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Deficiency of muscle basement membrane (MBM) component laminin-α2 leads to muscular dystrophy congenital type 1A (MDC1A), a currently untreatable myopathy. Laminin--α2 has two main binding partners within the MBM, dystroglycan and integrin. Integrins coordinate both cell adhesion and signalling; however, there is little mechanistic insight into integrin's function at the MBM. In order to study integrin's role in basement membrane development and how this relates to the MBM's capacity to handle force, an itgβ1.b-/- zebrafish line was created. Histological examination revealed increased extracellular matrix (ECM) deposition at the MBM in the itgβ1.b-/- fish when compared with controls. Surprisingly, both laminin and collagen proteins were found to be increased in expression at the MBM of the itgβ1.b-/- larvae when compared with controls. This increase in ECM components resulted in a decrease in myotomal elasticity as determined by novel passive force analyses. To determine if it was possible to control ECM deposition at the MBM by manipulating integrin activity, RGD peptide, a potent inhibitor of integrin-β1, was injected into a zebrafish model of MDC1A. As postulated an increase in laminin and collagen was observed in the lama2-/- mutant MBM. Importantly, there was also an improvement in fibre stability at the MBM, judged by a reduction in fibre pathology. These results therefore show that blocking ITGβ1 signalling increases ECM deposition at the MBM, a process that could be potentially exploited for treatment of MDC1A.

Congenital myopathies are muscle degenerative disorders with a broad clinical spectrum. A number of myopathies have been associated with molecular defects within sarcomeres, the force-generating component of the muscle cell. Whereas the highly regular organization of the myofibril has been studied in detail, in vivo assembly of sarcomeres remains a poorly understood process. Therefore, a more detailed knowledge of sarcomere assembly is crucial to better understand the pathogenic mechanisms within myopathies. Recently, mutations in myosin XVIIIB (MYO18B) have been associated with cases of myopathies, although the underlying mechanism for the resulting pathology remains to be defined. To analyze the role of myosin XVIIIB in skeletal muscle disease, zebrafish mutants for myo18b were generated. Full loss of myo18b function results in a complete lack of sarcomeric structure, revealing a highly surprising and essential role for myo18b in sarcomere assembly. Importantly, scattered thin and thick filaments assemble throughout the sarcoplasm; but fail to organize into recognizable sarcomeric structures in myo18b null mutants. In myo18b partial loss-of-function mutants sarcomeric structures are assembled, but thin and thick filaments remain misaligned within these structures. These observations suggest a novel model of sarcomere assembly where Myo18b coordinates the integration of preformed thick and thin filaments into the sarcomere. Disruption of this highly coordinated process results in a block in sarcomere biogenesis and the onset of myopathic pathology.

Laminins form essential components of the basement membrane and are integral to forming and maintaining muscle integrity. Mutations in the human Laminin-alpha2 (LAMA2) gene result in the most common form of congenital muscular dystrophy, MDC1A. We have previously identified a zebrafish model of MDC1A called candyfloss (caf), carrying a loss-of-function mutation in the zebrafish lama2 gene. In the skeletal muscle, laminins connect the muscle cell to the extracellular matrix (ECM) by binding either dystroglycan or integrins at the cell membrane. Through epistasis experiments, we have established that both adhesion systems individually contribute to the maintenance of fibre adhesions and exhibit muscle detachment phenotypes. However, larval zebrafish in which both adhesion systems are simultaneously genetically inactivated possess a catastrophic failure of muscle attachment that is far greater than a simple addition of individual phenotypes would predict. We provide evidence that this is due to other crucial laminins present in addition to Lama2, which aid muscle cell attachments and integrity. We have found that lama1 is important for maintaining attachments, whereas lama4 is localized and up-regulated in damaged fibres, which appears to contribute to fibre survival. Importantly, our results show that endogenous secretion of laminins from the surrounding tissues has the potential to reinforce fibre attachments and strengthen laminin-ECM attachments. Collectively these findings provide a better understanding of the cellular pathology of MDC1A and help in designing effective therapies.

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

The muscular dystrophies and congenital myopathies are inherited diseases of the skeletal muscle, which lead to a loss of muscle function and are often fatal. While many of the loci involved are already known, these conditions remain incurable, and genetic models are being developed in an effort to understand the pathological mechanisms involved. Recently several papers have shown that the zebrafish, which is now widely used in developmental genetic studies, will provide a useful addition to our toolkit in this regard. Here we describe these studies, including a zebrafish model of what is potentially the novel pathological mechanism of muscle attachment failure in Duchenne and other muscular dystrophies.

International audience; Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. The subcellular mechanisms of DMD remain poorly understood and there is currently no curative treatment available. Using a Caenorhabditis elegans model for DMD as a pharmacologic and genetic tool, we found that cyclosporine A (CsA) reduces muscle degeneration at low dose and acts, at least in part, through a mitochondrial cyclophilin D, CYN-1. We thus hypothesized that CsA acts on mitochondrial permeability modulation through cyclophilin D inhibition. Mitochondrial patterns and dynamics were analyzed, which revealed dramatic mitochondrial fragmentation not only in dystrophic nematodes, but also in a zebrafish model for DMD. This abnormal mitochondrial fragmentation occurs before any obvious sign of degeneration can be detected. Moreover, we demonstrate that blocking/delaying mitochondrial fragmentation by knocking down the fission-promoting gene drp-1 reduces muscle degeneration and improves locomotion abilities of dystrophic nematodes. Further experiments revealed that cytochrome c is involved in muscle degeneration in C. elegans and seems to act, at least in part, through an interaction with the inositol trisphosphate receptor calcium channel, ITR-1. Altogether, our findings reveal that mitochondria play a key role in the early process of muscle degeneration and may be a target of choice for the design of novel therapeutics for DMD. In addition, our results provide the first indication in the nematode that (i) mitochondrial permeability transition can occur and (ii) cytochrome c can act in cell death.

Myofibrillar myopathies are a group of muscle disorders characterized by the disintegration of skeletal muscle fibers and formation of sarcomeric protein aggregates. All the proteins known to be involved in myofibrillar myopathies localize to a region of the sarcomere known as the Z-disk, the site at which defects are first observed. Given the common cellular phenotype observed in this group of disorders, it is thought that there is a common mechanism of pathology. Mutations in filamin C, which has several proposed roles in the development and function of skeletal muscle, can result in filamin-related myofibrillar myopathy. The lack of a suitable animal model system has limited investigation into the mechanism of pathology in this disease and the role of filamin C in muscle development. Here, we characterize stretched out (sot), a zebrafish filamin Cb mutant, together with targeted knockdown of zebrafish filamin Ca, revealing fiber dissolution and formation of protein aggregates strikingly similar to those seen in filamin-related myofibrillar myopathies. Through knockdown of both zebrafish filamin C homologues, we demonstrate that filamin C is not required for fiber specification and that fiber damage is a consequence of muscle activity. The remarkable similarities in the myopathology between our models and filamin-related myofibrillar myopathy makes them suitable for the study of these diseases and provides unique opportunities for the investigation of the function of filamin C in muscle and development of therapies.