Knockdown of desmin in zebrafish larvae affects interfilament spacing and mechanical properties of skeletal muscle

Skeletal muscle was examined in zebrafish larvae in order to address questions related to the function of the intermediate filament protein desmin and its role in the pathogenesis of human desminopathy. A novel approach including mechanical and structural studies of 4–6-d-old larvae was applied. Morpholino antisense oligonucleotides were used to knock down desmin. Expression was assessed using messenger RNA and protein analyses. Histology and synchrotron light–based small angle x-ray diffraction were applied. Functional properties were analyzed with in vivo studies of swimming behavior and with in vitro mechanical examinations of muscle. The two desmin genes normally expressed in zebrafish could be knocked down by ∼50%. This resulted in a phenotype with disorganized muscles with altered attachments to the myosepta. The knockdown larvae were smaller and had diminished swimming activity. Active tension was lowered and muscles were less vulnerable to acute stretch-induced injury. X-ray diffraction revealed wider interfilament spacing. In conclusion, desmin intermediate filaments are required for normal active force generation and affect vulnerability during eccentric work. This is related to the role of desmin in anchoring sarcomeres for optimal force transmission. The results also show that a partial lack of desmin, without protein aggregates, is sufficient to cause muscle pathology resembling that in human desminopathy.     

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

Background

Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations.

Results

Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.

Conclusions

Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.     

The myosin co-chaperone UNC-45 is required for skeletal and cardiac muscle function in zebrafish

The assembly of myosin into higher order structures is dependent upon accessory factors that are often tissue-specific. UNC-45 acts as such a molecular chaperone for myosin in the nematode Caenorhabditis elegans, in both muscle and non-muscle contexts. Although vertebrates contain homologues of UNC-45, their requirement for muscle function has not been assayed. We identified a zebrafish gene, unc45b, similar to a mammalian unc-45 homologue, expressed exclusively in striated muscle tissue, including the somites, heart and craniofacial muscle. Morpholino-oligonucleotide-mediated knockdown of unc45b results in paralysis and cardiac dysfunction. This paralysis is correlated with a loss of myosin filaments in the sarcomeres of the trunk muscle. Morphants lack circulation, heart looping and display severe cardiac and yolk-sac edema and also demonstrate ventral displacement of several jaw cartilages. Overall, this confirms a role for unc45b in zebrafish motility consistent with a function in myosin thick filament assembly and stability and uncovers novel roles for this gene in the function and morphogenesis of the developing heart and jaw. These results suggest that Unc45b acts as a chaperone that aids in the folding of myosin isoforms required for skeletal, cranial and cardiac muscle contraction.     

Xirp proteins mark injured skeletal muscle in zebrafish

Myocellular regeneration in vertebrates involves the proliferation of activated progenitor or dedifferentiated myogenic cells that have the potential to replenish lost tissue. In comparison little is known about cellular repair mechanisms within myocellular tissue in response to small injuries caused by biomechanical or cellular stress. Using a microarray analysis for genes upregulated upon myocellular injury, we identified zebrafish Xin-actin-binding repeat-containing protein1 (Xirp1) as a marker for wounded skeletal muscle cells. By combining laser-induced micro-injury with proliferation analyses, we found that Xirp1 and Xirp2a localize to nascent myofibrils within wounded skeletal muscle cells and that the repair of injuries does not involve cell proliferation or Pax7(+) cells. Through the use of Xirp1 and Xirp2a as markers, myocellular injury can now be detected, even though functional studies indicate that these proteins are not essential in this process. Previous work in chicken has implicated Xirps in cardiac looping morphogenesis. However, we found that zebrafish cardiac morphogenesis is normal in the absence of Xirp expression, and animals deficient for cardiac Xirp expression are adult viable. Although the functional involvement of Xirps in developmental and repair processes currently remains enigmatic, our findings demonstrate that skeletal muscle harbours a rapid, cell-proliferation-independent response to injury which has now become accessible to detailed molecular and cellular characterizations.     

Mutations affecting skeletal muscle myofibril structure in the zebrafish

We describe embryonic lethal mutations in the zebrafish, Brachydanio rerio, which affect organization of skeletal muscle myofibrils. The mutations, fub-1(b45) and fub-1(b126), were independently isolated from progeny of gamma-irradiated females. Each segregates as a single recessive gene: b45 is located about 23 map units from its centromere. The b126 mutation has a similar but slightly larger apparent gene-centromere distance and a less severe phenotype. The two mutations fail to complement, suggesting that they are allelic. Homozygous b45 mutant embryos are paralyzed, and their axial skeletal muscle cells are unstriated, containing severely disorganized myofibrillar components. Gel-electrophoretic comparisons of b45 mutant and wild-type muscle proteins failed to reveal absent or altered major myofibrillar proteins. Embryos genetically mosaic for b45 were also phenotypically mosaic, suggesting that the defect is cell-autonomous. We suggest that these mutations identify a gene required for proper organization of skeletal muscle myofibrils, and that the more severe mutation may represent a null allele.     

