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Sneathia is an emerging pathogen implicated in adverse reproductive and perinatal outcomes. Although scarce, emerging data suggest that vaginally residing Sneathia becomes pathogenic following its ascension into the upper urogenital tract, amniotic fluid, placenta, and fetal membranes. The role of Sneathia in women’s health and disease is generally underappreciated because the cultivation of these bacteria is limited by their complex nutritional requirements, slow growth patterns, and anaerobic nature. For this reason, molecular methods are typically required for the detection and differential diagnosis of Sneathia infections. Here, we review the laboratory methods used for the diagnosis of Sneathia infections, the molecular mechanisms underlying its virulence, and its sensitivity to antibiotics. We further review the evidence of Sneathia’s contributions to the pathogenesis of bacterial vaginosis, chorioamnionitis, preterm prelabor rupture of membranes, spontaneous preterm labor, stillbirth, maternal and neonatal sepsis, HIV infection, and cervical cancer. Collectively, growing evidence indicates that Sneathia represents an important yet underappreciated pathogen affecting the development and progression of several adverse clinical conditions diagnosed in pregnant and non-pregnant women, as well as in their neonates.

Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this study was to investigate the process of assembly of obscurin into nascent sarcomeres during the transition from non-striated myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes. Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of obscurin in the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral fusion of nascent sarcomere bundles. We found that obscurin rapidly and precisely accumulated in the middle of the A-band regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern of sarcomeric alpha-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern of obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of obscurin in the areas of the terminal Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many non-striated fibers with very low concentration of obscurin were already immunopositive for sarcomeric actin and myosin. This suggests that obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison of obscurin and titin localization in these areas showed that obscurin assembly into the A-bands occurred soon after or concomitantly with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin filaments into the "open" half-assembled sarcomeres in the areas of the terminal Z-I structures and at the lateral surfaces of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed, terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned pattern. The sarcoplasmic reticulum, the organelle known to interact with obscurin, assembled around each new sarcomere. These results suggest that obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent sarcomeres and in the establishment of the registered pattern of newly assembled myofibrils and the sarcoplasmic reticulum at advanced stages of myofibrillogenesis.

Obscurin and obscurin-associated kinase are two products of the obscurin transcriptional unit that encodes a recently identified giant muscle-specific protein obscurin. In this study, we characterized the developmental expression and cellular localization of obscurin and obscurin-associated kinase in cardiac muscle cells. We cloned murine obscurin-associated kinase and found that it is abundantly expressed in the heart as two isotypes encoded by 2.2 and 4.9 kb sequences. The 2.2 kb isotype of the kinase was more prominently expressed than the 4.9 kb isotype. Both obscurin and the kinase-like domains were progressively upregulated since the early stages of cardiac development. Obscurin-associated kinase was expressed at higher levels than obscurin at early stages of cardiomyogenesis. Increasing intensity of obscurin expression in the developing heart positively correlated with progressive cell differentiation and was higher in the ventricles compared to the atria. These data were supported by the results of experiments with primary cardiac cell cultures. Obscurin localization changed from a weakly immunopositive diffuse pattern in poorly differentiated cells to an intensely immunolabeled cross-striated distribution at the level of mid-A-bands and Z-disks during the assembly of the myofibrillar contractile apparatus. In dividing myocytes, unlike the interphase cells, obscurin translocated from disassembling myofibrils into a diffuse granulated pattern segregated separately from alpha-actinin-immunopositive aggregates. Obscurin-associated kinase was localized mainly to cell nuclei with increasing incorporation into the Z-disks during differentiation. Our results suggest that these two novel proteins are involved in the progression of cardiac myogenesis during the transition to advanced stages of heart development.

Obscurin is a recently identified giant multidomain muscle protein (approximately 800 kDa) whose structural and regulatory functions remain to be defined. The goal of this study was to examine the effect of obscurin gene silencing induced by RNA interference on the dynamics of myofibrillogenesis and hypertrophic response to phenylephrine in cultured rat cardiomyocytes. We found that that the adenoviral transfection of short interfering RNA (siRNA) constructs targeting the first coding exon of obscurin sequence resulted in progressive depletion of cellular obscurin. Confocal microscopy demonstrated that downregulation of obscurin expression led to the impaired assembly of new myofibrillar clusters and considerable aberrations of the normal structure of the contractile apparatus. While the establishment of the initial periodic pattern of alpha-actinin localization remained mainly unaffected in siRNA-transfected cells, obscurin depletion did cause the defective lateral alignment of myofibrillar bundles, leading to their abnormal bifurcation, dispersal and multiple branching. Bending of immature myofibrils, apparently associated with the loss of their rigidity, a modified titin pattern, the absence of well-formed A-bands in newly formed contractile structures as documented by a diffuse localization of sarcomeric myosin labeling, and an occasional irregular periodicity of sarcomere spacing were typical of obscurin siRNA-treated cells. These results suggest that obscurin is indispensable for spatial positioning of contractile proteins and for the structural integration and stabilization of myofibrils, especially at the stage of myosin filament incorporation and A-band assembly. This demonstrates a vital role for obscurin in myofibrillogenesis and hypertrophic growth.

