Ann E. Davidson

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies.

The β-tropomyosin gene encodes a component of the sarcomeric thin filament. Rod-shaped dimers of tropomyosin regulate actin-myosin interactions and β-tropomyosin mutations have been associated with nemaline myopathy, cap myopathy, Escobar syndrome and distal arthrogryposis types 1A and 2B. In this study, we expand the allelic spectrum of β-tropomyosin-related myopathies through the identification of a novel β-tropomyosin mutation in two clinical contexts not previously associated with β-tropomyosin. The first clinical phenotype is core-rod myopathy, with a β-tropomyosin mutation uncovered by whole exome sequencing in a family with autosomal dominant distal myopathy and muscle biopsy features of both minicores and nemaline rods. The second phenotype, observed in four unrelated families, is autosomal dominant trismus-pseudocamptodactyly syndrome (distal arthrogryposis type 7; previously associated exclusively with myosin heavy chain 8 mutations). In all four families, the mutation identified was a novel 3-bp in-frame deletion (c.20_22del) that results in deletion of a conserved lysine at the seventh amino acid position (p.K7del). This is the first mutation identified in the extreme N-terminus of β-tropomyosin. To understand the potential pathogenic mechanism(s) underlying this mutation, we performed both computational analysis and in vivo modelling. Our theoretical model predicts that the mutation disrupts the N-terminus of the α-helices of dimeric β-tropomyosin, a change predicted to alter protein-protein binding between β-tropomyosin and other molecules and to disturb head-to-tail polymerization of β-tropomyosin dimers. To create an in vivo model, we expressed wild-type or p.K7del β-tropomyosin in the developing zebrafish. p.K7del β-tropomyosin fails to localize properly within the thin filament compartment and its expression alters sarcomere length, suggesting that the mutation interferes with head-to-tail β-tropomyosin polymerization and with overall sarcomeric structure. We describe a novel β-tropomyosin mutation, two clinical-histopathological phenotypes not previously associated with β-tropomyosin and pathogenic data from the first animal model of β-tropomyosin-related myopathies.

Fluoxetine prevents dystrophic changes in a zebrafish model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a common and relentlessly progressive muscle disease. Some interventions have been identified that modestly slow progression and prolong survival, but more meaningful therapies are lacking. The goal of this study is to identify new therapeutic pathways for DMD using a zebrafish model of the disease. To accomplish this, we performed a non-biased drug screen in sapje, a zebrafish line with a recessive nonsense mutation in dystrophin. We identified 6 positive hits (out of 640 total drugs tested) by their ability to prevent abnormal birefringence in sapje. Follow-up analyses demonstrated that fluoxetine, a selective serotonin reuptake inhibitor (SSRI), provided the most substantial benefit. Morpholino-based experimentation confirmed that modulation of the serotonin pathway alone can prevent the dystrophic phenotype, and transcriptomic analysis revealed changes in calcium homeostasis as a potential mechanism. In all, we demonstrate that monoamine agonists can prevent disease in a vertebrate model of DMD. Given the safe and widespread use of SSRIs in clinical practice, our study identifies an attractive target pathway for therapy development.

PIK3C2B inhibition improves function and prolongs survival in myotubular myopathy animal models

Myotubular myopathy (MTM) is a devastating pediatric neuromuscular disorder of phosphoinositide (PIP) metabolism resulting from mutations of the PIP phosphatase MTM1 for which there are no treatments. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in animal models of MTM. Here, we tested the hypothesis that lowering PI3P levels may prevent or reverse the MTM disease process. To test this, we targeted class II and III PI3 kinases (PI3Ks) in an MTM1-deficient mouse model. Muscle-specific ablation of Pik3c2b, but not Pik3c3, resulted in complete prevention of the MTM phenotype, and postsymptomatic targeting promoted a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that certain PI3K inhibitors prevented development of the zebrafish mtm phenotype. Finally, the PI3K inhibitor wortmannin improved motor function and prolonged lifespan of the Mtm1-deficient mice. In all, we have identified Pik3c2b as a genetic modifier of Mtm1 mutation and demonstrated that PIK3C2B inhibition is a potential treatment strategy for MTM. In addition, we set the groundwork for similar reciprocal inhibition approaches for treating other PIP metabolic disorders and highlight the importance of modifier gene pathways as therapeutic targets.

