NRAP reduction rescues sarcomere defects in nebulin-related nemaline myopathy.

Nemaline myopathy (NM) is a rare neuromuscular disorder associated with congenital or childhood-onset of skeletal muscle weakness and hypotonia, which results in limited motor function. NM is a genetic disorder and mutations in 12 genes are known to contribute to autosomal dominant or recessive forms of the disease. Recessive mutations in nebulin (NEB) are the most common cause of NM affecting about 50% of patients. Because of the large size of the NEB gene and lack of mutational hot spots, developing therapies that can benefit a wide group of patients is challenging. Although there are several promising therapies under investigation, there is no cure for NM. Therefore, targeting disease modifiers that can stabilize or improve skeletal muscle function may represent alternative therapeutic strategies. Our studies have identified Nrap upregulation in nebulin deficiency that contributes to structural and functional deficits in NM. We show that genetic ablation of nrap in nebulin deficiency restored sarcomeric disorganization, reduced protein aggregates and improved skeletal muscle function in zebrafish. Our findings suggest that Nrap is a disease modifier that affects skeletal muscle structure and function in NM; thus, therapeutic targeting of Nrap in nebulin-related NM and related diseases may be beneficial for patients.

Prednisolone rescues Duchenne muscular dystrophy phenotypes in human pluripotent stem cell-derived skeletal muscle in vitro.

Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

Dysregulation of NRAP degradation by KLHL41 contributes to pathophysiology in nemaline myopathy.

Nemaline myopathy (NM) is the most common form of congenital myopathy that results in hypotonia and muscle weakness. This disease is clinically and genetically heterogeneous, but three recently discovered genes in NM encode for members of the Kelch family of proteins. Kelch proteins act as substrate-specific adaptors for Cullin 3 (CUL3) E3 ubiquitin ligase to regulate protein turnover through the ubiquitin-proteasome machinery. Defects in thin filament formation and/or stability are key molecular processes that underlie the disease pathology in NM; however, the role of Kelch proteins in these processes in normal and diseases conditions remains elusive. Here, we describe a role of NM causing Kelch protein, KLHL41, in premyofibil-myofibil transition during skeletal muscle development through a regulation of the thin filament chaperone, nebulin-related anchoring protein (NRAP). KLHL41 binds to the thin filament chaperone NRAP and promotes ubiquitination and subsequent degradation of NRAP, a process that is critical for the formation of mature myofibrils. KLHL41 deficiency results in abnormal accumulation of NRAP in muscle cells. NRAP overexpression in transgenic zebrafish resulted in a severe myopathic phenotype and absence of mature myofibrils demonstrating a role in disease pathology. Reducing Nrap levels in KLHL41 deficient zebrafish rescues the structural and function defects associated with disease pathology. We conclude that defects in KLHL41-mediated ubiquitination of sarcomeric proteins contribute to structural and functional deficits in skeletal muscle. These findings further our understanding of how the sarcomere assembly is regulated by disease-causing factors in vivo, which will be imperative for developing mechanism-based specific therapeutic interventions.

Transgenic zebrafish model of DUX4 misexpression reveals a developmental role in FSHD pathogenesis.

Facioscapulohumeral dystrophy type 1 (FSHD-1) is the most common autosomal dominant form of muscular dystrophy with a prevalence of ∼1 in 8000 individuals. It is considered a late-onset form of muscular dystrophy and leads to asymmetric muscle weakness in the facial, scapular, trunk and lower extremities. The prevalent hypothesis on disease pathogenesis is explained by misexpression of a germ line, primate-specific transcription factor DUX4-fl (double homeobox 4, full-length isoform) linked to the chromosome 4q35. In vitro and in vivo studies have demonstrated that very low levels of DUX4-fl expression are sufficient to induce an apoptotic and/or lethal phenotype, and therefore modeling of the disease has proved challenging. In this study, we expand upon our previously established injection model of DUX4 misexpression in zebrafish and describe a DUX4-inducible transgenic zebrafish model that better recapitulates the expression pattern and late onset phenotype characteristic of FSHD patients. We show that an induced burst of DUX4 expression during early development results in the onset of FSHD-like phenotypes in adulthood, even when DUX4 is no longer detectable. We also utilize our injection model to study long-term consequences of DUX4 expression in those that fail to show a developmental phenotype. Herein, we introduce a hypothesis that DUX4 expression during developmental stages is sufficient to induce FSHD-like phenotypes in later adulthood. Our findings point to a developmental role of DUX4 misexpression in the pathogenesis of FSHD and should be factored into the design of future therapies.