Little is known about the biological properties of myogenic satellite cells during post-denervation muscle atrophy. The present study investigated the differentiative capacity of satellite cells and their involvement in the compensatory regenerative process in long-term denervated rat muscle. Electron microscopy and immunocytochemical labeling of muscle tissue 1-18 months following denervation demonstrated that despite activation of satellite cells, myogenesis in denervated muscle is abortive and does not lead to the formation of normal muscle fibers. Small sizes, poor development of the contractile system in newly formed denervated myotubes, and the absence of satellite cells on the surface indicate that their differentiation typically does not progress to terminal stages. Many immature myotubes degenerate, and others survive but are embedded in a collagen lattice near their parent fibers. Interestingly, newly formed myotubes located on the surface of parent muscle fibers beneath the basal lamina typically did not contain developed myofibrils. This suggests that the contacts of daughter and parent muscle fibers block myofibrillogenesis. Assembly of sarcomeres in most cases occurs following complete spatial separation of daughter and parent muscle fibers. Another manifestation of the involvement of myogenic precursors in abortive myogenesis is the formation of clusters of underdeveloped branching myotubes surrounded by a common basal lamina. We found that myoblasts can also fuse directly with differentiated muscle fibers. The presence of satellite cells near the openings in the basal lamina and in the interstitial space indicates that myogenic precursors can migrate through the basal lamina and form myotubes at a distance from parent fibers. Our data may explain why long-term denervated skeletal muscle has a poor capacity for regeneration and functional restoration.

Obscurin is a newly identified giant muscle protein whose functions remain to be elucidated. In this study we used high-resolution confocal microscopy to examine the dynamics of obscurin localization in cultures of rat cardiac myocytes during the assembly and disassembly of myofibrils. Double immunolabeling of neonatal and adult rat cells for obscurin and sarcomeric α-actinin, the major protein of Z-lines, demonstrated that, during myofibrillogenesis, obscurin is intensely incorporated into M-band areas of A-bands and, to a lesser extent, in Z-lines of newly formed sarcomeres. Presarcomeric structural precursors of myofibrils were intensely immunopositive for α-actinin and, unlike mature myofibrils, weakly immunopositive or immunonegative for obscurin. This indicates that most of the obscurin assembles in developing myofibrils after abundant incorporation of α-actinin and that massive integration of obscurin occurs at more advanced stages of sarcomere assembly. Immunoreactivity for obscurin in the middle of A-bands and in Z-lines of sarcomeres bridged the gaps between individual bundles of newly formed myofibrils, suggesting that this protein appears to be directly involved in their primary lateral connection and registered alignment into larger clusters. Close sarcomeric localization of obscurin and titin suggests that they may interact during myofibril assembly. Interestingly, the laterally aligned striated pattern of obscurin formed at a stage when desmin, traditionally considered as a molecular linker responsible for the lateral binding and stabilization of myofibrils at the Z-bands, was still diffusely localized. During the disassembly of the contractile system in adult myocytes, disappearance of the cross-striated pattern of obscurin preceded the disorganization of registered alignment and intense breakdown of myofibrils. The cross-striated pattern of desmin typical of terminally differentiated myocytes disappeared before or simultaneously with obscurin. During redifferentiation, as in neonatal myocytes, sarcomeric incorporation of obscurin closely followed that of α-actinin and occurred earlier than the striated arrangement of desmin intermediate filaments. The presence of obscurin in the Z-lines and its later assembly into the A/M-bands indicate that it may serve to stabilize and align sarcomeric structure when myosin filaments are incorporated. Our data suggest that obscurin, interacting with other muscle proteins and possibly with the sarcoplasmic reticulum, may have a role as a flexible structural integrator of myofibrils during assembly and adaptive remodeling of the contractile apparatus.

Mutations in sarcomeric proteins can lead to either hypertrophic or dilated cardiomyopathy depending on their effects on the structural and functional properties of the contractile unit of the heart. Mutations in cardiac troponin T, which binds the calcium-responsive troponin complex to α-tropomyosin, have been shown to result in cardiac hypertrophy or cardiac dilatation and heart failure, depending on the nature of the specific mutation. In this study, we report the identification of a novel cardiac troponin T mutation (A171S) leading to dilated cardiomyopathy and sudden cardiac death. In contrast to prior described mutations, the A171S mutation results in a significant gender difference in the severity of the observed phenotype with adult males (over 20 years of age) demonstrating more severe ventricular dilatation [left ventricular end diastolic dimension (LVEDD) 7.1 vs. 5.1cm; P =0.01, t test] and left ventricular dysfunction [left ventricular shortening fraction (LVSF) 21 vs. 34%; P =0.04, t test] than adult females. The described mutation substitutes a hydrophilic amino acid for a hydrophobic one in a highly conserved domain involved in the interaction between troponin T and α-tropomyosin. Interestingly, four previously described mutations within 12 amino acids of A171 lead to a hypertrophic phenotype, suggesting that further characterization of the functional consequences of the A171S mutation may lead to a better understanding of the pathophysiology of DCM and of the functional differences between HCM- and DCM-causing mutations in cardiac troponin T.

We investigated the dynamics of muscle fiber atrophy in denervated fast and slow muscles of young and old rats. Hind limbs of 4-month-old and 24-month-old male rats were denervated, and soleus and tibialis anterior muscles were examined morphometrically 1 and 2 months after denervation. In all denervated muscles, type II muscle fibers underwent rapid atrophy, although muscle-specific differences in rate were observed. In both young and old denervated soleus muscles, the type I fibers underwent a pattern of atrophy closely paralleling that of the type II fibers, but in the tibialis anterior muscle, the mean cross-sectional area of the type I fibers actually increased during the first 2 months postdenervation. This study has shown that, among different muscles and between young and old rats, there is considerable variation in the response of the muscle fibers to denervation and that one cannot generalize from one muscle or one age to another.

