Broad spectrum of anomalies including quadricuspid aortic valve associated with a novel frameshift SALL4 variant.

Each family member had a SALL4 variant. This is the first report of quadricuspid aortic valve and a genetic variant. The variation in phenotype caused by SALL4 mutations questions the division of SALL4-related phenotypes in three different entities.

Expanding genotypic and phenotypic spectrums of LTBP3 variants in dental anomalies and short stature syndrome.

Mutations in LTBP3 are associated with Dental Anomalies and Short Stature syndrome (DASS; MIM 601216), which is characterized by hypoplastic type amelogenesis imperfecta, hypodontia, underdeveloped maxilla, short stature, brachyolmia, aneurysm and dissection of the thoracic aorta. Here we report a novel (p.Arg545ProfsTer22) and a recurrent (c.3107-2A > G) LTBP3 variants, in a Turkish family affected with DASS. The proband, who carried compound heterozygous variant c.3107-2A > G, p.Arg545ProfsTer22, was most severely affected with DASS. The proband's father, who carried the heterozygous variant c.3107-2A > G had short stature and prognathic mandible. The mother and brother of the proband carried the heterozygous variant p.Arg545ProfsTer22, but only the mother showed any DASS characteristics. The c.3107-2A > G and the p.Arg545ProfsTer22 variants are expected to result in abnormal LTPB3 protein, failure of TGFß-LAP-LTBP3 complex formation, and subsequent disruption of TGFß secretion and activation. This is the first report of heterozygous carriers of LTBP3 variants showing phenotypes. The new findings of DASS found in this family include taurodontism, single-rooted molars, abnormal dentin, calcified dental pulp blood vessels, prognathic mandible, failure of mandibular tooth eruption, interatrial septal aneurysm, secundum atrial septal defect, tricuspid valve prolapse, and a recurrent glenohumeral joint dislocation.

Treacher Collins syndrome: A novel TCOF1 mutation and monopodial stapes.

Treacher Collins syndrome (TCS: OMIM 154500) is an autosomal dominant craniofacial disorder belonging to the heterogeneous group of mandibulofacial dysostoses. OBJECTIVE: To investigate four Treacher Collins syndrome patients of the Sgaw Karen family living in Thailand. METHOD: Clinical examination, hearing tests, lateral cephalometric analyses, Computed tomography, whole exome sequencing and Sanger direct sequencing were performed. RESULTS: All of the patients affected with Treacher Collins syndrome carried a novel TCOF1 mutation (c.4138_4142del; p.Lys1380GlufsTer12), but clinically they did not have the typical facial gestalt of Treacher Collins syndrome, which includes downward-slanting palpebral fissures, colobomas of the lower eyelids, absence of eyelashes medial to the colobomas, malformed pinnae, hypoplastic zygomatic bones and mandibular hypoplasia. Lateral cephalometric analyses identified short anterior and posterior cranial bases, and hypoplastic maxilla and mandible. Computed tomography showed fusion of malleus and incus, sclerotic mastoid, hypoplastic middle ear space with a soft tissue remnant, dehiscence of facial nerve and monopodial stapes. CONCLUSION: Treacher Collins syndrome in Sgaw Karen patients has not been previously documented. This is the first report of monopodial stapes in a TCS patient who had a TCOF1 mutation. The absence of a common facial phenotype and/or the presence of monopodial stapes may be the effects of this novel TCOF1 mutation.

WNT1-associated osteogenesis imperfecta with atrophic frontal lobes and arachnoid cysts.

A rare form of osteogenesis imperfecta (OI) caused by Wingless-type MMTV integration site family 1 (WNT1) mutations combines central nervous system (CNS) anomalies with the characteristic increased susceptibility to fractures. We report an additional case where arachnoid cysts extend the phenotype, and that also confirms the association of intellectual disabilities with asymmetric cerebellar hypoplasia here. Interestingly, if the cerebellum is normal in this disorder, intelligence is as well, analogous to an association with similar delays in a subset of patients with sporadic unilateral cerebellar hypoplasia. Those cases typically appear to represent vascular disruptions, and we suggest that most brain anomalies in WNT1-associated OI have vascular origins related to a role for WNT1 in CNS angiogenesis. This unusual combination of benign cerebellar findings with effects on higher functions in these two situations raises the possibility that WNT1 is involved in the pathogenesis of the associated sporadic cases as well. Finally, our patient reacted poorly to pamidronate, which appears ineffective with this form of OI, so that a lack of improvement is an indication for molecular testing that includes WNT1.

Genetic regulatory pathways of split-hand/foot malformation.

