis. These muscular changes were particularly prominent in the inner layer of the lower esophagus. Immunohistochemically, degenerated muscular tissues of the esophagus were positive for anti-IgG and anti-C3 antibodies, but not for anti-IgM antibodies. IgG fractions extracted from three MCTD patients were immunohistochemically used to examine whether some antibodies in MCTD patients showed reactivity for esophageal components. The IgG fractions isolated from MCTD patients reacted with smooth muscle from non-connective tissue disease cases, suggesting that some serum antibodies may trigger esophageal changes. These findings suggest that esophageal lesions associated with clinical dysphagia in MCTD may be related to autoantibodies.     

Evaluation of decellularized esophagus as a scaffold for cultured esophageal epithelial cells

Recently, decellularized tissue has been reported to have the potential to regenerate a variety of tissues. However, the optimal protocol for a decellularized esophagus has not been studied. Here, we investigated the effect of different decellularization protocols on the histology and biocompatibility of decellularized esophagi in view of future applications to tissue engineering. The esophageal mucosal epithelium (EP) from 4-week-old Wistar rats was enzymatically dissociated and cultured with growth-arrested feeder cells. Two methods for decellularization using deoxycholic acid (DEOX) or Triton X-100 (TRITON) were compared on esophagi from adult Wistar rats. Those treated with DEOX showed superior mechanical properties, maintenance of extracellular matrix, and lower DNA content than those treated with TRITON. To evaluate the biocompatibility of the scaffold, cultured (passage 3) esophageal epithelial cells were seeded inside the decellularized esophagus and cultured for 7 days. The cells seeded onto the decellularized esophagus were examined histologically and immunocytochemically. Esophageal epithelial cells were stratified into three to four cellular layers in vitro inside the decellularized esophagus, to show polarity. The results from immunocytochemistry indicated that the seeded epithelial cells expressed characteristic marker proteins for native esophageal EP. Decellularized esophagus showed suitable compatibility as a scaffold material for esophageal tissue engineering.     

Developing rat lung has a sided pacemaker region for morphogenesis-related airway peristalsis

Prenatal airways from diverse species are capable of spontaneous peristaltic contractions in each trimester. The function of this smooth muscle activity is unknown. We demonstrate that peristalsis of the embryonic airway originates from a sided pacemaker focus, is stimulated in a calcium-dependent fashion by the pulmonary morphogen fibroblast growth factor-10 (FGF-10), and appears coupled to lung growth. Airway peristalsis may be crucial for lung development (thereby providing a physiologic role for airway smooth muscle) and play a hitherto unanticipated role in reported transgenic mutant lung phenotypes.     

Effect of atropine on esophageal motor function in humans

In this study, we used a high-fidelity manometric recording system to quantitate the effects of atropine on lower esophageal sphincter (LES) pressure and primary peristalsis (1 degree P). A sleeve sensor recorded LES pressure, and seven recording orifices spaced at 3-cm intervals registered motor activity in the esophageal body. Five randomized manometric studies were done in each of five normal subjects. LES pressure and 1 degree P with wet swallows were recorded for 30 min before and 70 min after intravenous injection of saline or atropine, 3, 6, 12, and 24 micrograms/kg. We also studied the effect of atropine on LES pressure in five additional subjects, four dogs, four opossums, and six monkeys. In humans, saline and 3 micrograms/kg atropine caused no significant change in pulse rate, LES pressure, or the incidence of complete peristaltic sequences. The 6, 12, and 24 micrograms/kg atropine doses caused significant inhibition of LES pressure and the incidence of intact 1 degree P. Only the 12 and 24 micrograms/kg doses increased pulse rate. When 1 degree P occurred in the smooth muscle portion of the esophagus its appearance in the proximal portion of the smooth muscle segment was delayed for several seconds. The amplitude of 1 degree P was decreased 30-60% in the smooth muscle segment, but 1 degree P was not affected in the proximal striated muscle esophageal segment. Atropine reduced canine LES pressure substantially but caused no change in opossums or monkeys. We conclude that 1) basal LES tone in humans and dogs, unlike that of the opossum and monkey, is partially generated by cholinergic neural input, 2) cholinergic nerves elicit 1 degree P in human esophageal smooth muscle, and 3) species variation exists in esophageal responses to atropine.     

Evidence for the involvement of neurotrophins in muscle transdifferentiation and acetylcholine receptor transformation in the esophagus of Myf5(-/-):MyoD(-/-) and NT-3(-/-) embryos.