We compared the reactions to denervation of limb muscles between young adult and old rats. After denervation for up to 4 months in 24-month-old rats, limb muscles were removed and analyzed for contractile properties, morphology, and levels of several key molecules, including the peptide elongation factors eEF1A-1 and eEF1A-2/S1, myogenin, gamma-subunit of the acetylcholine receptor, and cyclin D3. The principal difference between denervated old and young muscle is a somewhat slower rate of atrophy in denervated older muscle, especially among the type II fibers. Expression levels of certain molecules were higher in old than in young control muscle, but after denervation, levels of these molecules increased to the same absolute values in both young and old rats. Although many aspects of postdenervation reactions do not differ greatly between young and old animals, the lesser degree of atrophy in the old rats may reflect significant age-based mechanisms.

Denervation of skeletal muscle is followed by the progressive loss of tissue mass and impairment of its functional properties. The purpose of the present study was to investigate the occurrence of cell death and its mechanism in rat skeletal muscle undergoing post-denervation atrophy. We studied the expression of specific markers of apoptosis and necrosis in experimentally denervated tibialis anterior, extensor digitorum longus and soleus muscles of adult rats. Fluorescent staining of nuclear DNA with propidium iodide revealed the presence of nuclei with hypercondensed chromatin and fragmented nuclei typical of apoptotic cells in the muscle tissue 2, 4 and to a lesser extent 7 months after denervation. This finding was supported by electron microscopy of the denervated muscle. We found clear morphological manifestations of muscle cell death, with ultrastructural characteristics very similar if not identical to those considered as nuclear and cytoplasmic markers of apoptosis. With increasing time of denervation, progressive destabilization of the differentiated phenotype of muscle cells was observed. It included disalignment and spatial disorganization of myofibrils as well as their resorption and formation of myofibril-free zones. These changes initially appeared in subsarcolemmal areas around myonuclei, and by 4 months following nerve transection they were spread throughout the sarcoplasm. Despite an increased number of residual bodies and secondary lysosomes in denervated muscle, we did not find any evidence of involvement of autophagocytosis in the resorption of the contractile system. Dead muscle fibers were usually surrounded by a folded intact basal lamina; they had an intact sarcolemma and highly condensed chromatin and sarcoplasm. Folds of the basal lamina around the dead cells resulted from significant shrinkage of cell volume. Macrophages were occasionally found in close proximity to dead myocytes. We detected no manifestations of inflammation in the denervated tissue. Single myocytes expressing traits of the necrotic phenotype were very rare. A search for another marker of apoptosis, nuclear DNA fragmentation, using terminal deoxyribonucleotidyl transferase mediated dUTP nick end labeling (the TUNEL method) in situ, revealed the presence of multiple DNA fragments in cell nuclei in only a very small number of cell nuclei in 2 and 4 month denervated muscle and to less extent in 7 month denervated muscle. Virtually no TUNEL reactivity was found in normal muscle. Double labeling of tissue denervated for 2 and 4 months for genome fragmentation with the TUNEL method and for total nuclear DNA with propidium iodide demonstrated co-localization of the TUNEL-positive fragmented DNA in some of the nuclei containing condensed chromatin and in fragmented nuclei. However, the numbers of nuclei of abnormal morphology containing condensed and/or irregular patterns of chromatin distribution, as revealed by DNA staining and electron microscopy, exceeded by 33-38 times the numbers of nuclei positive for the TUNEL reaction. Thus, we found a discrepancy between the frequences of expression of morphological markers of apoptosis and DNA fragmentation in denervated muscle. This provides evidence that fragmentation of the genomic DNA is not an obligatory event during atrophy and death of muscle cells, or, alternatively, it may occur only for a short period of time during this process. Unlike classical apoptosis described in mammalian thymocytes and lymphoid cells, non-inflammatory death of muscle fibers in denervated muscle occurs a long time after the removal of myotrophic influence of the nerve and is preceded by the progressive imbalance of the state of terminal differentiation. Our results indicate that apoptosis appears to be represented by a number of distinct isotypes in animals belonging to different taxonomic groups and in different cell lineages of the same organism.