Laminin-211 in skeletal muscle function

A chain is no stronger than its weakest link is an old idiom that holds true for muscle biology. As the name implies, skeletal muscle’s main function is to move the bones. However, for a muscle to transmit force and withstand the stress that contractions give rise to, it relies on a chain of proteins attaching the cytoskeleton of the muscle fiber to the surrounding extracellular matrix. The importance of this attachment is illustrated by a large number of muscular dystrophies caused by interruption of the cytoskeletal-extracellular matrix interaction. One of the major components of the extracellular matrix is laminin, a heterotrimeric glycoprotein and a major constituent of the basement membrane. It has become increasingly apparent that laminins are involved in a multitude of biological functions, including cell adhesion, differentiation, proliferation, migration and survival. This review will focus on the importance of laminin-211 for normal skeletal muscle function.     

Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish

The green fluorescent protein (GFP) acts as a vital dye upon the absorption of blue light. When the gfp gene is expressed in bacteria, flies or nematodes, green fluorescence can be directly observed in the living organism. We inserted the cDNA encoding this 238-amino-acid (aa) jellyfish protein into an expression vector containing the rat myosin light-chain enhancer (MLC-GFP) to evaluate its ability to serve as a muscle-specific marker. Transiently, as well as stably, transfected C2C12 cell lines produced high levels of GFP distributed homogeneously throughout the cytoplasm and was not toxic through several cell passages. Expression of MLC-GFP was strictly muscle-specific, since Cos 7 fibroblasts transfected with MLC-GFP did not fluoresce. When GFP and βGal markers were compared, the GFP signal was visible in the cytoplasm of the living cell, whereas visualization of βGal required fixation and resulted in deformation of the cells. When the MLC-GFP construct was injected into zebrafish embryos, muscle-specific gfp expression was apparent within 24 h of development, gfp expression was never observed in non-muscle tissues using the MLC-GFP construct. Transgenic fish continued to express high levels of gfp in skeletal muscle at 1.5 months, demonstrating that GFP is an effective marker of muscle cells in vivo.     

骨骼肌的内分泌功能

长期以来,骨骼肌被认为是一种效应器官,接受神经和体液的调节。近年大量实验研究资料发现骨骼肌也具有分泌活性物质的功能,能表达、合成和分泌多种生物信号分子,包括调节肽、细胞因子和生长因子等,也是一种重要的内分泌器官。骨骼肌分泌的活性物质以旁分泌和／或自分泌方式调节骨骼肌的生长、代谢和运动功能;甚至以血液循环内分泌的方式调节机体远隔器官组织的功能。骨骼肌生成和分泌的活性物质在运动系统疾病和某些全身性疾病的发病中具有重要的作用。本文将对骨骼肌分泌的主要活性物质及其生理和病理生理学意义进行综述。     

Delta-sarcoglycan is required for early zebrafish muscle organization

Mutations in sarcoglycans (alpha-, beta-, gamma-, and delta-) have been linked with limb girdle muscular dystrophy (LGMD) types 2C-F in humans. We have cloned the zebrafish orthologue encoding delta-sarcoglycan and mapped the gene to linkage group 21. The predicted zebrafish delta-sarcoglycan protein is highly homologous with its human orthologue including conservation of two of the three predicted glycosylation sites. Like other members of the dystrophin-associated protein complex (DAPC), delta-sarcoglycan localizes to the sarcolemmal membrane of the myofiber in adult zebrafish, but is more apparent at the myosepta in developing embryos. Zebrafish embryos injected with morpholinos against delta-sarcoglycan were relatively inactive at 5 dpf, their myofibers were disorganized, and swim bladders uninflated. Immunohistochemical and immunoblotting experiments show that delta-, beta-, and gamma-sarcoglycans were all downregulated in the morphants, whereas dystrophin expression was unaffected. Whereas humans lacking delta-sarcoglycan primarily show adult phenotypes, our results suggest that delta-sarcoglycan plays a role in early zebrafish muscle development.     

Development of ionic currents of zebrafish slow and fast skeletal muscle fibers

Voltage-gated Na+ and K+ channels play key roles in the excitability of skeletal muscle fibers. In this study we investigated the steady-state and kinetic properties of voltage-gated Na+ and K+ currents of slow and fast skeletal muscle fibers in zebrafish ranging in age from 1 day postfertilization (dpf) to 4-6 dpf. The inner white (fast) fibers possess an A-type inactivating K+ current that increases in peak current density and accelerates its rise and decay times during development. As the muscle matured, the V50s of activation and inactivation of the A-type current became more depolarized, and then hyperpolarized again in older animals. The activation kinetics of the delayed outward K+ current in red (slow) fibers accelerated within the first week of development. The tail currents of the outward K+ currents were too small to allow an accurate determination of the V50s of activation. Red fibers did not show any evidence of inward Na+ currents; however, white fibers expressed Na+ currents that increased their peak current density, accelerated their inactivation kinetics, and hyperpolarized their V50 of inactivation during development. The action potentials of white fibers exhibited significant changes in the threshold voltage and the half width. These findings indicate that there are significant differences in the ionic current profiles between the red and white fibers and that a number of changes occur in the steady-state and kinetic properties of Na+ and K+ currents of developing zebrafish skeletal muscle fibers, with the most dramatic changes occurring around the end of the first day following egg fertilization.     