The myopathy-causing mutation DNM2-S619L leads to defective tubulation in vitro and in developing zebrafish

DNM2 is a ubiquitously expressed GTPase that regulates multiple subcellular processes. Mutations in DNM2 are a common cause of centronuclear myopathy, a severe disorder characterized by altered skeletal muscle structure and function. The precise mechanisms underlying disease-associated DNM2 mutations are unresolved. We examined the common DNM2-S619L mutation using both in vitro and in vivo approaches. Expression of DNM2-S619L in zebrafish led to the accumulation of aberrant vesicular structures and to defective excitation-contraction coupling. Expression of DNM2-S619L in COS7 cells resulted in defective BIN1-dependent tubule formation. These data suggest that DNM2-S619L causes disease, in part, by interfering with membrane tubulation.

Two dynamin-2 genes are required for normal zebrafish development.

Dynamin-2 (DNM2) is a large GTPase involved in clathrin-mediated endocytosis and related trafficking pathways. Mutations in human DNM2 cause two distinct neuromuscular disorders: centronuclear myopathy and Charcot-Marie-Tooth disease. Zebrafish have been shown to be an excellent animal model for many neurologic disorders, and this system has the potential to inform our understanding of DNM2-related disease. Currently, little is known about the endogenous zebrafish orthologs to human DNM2. In this study, we characterize two zebrafish dynamin-2 genes, dnm2 and dnm2-like. Both orthologs are structurally similar to human DNM2 at the gene and protein levels. They are expressed throughout early development and in all adult tissues examined. Knockdown of dnm2 and dnm2-like gene products resulted in extensive morphological abnormalities during development, and expression of human DNM2 RNA rescued these phenotypes. Our findings suggest that dnm2 and dnm2-like are orthologs to human DNM2, and that they are required for normal zebrafish development.

Analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations.

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 μm) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Use of the Sleeping Beauty transposon system for stable gene expression in mouse embryonic stem cells.

Sleeping Beauty (SB) transposon-based transfection is a two-component system consisting of a transposase and a transposon containing inverted repeat/direct repeat (IR/DR) sequences that result in precise integration into a TA dinucleotide. The transposon is designed with an expression cassette of interest flanked by IR/DRs, and SB transposase mediates stable integration and reliable long-term expression of the gene of interest. It has recently been demonstrated that SB efficiently mediates gene transfer and stable gene expression in human embryonic stem (ES) cells. Here, we describe a method for transfecting and establishing stable cell lines in mouse embryonic stem (mES) cells with the SB system.

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

P9.17 Function of CCDC78 in muscle development and muscle disease

Congenital myopathies are a heterogeneous group of diseases that typically present in childhood with hypotonia and weakness and are commonly defined by characteristic changes observed on muscle biopsy. Currently, no treatments or disease-modifying therapies have been identified. Therapy development is hindered because approximately 40% of congenital myopathies are genetically unresolved. An overarching goal of our laboratory is to identify and characterize new causes of congenital myopathy. To this end, using whole exome sequencing we recently identified a splice-acceptor variant in the coiled-coil domain containing 78 gene (CCDC78) in a family with autosomal dominant core myopathy. CCDC78 is a previously uncharacterized gene with no known function that is highly enriched in skeletal muscle and localized to the perinuclear compartment and to the excitation contraction coupling apparatus. The specific goals of this study are to determine how mutation in CCDC78 results in muscle disease, establish the relationship between CCDC78 and other congenital myopathies, and to elucidate the normal function of CCDC78. To accomplish these goals we have created both morpholino based and stable genetic models of CCDC78 mutation in the zebrafish, including characterization of a newly developed loss of function CCDC78 mutant (sa3254). We have combined analysis of these zebrafish models with cell culture studies and examination of a range of human muscle biopsies. Current data suggests that CCDC78 participates in both the regulation of excitation-contraction coupling and in the regulation of autophagy. Additional ongoing experimentation will examine expression of CCDC78 in other congenital muscle diseases, will determine interacting partners for CCDC78, and will establish the normal function for CCDC78 in skeletal muscle development and homeostasis. In all, we present the first extensive characterization of this newly identified congenital myopathy gene.