Split-hand/foot malformation (SHFM) is caused by mutations in TP63, DLX5, DLX6, FGF8, FGFR1, WNT10B, and BHLHA9. The clinical features of SHFM caused by mutations of these genes are not distinguishable. This implies that in normal situations these SHFM-associated genes share an underlying regulatory pathway that is involved in the development of the central parts of the hands and feet. The mutations in SHFM-related genes lead to dysregulation of Fgf8 in the central portion of the apical ectodermal ridge (AER) and subsequently lead to misexpression of a number of downstream target genes, failure of stratification of the AER, and thus SHFM. Syndactyly of the remaining digits is most likely the effects of dysregulation of Fgf-Bmp-Msx signaling on apoptotic cell death. Loss of digit identity in SHFM is hypothesized to be the effects of misexpression of HOX genes, abnormal SHH gradient, or the loss of balance between GLI3A and GLI3R. Disruption of canonical and non-canonical Wnt signaling is involved in the pathogenesis of SHFM. Whatever the causative genes of SHFM are, the mutations seem to lead to dysregulation of Fgf8 in AER cells of the central parts of the hands and feet and disruption of Wnt-Bmp-Fgf signaling pathways in AER.

The Biology of Long-Term Denervated Skeletal Muscle.

This review concentrates on the biology of long-term denervated muscle, especially as it relates to newer techniques for restoring functional mass. After denervation, muscle passes through three stages: 1) immediate loss of voluntary function and rapid loss of mass, 2) increasing atrophy and loss of sarcomeric organization, and 3) muscle fiber degeneration and replacement of muscle by fibrous connective tissue and fat. Parallel to the overall program of atrophy and degeneration is the proliferation and activation of satellite cells, and the appearance of neomyogenesis within the denervated muscle. Techniques such as functional electrical stimulation take advantage of this capability to restore functional mass to a denervated muscle.

Effects of high- and low-velocity resistance training on the contractile properties of skeletal muscle fibers from young and older humans.

A two-arm, prospective, randomized, controlled trial study was conducted to investigate the effects of movement velocity during progressive resistance training (PRT) on the size and contractile properties of individual fibers from human vastus lateralis muscles. The effects of age and sex were examined by a design that included 63 subjects organized into four groups: young (20-30 yr) men and women, and older (65-80 yr) men and women. In each group, one-half of the subjects underwent a traditional PRT protocol that involved shortening contractions at low velocities against high loads, while the other half performed a modified PRT protocol that involved contractions at 3.5 times higher velocity against reduced loads. Muscles were sampled by needle biopsy before and after the 14-wk PRT program, and functional tests were performed on permeabilized individual fiber segments isolated from the biopsies. We tested the hypothesis that, compared with low-velocity PRT, high-velocity PRT results in a greater increase in the cross-sectional area, force, and power of type 2 fibers. Both types of PRT increased the cross-sectional area, force, and power of type 2 fibers by 8-12%, independent of the sex or age of the subject. Contrary to our hypothesis, the velocity at which the PRT was performed did not affect the fiber-level outcomes substantially. We conclude that, compared with low-velocity PRT, resistance training performed at velocities up to 3.5 times higher against reduced loads is equally effective for eliciting an adaptive response in type 2 fibers from human skeletal muscle.

The Denervated Muscle: 45 years later.

Forty-five years after its publication, Ernest Gutmann's book, The Denervated Muscle, still stands as a landmark publication. It summarized the state of knowledge of the time and introduced many new researches that were continuing at the Institute of Physiology in Prague. At the time, the response of a muscle to denervation was viewed primarily through the lens of the neurotrophic theory. Advancements in our understanding of neurotrophic effects and mechanisms would now call into question some of the hypotheses and interpretations presented in the book, but many of the research findings have stood the test of time. This review will cover some of the questions asked and data presented in this book, and will place them into the context of contemporary muscle biology.

Electrical stimulation of denervated muscles of rats maintains mass and force, but not recovery following grafting.

PURPOSE: Denervated skeletal muscles lack contractile activity and subsequently lose mass and force generation. Prolonged periods of denervation prior to nerve-implant grafting limit the recovery of mass and force. We hypothesized that electrical stimulation during a period of denervation that maintains mass and force above the levels of denervated muscles enhances the recovery of mass and force following nerve-implant grafting. METHODS: The extensor digitorum longus (EDL) muscles of anesthetized rats were denervated, and a stimulator was implanted. Following 4 or 7 months of denervation, with or without electrical stimulation, the EDL muscles were removed, evaluated in vitro for mass and contractile properties, and then nerve-implant grafted into syngeneic rats. Unoperated, contralateral muscles were also evaluated and grafted. RESULTS: The hypothesis was not supported by the experimental data. Compared with values for 4- or 7-month denervated muscles, the stimulated-denervated muscles maintained higher mass and force, less prolonged time-to-peak tensions and half-relaxation times, and higher excitability. Nevertheless, the recovery of mass and force following grafting was not improved. CONCLUSION: The factors within long-term denervated muscles that hinder recovery following grafting appear to be related primarily to factors associated with the duration of denervation and not to the level of atrophy and weakness prior to grafting.

Some principles of regeneration in mammalian systems.