An electrically coupled network of skeletal muscle in zebrafish distributes synaptic current

Fast and slow skeletal muscle types are readily distinguished in larval zebrafish on the basis of differences in location and orientation. Additionally, both muscle types are compact, rendering them amenable to in vivo patch clamp study of synaptic function. Slow muscle mediates rhythmic swimming, but it does so purely through synaptic drive, as these cells are unable to generate action potentials. Our patch clamp recordings from muscle pairs of zebrafish reveal a network of electrical coupling in slow muscle that allows sharing of synaptic current within and between segmental boundaries of the tail. The synaptic current exhibits slow kinetics (tau(decay) approximately 4 ms), which further facilitates passage through the low pass filter, a consequence of the electrically coupled network. In contrast to slow muscle, fast skeletal muscle generates action potentials to mediate the initial rapid component of the escape response. The combination of very weak electrical coupling and synaptic kinetics (tau(decay) <1 ms) too fast for the network low pass filter minimizes intercellular sharing of synaptic current in fast muscle. These differences between muscle types provide insights into the physiological role(s) of electrical coupling in skeletal muscle. First, intrasegmental coupling among slow muscle cells allows effective transfer of synaptic currents within tail segments, thereby minimizing differences in synaptic depolarization. Second, a fixed intersegmental delay in synaptic current transit, resulting from the low pass filter properties of the slow muscle network, helps coordinate the rostral-caudal wave of contraction.     

Delta-sarcoglycan is necessary for early heart and muscle development in zebrafish

Delta-sarcoglycan, one member of the sarcoglycan complex, is a very conservative muscle-specific protein exclusively expressed in the skeletal and cardiac muscles of vertebrates. Mutations in sarcoglycans are known to be involved in limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy (DCM) in humans. To address the role of delta-sarcoglycan gene in zebrafish development, we have studied expression pattern of delta-sarcoglycan in zebrafish embryos and examined the role of delta-sarcoglycan in zebrafish embryonic development by morpholino. Strong expression of delta-sarcoglycan was observed in various muscles including those of the segment, heart, eye, jaw, pectoral fin, branchial arches, and swim bladder in zebrafish embryo. Delta-sarcoglycan was also expressed in midbrain and retina. Knockdown of delta-sarcoglycan resulted in severe abnormality in both the cardiac and skeletal muscles. Some severe ones displayed serious morphological abnormality such as hypoplastic head, linear heart, very weak heartbeats, and runtish trunk, all dead within 5 dpf. Whole-mount in situ hybridization analysis showed that adaxial cells and muscle pioneers were affected in delta-sarcoglycan knockdown embryos. In addition, absence of delta-sarcoglycan protein severely delayed the cardiac development and influenced the differentiation of cardiac muscle, and the cardiac left-right asymmetry was dramatically changed in morpholino-treated embryos. These data together suggest that delta-sarcoglycan plays an important role in early heart and muscle development.     

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions.     

Corticosteroids and skeletal muscle function in cystic fibrosis.

Patients with cystic fibrosis (CF) have reduced peripheral muscle strength. We tested the hypothesis that steroid treatment contributes to muscle weakness in adults with CF. Twenty-three stable CF patients were studied. Measurements included knee extensor (KE), knee flexor (KF), elbow flexor (EF), handgrip (HG), expiratory (Pemax), and inspiratory (Pimax) muscle strengths. Spirometry, body mass index (BMI), and days spent in hospital over the preceding 12 mo (DH) were also measured. Average daily dose of prednisolone over the preceding 12 mo (ADD) was 5.1 mg/day. Pearson's correlation analysis revealed that ADD correlated significantly with skeletal muscle strengths (KF%, r = -0.63, P < 0.01) with the exception of HG%. These findings are independent of age, BMI, pulmonary function, and DH. Multiple-regression analysis revealed that ADD was the most significant predictor of all measures of skeletal muscle function except HG%. It was independently responsible for 54% of the variance in Pimax%, for 46% of the variance in Pemax%, for 45% of the variance in KE%, for 39% of the variance in KF%, and for 41% of the variance in EF%. Concomitant medications (e.g., theophylline) were shown to have no causative effect. Corticosteroids contribute to the skeletal muscle weakness seen in CF patients. The correlation of proximal muscle strength, but not HG strength, with steroid dosage further supports a cause-effect relationship.