Very little is known regarding structural and functional responses of the vascular bed of skeletal muscle to denervation and about the role of microcirculatory changes in the pathogenesis of post-denervation muscle atrophy. The purpose of the present study was to investigate the changes of the anatomical pattern of vascularization of the extensor digitorum longus muscle in WI/HicksCar rats 1, 2, 4, 7, 12, and 18 months following denervation of the limb. We found that the number of capillaries related to the number of muscle fibers, i.e. the capillary-to-fiber ratio (CFR), decreased by 88%, from 1.55 +/- 0.35 to 0.19 +/- 0.04, during the first 7 months after denervation and then slightly declined at a much lower rate during the next 11 months of observation to 10% of the CFR in normal muscle. Between months 2 and 4 after denervation, the CRF decreased by 2.4 times, from 58% to 24% of the control value. The loss of capillaries during the first 4 months following nerve transection was nearly linear and progressed with an average decrement of 4.16% per week. Electron microscopy demonstrated progressive degeneration of capillaries following nerve transection. In muscle cells close to degenerating capillaries, the loss of subsarcolemmal and intermyofibrillar mitochondria, local disassembly of myofibrils and other manifestations of progressive atrophy were frequently observed. The levels of devascularization and the degree of degenerative changes varied greatly within different topographical areas, resulting in significant heterogeneity of intercapillary distances and local capillary densities within each sample of denervated muscle. Perivascular and interstitial fibrosis that rapidly developed after denervation resulted in the spatial separation of blood vessels from muscle cells and their embedment in a dense lattice of collagen. As a result of this process, diffusion distances between capillaries and the surfaces of muscle fibers increased 10-400 times. Eighteen months after denervation most of the capillaries were heavily cushioned with collagen, and on the average 40% of the muscle cells were completely avascular. Devascularization of the tissue was accompanied by degeneration and death of muscle cells that had become embedded in a dense lattice of collagen. Immunofluorescent staining for the vascular isoform of alpha-actin revealed preservation of major blood vessels and a greater variability in thickness of their medial layer. Hyperplastic growth of the medial layer in some blood vessels resulted in narrowing of their lumens. By the end of month 7 after denervation, large deposits of collagen around arterioles often exceeded their diameters. Identification of oxidative muscle fibers after immunostaining for slow-twitch myosin, as well as using ultrastructural criteria, has shown that after 2 months of denervation oxidative muscle fibers were less susceptible to atrophy than glycolytic fibers. The lower rate of atrophy of type I muscle fibers at early stages of denervation may be explained by their initially better vascularization in normal muscle and their higher capacity to retain capillaries shortly after denervation. Thus, degeneration and loss of capillaries after denervation occurs more rapidly than the loss of muscle fibers, which results in progressive decrease of the CFR in denervated muscle. The change of capillary number in denervated muscle is biphasic: the phase of a rapid decrease of the CFR during the first 7 months after nerve transection is followed by the phase of stabilization. The presence of areas completely devoid of capillaries in denervated muscle and the virtual absence of such areas in normal muscle indicate the development of foci of regional hypoxia during long-term denervation. The anatomical pattern of muscle microvascularization changes dramatically after nerve transection. Each muscle fiber in normal muscle directly contacts on average 3-5 capillaries. (ABSTRACT TRUNCATED)

Most of the available information regarding the regenerative potential and compensatory remodeling of mammalian tissues has been obtained from nonprimate animals, mainly rodent experimental models. The increasing use of transgenic mice for studies of the mechanisms controlling organogenesis and regeneration also requires a clear understanding of their applicability as experimental models for studies of similar processes in humans and other mammals. Application of modern cell biology methods to studies of regenerative processes has provided new insights into similarity and differences in cellular responses to injury in the tissues of different mammalian species. During more than 200-million years of progressive divergent evolution of mammals, cellular mechanisms of tissue regeneration and compensatory remodeling evolved together with increasingly adaptive functional specialization and structural complexity of mammalian tissues and organs. Rodents represent a phylogenetically ancient order of mammals that has conservatively retained a number of morphofunctional characteristics of early representatives of this class, which include enhanced regenerative capacity of tissues. A comparative analysis of regenerative processes in skeletal and cardiac muscle, as well as in several other mammalian tissues, shows that time courses and intensities of regeneration in response to the same type of injury vary even within taxonomically related species (e.g., rat, mouse, and hamster). The warm bloodedness of mammals facilitated the development of more complex mechanisms of metabolic, immune, and neurohumoral regulation, which resulted in a stronger dependence of regenerative processes on vascularization and innervation. For this reason, interspecies modifications of regenerative responses are limited by the capacity of the animal to resorb rapidly the foci of necrosis and to revascularize and reinnervate the volume of the regenerating tissue. These differences, among other factors, result in significantly lower rates of reparative regeneration in mammals possessing larger body sizes than rodents. A review of these data strongly indicates that the phylogenetic age and biological differences between different species should be taken into account before extrapolation of regenerative properties of nonprimate tissues on the regenerative responses in the primates.

The overall mortality of diabetic patients after myocardial infarction is 3-4 times higher than non-diabetics. The cellular mechanisms underlying such a poor clinical prognosis remain incompletely understood. Recent reports suggest that lipotoxicity associated with impaired liporegulation is among the leading factors in the pathogenesis of type 2 diabetes. The goal of this study was to investigate whether excess lipid accumulation specifically in heart muscle cells contributes to the expansion of myocardial infarction in type 2 diabetic patients. Comparative structural analysis of cardiac tissue was performed on autopsy samples from the infracted hearts of diabetic and non-diabetic individuals with special reference to the expansion of the infarction, degenerative changes, lipoatrophy, cell death, and replacement fibrosis. We found that progressive accumulation of lipids in cardiac myocytes was accompanied by considerable loss of myofibrils and was frequently observed in the heart tissue of type 2 diabetic patients. This indicates that disassembly of the contractile apparatus in the cells infiltrated with lipids weakens their capability for functional activity. Analysis of degenerative changes in the diabetic tissue has shown that lipid-laden cardiac myocytes were more susceptible to necrotic and apoptotic cells death leading to expansion of the infarction and the development of progressive focal replacement fibrosis both in the perinecrotic zone and in the areas located far from the site of injury. Our data show that lipoatrophy and loss of muscle cells during the post-infarction period aggravate the functional impairment in the diabetic heart and limits its adaptive capacity for compensatory remodeling. This suggests that lipotoxic myocardial injury associated with defects of lipid metabolism in type 2 diabetes predisposes its evolution toward congestive heart failure and is an important factor contributing to a high mortality following infarction.