This article presents some general principles underlying regenerative phenomena in vertebrates, starting with the epimorphic regeneration of the amphibian limb and continuing with tissue and organ regeneration in mammals. Epimorphic regeneration following limb amputation involves wound healing, followed shortly by a phase of dedifferentiation that leads to the formation of a regeneration blastema. Up to the point of blastema formation, dedifferentiation is guided by unique regenerative pathways, but the overall developmental controls underlying limb formation from the blastema generally recapitulate those of embryonic limb development. Damaged mammalian tissues do not form a blastema. At the cellular level, differentiation follows a pattern close to that seen in the embryo, but at the level of the tissue and organ, regeneration is strongly influenced by conditions inherent in the local environment. In some mammalian systems, such as the liver, parenchymal cells contribute progeny to the regenerate. In others, e.g., skeletal muscle and bone, tissue-specific progenitor cells constitute the main source of regenerating cells. The substrate on which regeneration occurs plays a very important role in determining the course of regeneration. Epimorphic regeneration usually produces an exact replica of the structure that was lost, but in mammalian tissue regeneration the form of the regenerate is largely determined by the mechanical environment acting on the regenerating tissue, and it is normally an imperfect replica of the original. In organ hypertophy, such as that occurring after hepatic resection, the remaining liver mass enlarges, but there is no attempt to restore the original form.

Differentiation of activated satellite cells in denervated muscle following single fusions in situ and in cell culture.

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.

Abortive myogenesis in denervated skeletal muscle: differentiative properties of satellite cells, their migration, and block of terminal differentiation.

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.

Dynamics of postdenervation atrophy of young and old skeletal muscles: differential responses of fiber types and muscle types.

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.

Concentration of caveolin-3 at the neuromuscular junction in young and old rat skeletal muscle fibers.

Caveolin-3, a muscle-specific member of the caveolin family, is strongly localized to the neuromuscular junction (NMJ) in adult rat muscle fibers, where it co-localizes with alpha-bungarotoxin staining. In 24-month-old rats, less distinct staining corresponds with the normal aging changes in the NMJ. After denervation, the pattern and intensity of staining begin to break up as early as 3 days, and by 10 days little staining remains. The functional implications of this concentration of caveolin-3 at the NMJ remain obscure, but it is possible that its absence could account for some of the phenotypic characteristics of individuals with caveolin-3 mutations.

Aging of skeletal muscle does not affect the response of satellite cells to denervation.

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 x 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.

Changes in protein levels of elongation factors, eEF1A-1 and eEF1A-2/S1, in long-term denervated rat muscle.

PURPOSE: This study was designed to determine whether the quantitative relationship between the levels of the eEF1A-1(developmental) and eEF1A-2/S1 (adult) isoforms of peptide elongation factor remains stable after denervation of skeletal muscle or whether in response to denervation the relative amount of the developmental form would increase. In normal postnatal rat muscle, eEF1A-2/S1 is the dominant form represented, and levels of eEF1A-1 are extremely low. METHODS: One hind limb in young adult rats was permanently denervated. Denervated and corresponding contralateral control muscles were removed for biochemical and morphological analysis from 2 days to 25 months after denervation. RESULTS: By one month after denervation, relative levels of eEF1A-1 rose dramatically in relation to those of eEF1A-2/S1, and they remained high throughout the remainder of the 25-month denervation period. Ultrastructural analysis showed a complex mix of muscle fiber atrophy, dying muscle nuclei and muscle fibers, and newly forming muscle fibers in the same tissue. CONCLUSIONS: As during muscle regeneration, levels of the developmental eEF1A-1 isoform of peptide elongation factor greatly increased relative to those of the adult eEF1A-2/S1 adult isoform following denervation in rat muscles. However, in contrast to regeneration, the elevated level of eEF1A-1 did not return to the basal minimal level. Since switching from eEF1A-1 to eEF1A-2/S1 is an indicator that terminal differentiated is completed, the failure of eEF1A-1 to return to basal level may be indicative of the persistence of an unstable tissue environment that includes muscle fiber atrophy, degeneration and neomyogenesis. The specific cellular basis for the increase in eEF1A-1 could not be determined from this study.

MyoD and myogenin protein expression in skeletal muscles of senile rats.

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.

Muscle regeneration in amphibians and mammals: passing the torch.

Skeletal muscle in both amphibians and mammals possesses a high regenerative capacity. In amphibians, a muscle can regenerate in two distinct ways: as a tissue component of an entire regenerating limb (epimorphic regeneration) or as an isolated entity (tissue regeneration). In the absence of epimorphic regenerative ability, mammals can regenerate muscles only by the tissue mode. This review focuses principally on the regeneration of entire muscles and covers what is known and what remains to be elucidated about fundamental mechanisms underlying muscle regeneration at this level.

Effects of long-term denervation on skeletal muscle in old rats.

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.