The primary aim of our study was to determine whether the esophageal innervation (i.e., vagal and enteric) and the skeletal muscle-secreted neurotrophins have a role in smooth-to-skeletal muscle transdifferentiation and in the muscarinic-to-nicotinic acetylcholine receptor type transition. To that end, we used genetically engineered embryos and immunohistochemistry. We found that, in the absence of Myf5 and MyoD, the esophageal muscle cells failed to develop the striated phenotype of acetylcholine receptors. In addition, the development of vagal and enteric innervation was delayed in Myf5(-/-):MyoD(-/-) and NT-3(-/-) mutants, but it was reestablished 2 days before the end of gestation. The smooth muscle cells in the esophagus appeared to be a distinct subpopulation of cells and their ability to transdifferentiate was based on their competence to express neurotrophins and their receptors. Finally, our data suggest a role for NT-3 in the esophageal muscle transdifferentiation.     

Filament formation in smooth muscle homogenates

Summary To provide more detailed information on the aggregation properties of smooth muscle myosin, we have extended earlier work on the formation of thick filaments when homogenates of guinea-pig taenia coli and chicken gizzard muscle are diluted. In both preparations there is a slow and a fast phase of filament formation. The slow phase, which generally develops over several hours, appears to depend primarily on the ATP concentration while the rapid phase, which develops over 5–15 min, is influenced by the extent of dilution, homogenization conditions, divalent cation concentration, ATP concentration and presence of chicken gizzard tropomyosin. Many of these effects on the rapid phase can be explained by postulating that filament formation only takes place when the ATP concentration is reduced.There are significant differences between the filament populations formed from each muscle, with those from taenia coli being shorter than those from gizzard. Two types of filament are present in preparations from each muscle, the first being characterized by the presence of a central bare zone and cross striations at both ends, whilst the second have cross striations along their entire length; the periodicity of the cross striations appears to be 14.5 nm. The bare zone filaments have an average length and width of 325 nm and 17.6 nm respectively, while the corresponding values for the cross striated filaments are 553 nm and 29.0 nm. The proportion of bare zone to cross striated filaments is 3 : 1 for taenia coli and 1 : 3 for chicken gizzard, which accounts for the difference in average filament length observed between these preparations. The gizzard filaments appear to form more readily than those of taenia coli.     

Effect of thoracic vagotomy and vagal stimulation on esophageal function

The purpose of this study was to determine the effect of thoracic vagotomy and thoracic vagal stimulation upon esophageal peristalsis and lower esophageal sphincter (LES) function in the opossum. The thoracic portion of the vagus nerve was sectioned in the upper or lower thorax. Bilateral, but not unilateral, thoracic vagotomy above the level of the heart abolished peristalsis and LES relaxation in response to swallowing or cervical vagal electrical stimulation. Thoracic vagotomy at the level of the ventricle or below did not alter either peristalsis or LES relaxation during swallowing or cervical vagal stimulation. Secondary peristalsis and its associated LES relaxation was unaltered by thoracic vagotomy at any level. Electrical stimulation of the distal end of the upper thoracic vagus elicited both peristalsis and LES relaxation. Electrical stimulation of the distal end of the lower thoracic vagus elicited both peristalsis and LES relaxation. Electrical stimulation of the distal end of the lower thoracic vagus, as well as stimulation of the vagal branches to the terminal esophagus, gave only LES relaxation. These studies suggest that: a) the major extrinsic vagal innervation mediating primary peristalsis terminates in the upper portion of the esophagus, whereas the vagal innervation mediating LES relaxation responses are present throughout the length of the esophagus; and b) secondary peristalsis and its associated LES relaxation occurs independent of extrinsic vagal innervation.     

Enteric co-innervation of striated muscle fibers in the esophagus: just a "hangover"?