Obscurin (approximately 800 kDa) in striated muscle closely surrounds sarcomeres at the level of the M-band and Z-disk where, we hypothesize, it participates in the assembly of the contractile apparatus and membrane systems required for Ca2+ homeostasis. In this study, we used small inhibitory RNA (siRNA) technology to reduce the levels of obscurin in primary cultures of skeletal myotubes to study its role in myofibrillogenesis and the organization of the sarcoplasmic reticulum (SR). siRNA-treated myotubes showed a specific and dramatic reduction in the approximately 800 kDa form of obscurin by reverse transcription-polymerase chain reaction, immunoblotting, and immunofluorescence. M-bands and A-bands, but not Z-disks or I-bands, were disrupted when the synthesis of obscurin was inhibited. Small ankyrin 1, an integral protein of the network SR that binds to obscurin, also failed to align around developing sarcomeres in treated myotubes. Myosin and myomesin levels were significantly reduced in treated myotubes but alpha-actinin was not, suggesting that down-regulation of obscurin destabilizes proteins of the M-band and A-band but not of the Z-disk. Our findings suggest that obscurin is required for the assembly of the M-band and A-band and for the regular alignment of the network SR around the contractile apparatus.

Obscurin/obscurin-MLCK is a giant sarcomere-associated protein with multiple isoforms whose interactions with titin and small ankyrin-1 suggest that it has an important role in myofibril assembly, structural support, and the sarcomeric alignment of the sarcoplasmic reticulum. In this study, we characterized the zebrafish orthologue of obscurin and examined its role in striated myofibril assembly. Zebrafish obscurin was expressed in the somites and central nervous system by 24 hours post-fertilization (hpf) and in the heart by 48 hpf. Depletion of obscurin using two independent morpholino antisense oligonucleotides resulted in diminished numbers and marked disarray of skeletal myofibrils, impaired lateral alignment of adjacent myofibrils, disorganization of the sarcoplasmic reticulum, somite segmentation defects, and abnormalities of cardiac structure and function. This is the first demonstration that obscurin is required for vertebrate cardiac and skeletal muscle development. The diminished capacity to generate and organize new myofibrils in response to obscurin depletion suggests that it may have a vital role in the causation of or adaptation to cardiac and skeletal myopathies.

Leptofibrils, or leptomeres, remain the least studied cytoskeletal structures in muscle cells, and their function and mechanism of assembly are still poorly understood. Our ultrastructural study of the surviving cardiac myocytes located in the perinecrotic border zone of the infarcted left ventricle in rats revealed intense formation of leptofibrils and leptofibrillar clusters during 4-15 days following experimental myocardial infarction. In the perinecrotic myocytes, leptofibrils developed predominantly in the subsarcolemmal areas, near disassembled intercalated discs and at the sites of intense myofibrillogenesis in the peripheral zones of the sarcoplasm. We found that the development of these structures occurred before or at the time of assembly of myofibrils. In our material, leptofibrils consisted of longitudinally oriented filamentous bundles inserted in electron dense Z-band-like material and periodically crossed by 3-8 bands of this material with the period of cross-striation of 120-210 nm. The presence of leptofibrils in growing cytoplasmic processes and ruffles developing in the border zone in the areas of lost intercellular contacts indicates their formation de novo during post-infarction period. We observed four major morphological types of localization of these structures: (1) direct contact of one end of leptofibrils with Z bands of nascent, mature or disassembling myofibrils; (2) direct contact with the sarcolemma: (a) multifocal attachment of leptofibrils to the sarcolemma through the lateral surfaces of their minute Z band-like structures; (b) attachment of one or both ends of leptofibrils to the sarcolemma without contacts or in contact with myofibrils; (3) attachment of leptofibrils to subsarcolemmal accumulations of electron dense Z-band material in newly formed fasciae adherentes of the remodeled intercalated disks; (4) clustering and contacts of leptofibrils with one another predominantly at the level of their Z bands. Interestingly, most leptofibrils of all four types were topographically associated with the system of T-tubules, the sarcoplasmic reticulum and subsarcolemmal vesicles. Serial sections through the areas containing leptofibrils indicate their spindle-like or nearly cylindrical shape. Thus, we found that leptofibrils assemble in terminally differentiated cardiac myocytes following destabilization of their differentiated state and partial dedifferentiation induced by myocardial infarction. The results of this study demonstrate that formation of leptofibrils, earlier described mainly in the developing and malignant muscle, is temporally associated with adaptive structural remodelling and the activation of myofibrillogenesis in functionally overloaded cardiac myocytes of adult animals. Our findings suggest that re-expression of some structural characteristics of the embryonic muscle appear to represent one of the mechanisms that underlie adaptive plasticity of the myocardium following injury and under conditions of hyperfunction.

Satellite cells represent a cellular source of regeneration in adult skeletal muscle. It remains unclear why a large pool of stem myoblasts in denervated muscle does not compensate for the loss of muscle mass during post-denervation atrophy. In this study, we present evidence that satellite cells in long-term denervated rat muscle are able to activate synthesis of contractile proteins after single fusions in situ. This process of early differentiation leads to formation of abnormally diminutive myotubes. The localization of such dwarf myotubes beneath the intact basal lamina on the surface of differentiated muscle fibers shows that they form by fusion of neighboring satellites or by the progeny of a single satellite cell following one or two mitotic divisions. We demonstrated single fusions of myoblasts using electron microscopy, immunocytochemical labeling and high resolution confocal digital imaging. Sequestration of nascent myotubes by the rapidly forming basal laminae creates a barrier that limits further fusions. The recruitment of satellite cells in the formation of new muscle fibers results in a progressive decrease in their local densities, spatial separation and ultimate exhaustion of the myogenic cell pool. To determine whether the accumulation of aberrant dwarf myotubes is explained by the intrinsic decline of myogenic properties of satellite cells, or depends on their spatial separation and the environment in the tissue, we studied the fusion of myoblasts isolated from normal and denervated muscle in cell culture. The experiments with a culture system demonstrated that the capacity of myoblasts to synthesize contractile proteins without serial fusions depended on cell density and the availability of partners for fusion. Satellite cells isolated from denervated muscle and plated at fusion-permissive densities progressed through the myogenic program and actively formed myotubes, which shows that their myogenic potential is not considerably impaired. The results of this study suggest that under conditions of denervation, progressive spatial separation and confinement of many satellite cells within the endomysial tubes of atrophic muscle fibers and progressive interstitial fibrosis are the important factors that prevent their normal differentiation. Our findings also provide an explanation of why denervated muscle partially and temporarily is able to restore its functional capacity following injury and regeneration: the release of satellite cells from their sublaminal location provides the necessary space for a more active regenerative process.

