Elizabeth M. Gibbs

Loss of Myotubularin Function Results in T-Tubule Disorganization in Zebrafish and Human Myotubular Myopathy

Myotubularin is a lipid phosphatase implicated in endosomal trafficking in vitro, but with an unknown function in vivo. Mutations in myotubularin cause myotubular myopathy, a devastating congenital myopathy with unclear pathogenesis and no current therapies. Myotubular myopathy was the first described of a growing list of conditions caused by mutations in proteins implicated in membrane trafficking. To advance the understanding of myotubularin function and disease pathogenesis, we have created a zebrafish model of myotubular myopathy using morpholino antisense technology. Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle. These changes include abnormally shaped and positioned nuclei and myofiber hypotrophy. These findings are consistent with those observed in the human disease. We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin. Finally, we identify abnormalities in the tubulo-reticular network in muscle from myotubularin zebrafish morphants and correlate these changes with abnormalities in T-tubule organization in biopsies from patients with myotubular myopathy. In all, we have generated a new model of myotubular myopathy and employed this model to uncover a novel function for myotubularin and a new pathomechanism for the human disease that may explain the weakness associated with the condition (defective excitation–contraction coupling). In addition, our findings of tubuloreticular abnormalities and defective excitation-contraction coupling mechanistically link myotubular myopathy with several other inherited muscle diseases, most notably those due to ryanodine receptor mutations. Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis.

Kindlin-2 Is an Essential Component of Intercalated Discs and Is Required for Vertebrate Cardiac Structure and Function

Integrins and proteins that associate with integrins are implicated in normal cardiac muscle function and development. Unc-112 is a cytoplasmic adaptor protein required for the proper establishment of integrin junctions in Caenorhabditis elegans muscle. A vertebrate homolog of unc-112, kindlin-2, is an integrin-binding protein that is expressed in cardiac muscle, but its function is unknown. We sought to understand the role of kindlin-2 in the development and function of the mouse and zebrafish heart. In the mouse, we found that kindlin-2 is highly expressed in the heart and is enriched at intercalated discs and costameres. Targeted disruption of the murine kindlin-2 gene resulted in embryonic lethality before cardiogenesis. To better assess the role of kindlin-2 in cardiac muscle development, we used morpholinos to knockdown the kindlin-2 homolog in zebrafish (z-kindlin-2), which resulted in severe abnormalities of heart development. Morphant hearts were hypoplastic and dysmorphic and exhibited significantly reduced ventricular contractility. Ultrastructural analysis of these hearts revealed disrupted intercalated disc formation and a failure in the attachment of myofibrils to membrane complexes. We conclude that kindlin-2 is an essential component of the intercalated disc, is necessary for cytoskeletal organization at sites of membrane attachment, and is required for vertebrate myocardial formation and function. These findings provide the first characterization of the in vivo functions of this novel and critical regulator of cardiogenesis.

Congenital Myopathies: An Update

Congenital myopathy is a clinicopathological concept of characteristic histopathological findings on muscle biopsy in a patient with early-onset weakness. Three main categories are recognized within the classical congenital myopathies: nemaline myopathy, core myopathy, and centronuclear myopathy. Recent evidence of overlapping clinical and histological features between the classical forms and their different genetic entities suggests that there may be shared pathomechanisms between the congenital myopathies. Animal models, especially mouse and zebrafish, have been especially helpful in elucidating such pathomechanisms associated with the congenital myopathies and provide models in which future therapies can be investigated.

Swimming into prominence: the zebrafish as a valuable tool for studying human myopathies and muscular dystrophies.

A new and exciting phase of muscle disease research has recently been entered. The application of next generation sequencing technology has spurred an unprecedented era of gene discovery for both myopathies and muscular dystrophies. Gene‐based therapies for Duchenne muscular dystrophy have entered clinical trial, and several pathway‐based therapies are doing so as well for a handful of muscle diseases. While many factors have aided the extraordinary developments in gene discovery and therapy development, the zebrafish model system has emerged as a vital tool in these advancements. In this review, we will highlight how the zebrafish has greatly aided in the identification of new muscle disease genes and in the recognition of novel therapeutic strategies. We will start with a general introduction to the zebrafish as a model, discuss the ways in which muscle disease can be modeled and analyzed in the fish, and conclude with observations from recent studies that highlight the power of the fish as a research tool for muscle disease.

Neuromuscular junction abnormalities in DNM2-related centronuclear myopathy

Dynamin-2-related centronuclear myopathy (DNM2-CNM) is a clinically heterogeneous muscle disorder characterized by muscle weakness and centralized nuclei on biopsy. There is little known about the muscle dysfunction underlying this disorder, and there are currently no treatments. In this study, we establish a novel zebrafish model for DNM2-CNM by transiently overexpressing a mutant version of DNM2 (DNM2-S619L) during development. We show that overexpression of DNM2-S619L leads to pathological changes in muscle and a severe motor phenotype. We further demonstrate that the muscle weakness seen in these animals can be significantly alleviated by treatment with an acetylcholinesterase inhibitor. Based on these results, we reviewed the clinical history of five patients with two different DNM2-CNM mutations (S619L and E368K) and found electrophysiological evidence of abnormal neuromuscular transmission in two of the individuals. All five patients showed improved muscle strength and motor function, and/or reduced fatigability following acetylcholinesterase inhibitor treatment. Together, our results suggest that deficits at the neuromuscular junction may play an important role in the pathogenesis of DNM2-CNM and that treatments targeting this dysfunction can provide an effective therapy for patients with this disorder.

Membrane Traffic and Muscle: Lessons from Human Disease

Like all mammalian tissues, skeletal muscle is dependent on membrane traffic for proper development and homeostasis. This fact is underscored by the observation that several human diseases of the skeletal muscle are caused by mutations in gene products of the membrane trafficking machinery. An examination of these diseases and the proteins that underlie them is instructive both in terms of determining disease pathogenesis and of understanding the normal aspects of muscle biology regulated by membrane traffic. This review highlights our current understanding of the trafficking genes responsible for human myopathies.

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.

The role of MTMR14 in autophagy and in muscle disease

Phosphoinositides (PIs) are a group of low abundance phospholipids that play a critical role in the control of organelle and membrane traffic. There is strong evidence that specific PIs are also important for the regulation of autophagy. PIs are modified by a complex network of lipid kinases and phosphatases. A recent study from our laboratory focused on two PI phosphatases from the myotubularin related protein (MTMR) family, myotubularin (MTM1) and MTMR14. Using zebrafish as a model system, we found that dual knockdown of MTM1 and MTMR14 leads to an unexpectedly severe developmental motor phenotype. We found that this severe phenotype was mediated, at least in part, by dysregulation of autophagy, as demonstrated by the accumulation of autophagic vacuoles and increased levels of LC3-II. Our study provides the first in vivo evidence for a role of myotubularins, and in particular MTMR14, in the regulation of autophagy.

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.