Striated muscle of the esophagus was until recently considered to consist of "classical" skeletal muscle fibers innervated by cholinergic vagal motoneurons. The recently described co-innervation originating from enteric neurons expressing nNOS, VIP, NPY, and galanin added a new dimension of complexity. The aim of this study was to summarize current knowledge about, and to get further hints as to the possible function of enteric co-innervation of striated esophageal muscle fibers. Aldehyde fixed rat esophagi were processed for immunocytochemistry for CGRP or VAChT (to demonstrate vagal motor terminals), nNOS/NADPH-d, VIP, NPY, and galanin (to demonstrate enteric terminals), met-enkephalin, mu opiate receptor, muscarinic receptors m1-3, soluble guanylyl cyclase, and cGMP dependent kinase type I and II. Motor endplates were visualized using fluorochrome tagged alpha-bungarotoxin to label nicotinic receptors, or with AChE histochemistry. Besides light and confocal laser scanning microscopy, immuno electron microscopy was also employed. Up to 80% of motor endplates were co-innervated. In addition to nNOS, VIP, NPY, and galanin, many enteric terminals in esophageal motor endplates expressed met-enkephalin. Some appeared to stain for the muscarinic m(2) receptor. There was prominent immunostaining for the micro opioid receptor in the sarcolemma at both junctional and extrajunctional sites. Immunostaining for soluble guanylyl cyclase was prominent immediately beneath the clusters of nicotinic receptors. Enteric varicosities and vagal terminals intermingled in motor endplates often without intervening teloglial processes. During ontogeny, initially high co-innervation rates were reduced to adult levels in a cranio-caudally progressing manner. We conclude that, in addition to a possible nitrergic, VIP-, NPY-, and galaninergic modulation of neuromuscular transmission by enteric neurons, opioidergic mechanisms could play a role. On the other hand, cholinergic influence on enteric neurons may be exerted also by the nucleus ambiguus via motor endplates, in addition to the input from the dorsal motor nucleus. The observations that enteric nerve fibers contact striated muscle fibers at specialized sites, i.e., motor endplates, and that these contacts appear in an ordered cranio-caudal sequence after cholinergic motor endplates have been established point to a specific function in neuronal control of esophageal muscle rather than to be an unspecific "hangover" from the smooth muscle past of this organ.     

Characteristics of vagal esophageal tension-sensitive afferent fibers in the opossum

1. Single-unit vagal afferent activity was recorded from 35 fibers that demonstrated evoked response to distension in the smooth muscle portion of the esophagus in anesthetized opossums. 2. The conduction velocities, measured in 22 fibers, varied from 1.0 to 21.33 m/s. Eight fibers (36%) had conduction velocities in the range of C-fibers (less than 2.5 m/s), whereas 14 (64%) had velocities in the range of A delta-fibers (3.16-21.33 m/s). All fibers were spontaneously active with an average discharge rate of 7.3 +/- 1.0 imp/s (mean +/- SE; range, 1.2-23 imp/s). 3. Esophageal distension produced a reproducible increase in discharge rate that adapted slowly to sustained distension. The average threshold pressure of the endings was 10 mmHg. The saturation pressure was 70 mmHg with cumulative, stepwise distension and 56 mmHg with graded, discrete distensions, respectively. 4. The discharge rate at the saturation pressure was 46 +/- 7 imp/s with cumulative, stepwise distensions and was 59 +/- 4 imp/s with graded, discrete distensions. The difference in maximum discharge between these two modes of distension was not statistically significant (P greater than 0.05). 5. Esophageal peristaltic contraction was associated with bursts of spike discharge with an average rate of 53.6 +/- 4.7 imp/s. 6. Two types of fibers were identified based on their duration of spike discharge associated with peristaltic contraction. The short-activity fibers showed a short duration of response consisting of approximately 3 s of spike bursts in response to swallows. These short-activity fibers were not activated by either stretch or contraction of the longitudinal esophageal muscle. The long-activity fibers showed a long duration of response consisting of approximately 10 s of activity in response to swallows. These long-activity fibers could be activated by longitudinal muscle stretch or contraction. 7. It is concluded that esophageal tension-sensitive mechano-receptors associated with vagal afferents are activated by physiological peristalsis and are present "in series" with either circular or longitudinal muscle layers.     

Development of neuromuscular junctions in the mouse esophagus: focus on establishment and reduction of enteric co-innervation

The development of vagal and enteric innervation of esophageal motor endplates was examined in perinatal and adult BALB/c and NMRI mice using immunocytochemistry and confocal laser scanning microscopy. Nicotinic acetylcholine receptors were demonstrated with fluorochrome-tagged alpha-bungarotoxin, vagal motor terminals with antisera against vesicular acetylcholine transporter and calcitonin gene-related peptide, and enteric nerve terminals with antisera against neuronal nitric oxide synthase, vasoactive intestinal peptide and galanin. Results demonstrated that enteric and vagal innervations of striated esophageal muscle fibers develop in close spatiotemporal relationship, but with different courses. Connections between VAChT-positive vagal nerve terminals and growing acetylcholine receptor clusters were established from E17 to reach 100% motor endplate innervation at P14 and were maintained throughout adult life. CGRP immunoreactivity developed with a delay of several days after the appearance of VAChT in vagal terminals. From P14 to adulthood CGRP was colocalized with VAChT in almost all motor endplates. In contrast, enteric co-innervation rates increased from E17 to a maximum of 70-80% at P4, while their incidence at motor endplates progressively declined over the following 5 months to lower levels maintained throughout adulthood. Whereas adult enteric co-innervation rates in BALB/c and NMRI mice differed significantly (approximately 30% versus approximately 10%, respectively), their increase and reduction, respectively, during development showed an identical time course. These results suggest a well-ordered sequence of attraction of enteric nerve fibers to, and removal from motor endplates in the developing mouse esophagus. Thus, enteric co-innervation may subserve a functional role in the development and control of perinatal striated esophageal muscle rather than representing an unspecific "hangover" from the smooth muscle past of this organ.     