Myosin light chain kinases (MLCK) are a family of signaling proteins that are required for cytoskeletal remodeling in myocytes. Recently, two novel MLCK proteins, SPEG and obscurin-MLCK, were identified with the unique feature of two tandemly-arranged MLCK domains. In this study, the evolutionary origins of this MLCK subfamily were traced to a probable orthologue of obscurin-MLCK in Drosophila melanogaster, Drosophila Unc-89, and the MLCK kinase domains of zebrafish SPEG, zebrafish obscurin-MLCK, and human SPEG were characterized. Phylogenetic analysis of the MLCK domains indicates that the carboxy terminal kinase domains of obscurin-MLCK, SPEG and Unc-89 are more closely related to each other than to the amino terminal kinase domains or to other MLCKs, supporting the assertion that obscurin-MLCK is the vertebrate orthologue of Caenorhabditis elegans Unc-89, a giant multidomain protein that is required for normal myofibril assembly. The apparent lack of an invertebrate orthologue of SPEG and the conserved exon structure of the kinase domains between SPEG and obscurin-MLCK suggests that SPEG arose from obscurin-MLCK by a gene duplication event. The length of the primary amino acid sequence between the immunoglobulin (Ig) domains associated with the MLCK motifs is conserved in obscurin-MLCK, SPEG and C. elegans Unc-89, suggesting that these putative protein interaction domains may target the kinases to highly conserved intracellular sites. The conserved arrangement of the tandem MLCK domains and their relatively restricted expression in striated muscle indicates that further characterization of this novel MLCK subfamily may yield important insights into cardiac and skeletal muscle physiology.

Obscurin and obscurin myosin light chain kinase (MLCK) are two recently identified muscle proteins encoded by the same gene cluster. The production of obscurin, which contains a Rho-guanine exchange factor (GEF)-like sequence, and obscurin-MLCK by this cluster suggests that these novel genes may be involved in signal transduction cascades that control adaptive and compensatory responses of the heart. The goal of the present study was to investigate the transcriptional response of the obscurin gene cluster to the initiation of myocardial hypertrophy induced in mice by aortic constriction. The transcriptional activity of the obscurin genes was examined using reverse-transcriptase primed quantitative PCR. We found that the transcripts encoding the obscurin Rho-GEF and the obscurin-MLCK internal serine–threonine kinase II (SK II) domains were significantly upregulated following aortic constriction. The expression of Rho-GEF-containing transcripts at different stages of the hypertrophic growth exceeded the control levels by 2- to 6-fold. Following the induction of hypertrophy, the quantity of the SK II-encoding transcripts increased 10-fold by 24 h and 16-fold by 48 h, then decreased by day 7, and returned to the control level by day 56. The quantity of the carboxy terminal obscurin-MLCK transcripts encoding for SK I increased 2-fold by day 2 and returned to the control values at later stages. Immunolocalization of obscurin, which contains Rho-GEF domain, in cardiomyocytes during pharmacologically induced hypertrophic growth in vitro demonstrated that the expression was topographically associated with the growing myofibrils and with the sites of initiation and progression of myofibrillogenesis at the periphery of the sarcoplasm. This suggests that upregulation of obscurin synthesis is associated with the formation of additional amounts of contractile structures during cardiac hypertrophy. Thus, the obscurin gene cluster represents a new example of an operon that encodes differentially regulated structural and signaling proteins implicated in the control of assembly and adaptive remodeling of myofibrils during normal and hypertrophic growth.

Satellite cells (SCs) are the main source of new fibers in regenerating skeletal muscles and the key contributor to extra nuclei in growing fibers during postnatal development. Aging results in depletion of the SC population and in the reduction of its proliferative activity. Although it has been previously determined that under conditions of massive fiber death in vivo the regenerative potential of SCs is not impaired in old muscle, no studies have yet tested whether advanced age is a factor that may restrain the response of SCs to muscle denervation. The present study is designed to answer this question, comparing the changes of SC numbers in tibialis anterior (TA) muscles from young (4 months) and old (24 months) WI/HicksCar rats after 2 months of denervation. Immunostaining with antibodies against M-cadherin and NCAM was used to detect and count the SCs. The results demonstrate that the percentages of both M-cadherin- and NCAM-positive SCs (SC/Fibers × 100) in control TA muscles from young rats (5.6 ± 0.5% and 1.4 ± 0.2%, respectively) are larger than those in old rats (2.3 ± 0.3% and 0.5 ± 0.1%, respectively). At the same time, in 2-month denervated TA muscles the percentages of M-cadherin and NCAM positive SC are increased and reach a level that is comparable between young (16.2 ± 0.9% and 7.5 ± 0.5%, respectively) and old (15.9 ± 0.7% and 10.1 ± 0.5%, respectively) rats. Based on these data, we suggest that aging does not repress the capacity of SC to become activated and grow in the response to muscle denervation.

We analyzed the level of protein expression of two myogenic regulatory factors (MRFs), MyoD and myogenin, in senile skeletal muscles and determined the cellular source of their production in young adult (4 months old), old (24, 26, and 28 months old), and senile (32 months old) male rats. Immunoblotting demonstrated levels of myogenin approximately 3.2, approximately 4.0, and approximately 5.5 times higher in gastrocnemius muscles of 24-, 26-, and 32-month-old animals, respectively, than in those of young adult rats. Anti-MyoD antibody recognized two major areas of immunoreactivity in Western blots: a single MyoD-specific band (approximately 43-45 kDa) and a double (or triple) MyoD-like band (approximately 55-65 kDa). Whereas the level of MyoD-specific protein in the 43- to 45-kDa band remained relatively unchanged during aging compared with that of young adult rats, the total level of MyoD-like immunoreactivity within the 55- to 65-kDa bands was approximately 3.4, approximately 4.7, approximately 9.1, and approximately 11.7 times higher in muscles of 24-, 26-, 28-, and 32-month-old rats, respectively. The pattern of MRF protein expression in intact senile muscles was similar to that recorded in young adult denervated muscles. Ultrastructural analysis of extensor digitorum longus muscle from senile rats showed that, occasionally, the area of the nerve-muscle junction was partially or completely devoid of axons, and satellite cells with the features of activated cells were found on the surface of living fibers. Immunohistochemistry detected accumulated MyoD and myogenin proteins in the nuclei of both fibers and satellite cells in 32-month-old muscles. We suggest that the up-regulated production of MyoD and myogenin proteins in the nuclei of both fibers and satellite cells could account for the high level of MRF expression in muscles of senile rats.

In denervated skeletal muscles, atrophy of muscle fibers and interstitial fibrosis are associated with alterations within the vascular bed. Our study has placed particular emphasis on changes occurring in resistance vessels and the microcirculatory bed of rat hindlimb skeletal muscles that had been denervated for 25 months. We found that the tunica media of the majority of long-term denervated resistance vessels undergoes deterioration. In small intramuscular arteries and arterioles, atrophic vascular smooth muscle cells (vSMCs) enclosed in a thick basal lamina are separated by expanded extracellular space. The remodeling and sclerotic changes in the arterial wall occasionally result in deformation of the lumen. It was also found that the microcirculatory bed undergoes significant alterations. In 25-month denervated extensor digitorum longus muscle, the capillary-to-fiber ratio is only 0.13 +/- 0.01 and the mean number of capillaries per fascicle decreases almost ninefold compared to contralateral control muscle. Ultrastructural findings demonstrate that 24.67 +/- 0.48% of capillaries examined in the chronically denervated fascicles show structural features typical for capillary regeneration. In addition, long cytoplasmic extensions of pericytes might develop a layer completely encircling the capillary endothelium. In pre- and postcapillary segments of the microcirculatory bed, some perivascular cells possess a phenotype that is intermediate between that of pericytes and atrophic vSMCs. RT-PCR and/or Western blot analyses showed that molecules participating in angiogenesis are detected in 25-month denervated skeletal muscle. We hypothesize that despite the fact that the microcirculatory bed of chronically denervated muscle undergoes significant reduction it still sustains the capacity for reparative capillary growth.

Little is known concerning the time-course and structural dynamics of reactivation of compensatory myogenesis in denervated muscle, its initiating cellular mechanisms, and the relationship between this process and the progression of postdenervation atrophy. The purpose of this study was to investigate the interrelations between temporal and spatial patterns of the myogenic response in denervated muscle and progressive atrophy of muscle fibers. Another objective was to study whether reactivation of myogenesis correlates with destabilization of the differentiated state and death of denervated muscle cells. It has remained unclear whether muscle fiber atrophy was the primary factor activating the myogenic response, what levels of cellular atrophy were associated with its activation, and whether the initiation and intensity of myogenesis depended on the local and individual heterogeneity of atrophic changes among fibers. For this reason, our objective was also to identify the levels of atrophic and degenerative changes in denervated muscle fibers that are correlated with activation of the myogenic response. We found that the reactivation of myogenesis in the tibialis anterior and extensor digitorum longus muscles of the rat starts between days 10-21 following nerve transection, before atrophy has attained advanced level, long before dead cells are found in the tissue. Formation of new muscle fibers reaches its maximum between 2 and 4 months following denervation and gradually decreases with progressive postdenervation atrophy. The myogenic response is biphasic and includes two distinct processes. The first process resembles the formation of secondary and tertiary generations of myotubes during normal muscle development and dominates during the first 2 months of denervation. During this period, activated satellite cells form new myotubes on live differentiated muscle fibers. Most of the daughter myotubes in 1- and 2-month denervated muscle develop on the surface of fast type parent muscle fibers, and some of the newly formed muscle fibers express slow myosin. Some fast type parent fibers are weakly or, more rarely, moderately immunopositive for embryonic isomyosin. This indicates that reactivation of myogenesis may also depend on the fiber type. The level of atrophy, destabilization of the differentiated myofiber phenotype, and degenerative changes of individual fibers in denervated muscle are very heterogeneous. The myogenic response of the first type is associated predominantly with fibers of average and higher than average levels of atrophy. Muscle cells that undergo a lesser degree of atrophy also form daughter fibers, although with a lower incidence. We did not find any correlation between the size of newly formed fibers and the level of atrophy of parent fibers. The topographical distribution of new myotubes both in the peripheral and central areas of the mid-belly equatorial sections at the early stages following nerve transection indicates that myogenesis of the first type represents a systemic reaction of muscle to the loss of neural control. These data indicate that activation of the myogenic response does not depend on cell death and degenerative processes per se. The second type of myogenesis is a typical regenerative reaction that occurs mainly within the spaces surrounded by the basal laminae of dead muscle fibers. Myocytes of different sizes are susceptible to degeneration and death, which indicates that cell death in denervated muscle does not correlate with levels of muscle cell atrophy. The regenerative process frequently results in development of abnormal muscle cells that branch or form small clusters. Replacement of lost fibers becomes activated between 2 and 4 months following nerve transection, i.e., mainly at advanced stages of postdenervation atrophy, when cell death becomes a contributing factor of the atrophic process. In long-term denervated muscle, the first and second types of myogenesisoccur concurrently, and the topographical distribution of the myogenic response becomes more heterogeneous than during the first weeks following denervation. Thus, our data demonstrate differential temporal and spatial expression of two patterns of myogenesis in denervated muscle that appear to be controlled by different regulatory mechanisms during the postdenervation period. (c) 2001 Wiley-Liss, Inc.