Multiaxial mechanical response and constitutive modeling of esophageal tissues: Impact on esophageal tissue engineering

Congenital defects of the esophagus are relatively frequent, with 1 out of 2500 babies suffering from such a defect. A new method of treatment by implanting tissue engineered esophagi into newborns is currently being developed and tested using ovine esophagi. For the reconstruction of the biological function of native tissues with engineered esophagi, their cellular structure as well as their mechanical properties must be considered. Since very limited mechanical and structural data for the esophagus are available, the aim of this study was to investigate the multiaxial mechanical behavior of the ovine esophagus and the underlying microstructure. Therefore, uniaxial tensile, biaxial tensile and extension-inflation tests on esophagi were performed. The underlying microstructure was examined in stained histological sections through standard optical microscopy techniques. Moreover, the uniaxial ultimate tensile strength and residual deformations of the tissue were determined. Both the mucosa-submucosa and the muscle layers showed nonlinear and anisotropic mechanical behavior during uniaxial, biaxial and inflation testing. Cyclical inflation of the intact esophageal tube caused marked softening of the passive esophagi in the circumferential direction. The rupture strength of the mucosa-submucosa layer was much higher than that of the muscle layer. Overall, the ovine esophagus showed a heterogeneous and anisotropic behavior with different mechanical properties for the individual layers. The intact and layer-specific multiaxial properties were characterized using a well-known three-dimensional microstructurally based strain-energy function. This novel and complete set of data serves the basis for a better understanding of tissue remodeling in diseased esophagi and can be used to perform computer simulations of surgical interventions or medical-device applications.     

Immunocytochemical electron microscopic study and Western blot analysis of myosin, paramyosin and miniparamyosin in the striated muscle of the fruit fly Drosophila melanogaster and in obliquely striated and smooth muscles of the earthworm Eisenia foetida

Miniparamyosin is a paramyosin isoform (55--60 kDa) that has been isolated in insects (Drosophila) and immunolocalized in several species of arthropods, molluscs, annelids and nematodes. In this study, the presence and distribution of this protein, in comparison with that of paramyosin and myosin, has been examined in the striated muscle (tergal depressor of trochanter) of Drosophila melanogaster, and the obliquely striated muscle (body wall) and the smooth muscle (outer layer of the pseudoheart) of the earthworm Eisenia foetida by means of immunocytochemical electron microscopic study and Western blot analysis miniparamyosin, paramyosin and myosin antibodies from Drosophila. In the striated muscle of D. melanogaster, the three proteins were immunolocalized along the length of the thick filaments (A- bands). The distribution of immunogold particles along these filaments was uniform. The relative proportions miniparamyosin/paramyosin/myosin (calculated by counting the number of immunogold particles) were: 1/10/68. In the obliquely striated muscle of E. foetida, immunoreactions to the three proteins were also found in the thick filaments, and the relative proportions miniparamyosin/paramyosin/myosin were 1/2.4/6.9. However, whereas the distribution of both myosin and miniparamyosin along the thick filament length was uniform, paramyosin immunolabelling was more abundant in the extremes of thick filaments (the outer zones of A-bands in the obliquely striated muscle), where the thick filaments become thinner than in the centre (the central zone of A-bands), where these filaments are thicker. The relative proportions of paramyosin in the outer and of paramyosin in the central zones of A-bands were 4/1. This irregular distribution of paramyosin along the thick filament length might be actual but it may also be explained by the fusiform shape of thick filaments in the earthworm: assuming that paramyosin is covered by myosin, paramyosin antigens would be more exposed in the tips than in the centre of thick filaments. If miniparamyosin is, in turn, covered by paramyosin, the exposure of miniparamyosin antigens would be low even in the tips of thick filaments, and this might explain the scanty immunoreaction observed for this protein and the absence of a higher number of immunogold particles in the extremes of thick filaments. The distribution of the three proteins in the earthworm smooth muscle was as in the obliquely striated muscle, although the proportions miniparamyosin/paramyosin/myosin were 1/1.5/5.2; this is, immunoreactions to paramyosin and miniparamyosin were lower than in the obliquely striated muscle