It is well established that over time Schwann cells disappear from the endoneurial space of the distal stump of a chronically transected sciatic nerve trunk. Nevertheless, the status of the Schwann cells within terminal branches of the transected sciatic nerve remains poorly understood. To elucidate this issue we examined the endoneurial space of the intramuscular nerves in rat hindlimb skeletal muscles, which had been denervated for a 25-month period. Based on specific ultrastructural characteristics, we identified a small population of viable Schwann cells within the intramuscular nerve trunks. The surviving Schwann cells continued to be immunopositive for both S-100 protein and neural cell adhesion molecule. In addition, reverse transcription-polymerase chain reaction and/or Western blot analyses have shown that at least two molecules, brain-derived neurotrophic factor and a non-catalytic truncated form of tyrosine protein kinase receptor B, which could potentially participate in the process of nerve repair, were detectable in chronically denervated skeletal muscle. Our results demonstrate that Schwann cells can survive inside the intramuscular nerve trunks of denervated skeletal muscle for a 25-month period without axonal contact.

This study, conducted on 25-month denervated rat hindlimb muscles, was directed toward elucidating the basis for the poor regeneration that is observed in long-term denervated muscles. Despite a approximately 97.6% loss in mean cross-sectional area of muscle fibers, the muscles retained their fascicular arrangement, with the fascicles containing approximately 1.5 times more fibers than age-matched control muscles. At least three distinct types of muscle fibers were observed: degenerating, persisting (original), and newly formed (regenerated) fibers. A majority of newly formed fibers did not appear to undergo complete maturation, and morphologically they resembled myotubes. Sites of former motor end-plates remained identifiable in persisting muscle fibers. Nuclear death was seen in all types of muscle fibers, especially in degenerating fibers. Nevertheless, the severely atrophic skeletal muscles continued to express developmentally and functionally important proteins, such as MyoD, myogenin, adult and embryonic subunits of the nicotinic acetylcholine receptor, and neural-cell adhesion molecule. Despite the prolonged period of denervation, slow and fast types of myosin were found in surviving muscle fibers. The number of satellite cells was significantly reduced in long-term denervated muscles, as compared with age-matched control muscles. In 25-month denervated muscle, satellite cells were only attached to persisting muscle fibers, but were never seen on newly formed fibers. Our data suggest that the absence of satellite cells in a population of immature newly formed muscle fibers that has arisen as a result of continuous reparative myogenesis may be a crucial, although not necessarily the only, factor underlying the poor regenerative ability of long-term denervated muscle.

This study was undertaken to assess the regenerative capacity of skeletal muscle in rats near the end of their normal life span. Two experiments were performed. In the first, extensor digitorum longus (EDL) muscles were cross-age transplanted from 32-month-old male inbred Wistar (WI/HicksCar) rats in place of an EDL muscle in 4-month-old hosts. The other EDL muscle in the hosts was autotransplanted. After 60 days, the old-into-young muscle transplants regenerated as well as the young-into-young autotransplants. In the second experiment, EDL muscles in young adult (4 months) and old rats (32 and 34 months) of WI/HicksCar and Brown Norway (BN) were injected with a local anesthetic, bupivacaine, and allowed to regenerate for 41 days. In all cases, the masses and absolute maximum tetanic force of the regenerates equaled or exceeded those of untouched contralateral control muscles. These experiments showed that under appropriate conditions, very old muscles can regenerate to equal or exceed the contralateral control values, which in old rats are much less than those in muscles of young rats.

Cytoskeletal adaptor proteins serve vital functions in linking the internal cytoskeleton of cells to the cell membrane, particularly at sites of cell–cell and cell–matrix interactions. The importance of these adaptors to the structural integrity of the cell is evident from the number of clinical disease states attributable to defects in these networks. In the heart, defects in the cytoskeletal support system that surrounds and supports the myofibril result in dilated cardiomyopathy and congestive heart failure. In this study, we report the cloning and characterization of a novel cytoskeletal adaptor, obscurin-like 1 (OBSL1), which is closely related to obscurin, a giant structural protein required for sarcomere assembly. Multiple isoforms arise from alternative splicing, ranging in predicted molecular mass from 130 to 230 kDa. OBSL1 is located on human chromosome 2q35 within 100 kb of SPEG, another gene related to obscurin. It is expressed in a broad range of tissues and localizes to the intercalated discs, to the perinuclear region, and overlying the Z lines and M bands of adult rat cardiac myocytes. Further characterization of this novel cytoskeletal linker will have important implications for understanding the physical interactions that stabilize and support cell–matrix, cell–cell, and intracellular cytoskeletal connections.