Bi-allelic loss of function variants in GOLGA2 are associated with a complex neurological phenotype: Report of a second family.

GOGLA2/GM130 is a Golgin protein involved in vesicle tethering, cell proliferation and autophagy. Recessive loss of function mutation in GOLGA2 has been previously reported in a single family with muscular dystrophy and microcephaly. Here we describe a second consanguineous family with the bi-allelic loss of function mutations in GOLGA2. The patient exhibits microcephaly, seizures, and myopathy similar to the previously reported patient with GOLGA2 mutation. This report supports the critical developmental requirement of GOLGA2 and emphasizes a similar and severe clinical presentation with loss of function mutations in affected patients.

Novel Recessive TNNT1 Congenital Core-Rod Myopathy in French Canadians.

OBJECTIVE: Recessive null variants of the slow skeletal muscle troponin T1 (TNNT1) gene are a rare cause of nemaline myopathy that is fatal in infancy due to respiratory insufficiency. Muscle biopsy shows rods and fiber type disproportion. We report on 4 French Canadians with a novel form of recessive congenital TNNT1 core-rod myopathy. METHODS: Patients underwent full clinical characterization, lower limb magnetic resonance imaging (MRI), muscle biopsy, and genetic testing. A zebrafish loss-of-function model using morpholinos was created to assess the pathogenicity of the identified variant. Wild-type or mutated human TNNT1 mRNAs were coinjected with morpholinos to assess their abilities to rescue the morphant phenotype. RESULTS: Three adults and 1 child shared a novel missense homozygous variant in the TNNT1 gene (NM_003283.6: c.287T > C; p.Leu96Pro). They developed from childhood very slowly progressive limb-girdle weakness with rigid spine and disabling contractures. They suffered from restrictive lung disease requiring noninvasive mechanical ventilation in 3 patients, as well as recurrent episodes of rhabdomyolysis triggered by infections, which were relieved by dantrolene in 1 patient. Older patients remained ambulatory into their 60s. MRI of the leg muscles showed fibrofatty infiltration predominating in the posterior thigh and the deep posterior leg compartments. Muscle biopsies showed multiminicores and lobulated fibers, rods in half the patients, and no fiber type disproportion. Wild-type TNNT1 mRNA rescued the zebrafish morphants, but mutant transcripts failed to do so. INTERPRETATION: This study expands the phenotypic spectrum of TNNT1 myopathy and provides functional evidence for the pathogenicity of the newly identified missense mutation. ANN NEUROL 2020;87:568-583.

hnRNP L is essential for myogenic differentiation and modulates myotonic dystrophy pathologies.

INTRODUCTION: RNA-binding proteins (RBPs) play an important role in skeletal muscle development and disease by regulating RNA splicing. In myotonic dystrophy type 1 (DM1), the RBP MBNL1 (muscleblind-like) is sequestered by toxic CUG repeats, leading to missplicing of MBNL1 targets. Mounting evidence from the literature has implicated other factors in the pathogenesis of DM1. Herein we sought to evaluate the functional role of the splicing factor hnRNP L in normal and DM1 muscle cells. METHODS: Co-immunoprecipitation assays using hnRNPL and MBNL1 expression constructs and splicing profiling in normal and DM1 muscle cell lines were performed. Zebrafish morpholinos targeting hnrpl and hnrnpl2 were injected into one-cell zebrafish for developmental and muscle analysis. In human myoblasts downregulation of hnRNP L was achieved with shRNAi. Ascochlorin administration to DM1 myoblasts was performed and expression of the CUG repeats, DM1 splicing biomarkers, and hnRNP L expression levels were evaluated. RESULTS: Using DM1 patient myoblast cell lines we observed the formation of abnormal hnRNP L nuclear foci within and outside the expanded CUG repeats, suggesting a role for this factor in DM1 pathology. We showed that the antiviral and antitumorigenic isoprenoid compound ascochlorin increased MBNL1 and hnRNP L expression levels. Drug treatment of DM1 muscle cells with ascochlorin partially rescued missplicing of established early biomarkers of DM1 and improved the defective myotube formation displayed by DM1 muscle cells. DISCUSSION: Together, these studies revealed that hnRNP L can modulate DM1 pathologies and is a potential therapeutic target.

RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes.

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles.

ACTN2 mutations cause "Multiple structured Core Disease" (MsCD).

The identification of genes implicated in myopathies is essential for diagnosis and for revealing novel therapeutic targets. Here we characterize a novel subclass of congenital myopathy at the morphological, molecular, and functional level. Through exome sequencing, we identified de novo ACTN2 mutations, a missense and a deletion, in two unrelated patients presenting with progressive early-onset muscle weakness and respiratory involvement. Morphological and ultrastructural analyses of muscle biopsies revealed a distinctive pattern with the presence of muscle fibers containing small structured cores and jagged Z-lines. Deeper analysis of the missense mutation revealed mutant alpha-actinin-2 properly localized to the Z-line in differentiating myotubes and its level was not altered in muscle biopsy. Modelling of the disease in zebrafish and mice by exogenous expression of mutated alpha-actinin-2 recapitulated the abnormal muscle function and structure seen in the patients. Motor deficits were noted in zebrafish, and muscle force was impaired in isolated muscles from AAV-transduced mice. In both models, sarcomeric disorganization was evident, while expression of wild-type alpha-actinin-2 did not result in muscle anomalies. The murine muscles injected with mutant ACTN2 displayed cores and Z-line defects. Dominant ACTN2 mutations were previously associated with cardiomyopathies, and our data demonstrate that specific mutations in the well-known Z-line regulator alpha-actinin-2 can cause a skeletal muscle disorder.

Mutation-specific effects on thin filament length in thin filament myopathy.

OBJECTIVE: Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. METHODS: We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. RESULTS: Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force-sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin-thick filament overlap. INTERPRETATION: These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. Ann Neurol 2016;79:959-969.

Bridging integrator 1 (Bin1) deficiency in zebrafish results in centronuclear myopathy.

Autosomal recessive centronuclear myopathy (CNM2), caused by mutations in bridging integrator 1 (BIN1), is a mildly progressive neuromuscular disorder characterized by abnormally centralized myonuclei and muscle weakness. BIN1 is important for membrane sensing and remodeling in vitro in different cell types. However, to fully understand the biological roles of BIN1 in vivo and to answer critical questions concerning the muscle-specific function of BIN1 in vertebrates, robust small animal models are required. In this study, we create and characterize a novel zebrafish model of CNM2 using antisense morpholinos. Immunofluorescence and histopathological analyses of Bin1-deficient zebrafish skeletal muscle reveal structural defects commonly reported in human CNM2 biopsies. Live imaging of zebrafish embryos shows defective calcium release in bin1 morphants, linking the presence of abnormal triads to impairments in intracellular signaling. RNA-mediated rescue assays demonstrate that knockdown of zebrafish bin1 can reliably examine the pathogenicity of novel BIN1 mutations in vivo. Finally, our results strongly suggest that the phosphoinositide-binding domain of BIN1, present only in skeletal muscle isoforms, may be more critical for muscle maturation and maintenance than for early muscle development. Overall, our data support that BIN1 plays an important role in membrane tubulation and may promote skeletal muscle weakness in CNM2 by disrupting machinery necessary for excitation-contraction coupling in vertebrate organisms. The reproducible phenotype of Bin1-deficient zebrafish, together with the generalized advantages of the teleost system, makes this model readily adaptable to high-throughput screening strategies and may be used to identify therapies for CNM2 and related neuromuscular diseases.

ZBTB42 mutation defines a novel lethal congenital contracture syndrome (LCCS6).

Lethal congenital contracture syndrome (LCCS) is a lethal autosomal recessive form of arthrogryposis multiplex congenita (AMC). LCCS is genetically heterogeneous with mutations in five genes identified to date, all with a role in the innervation or contractile apparatus of skeletal muscles. In a consanguineous Saudi family with multiple stillbirths presenting with LCCS, we excluded linkage to all known LCCS loci and combined autozygome analysis and whole-exome sequencing to identify a novel homozygous variant in ZBTB42, which had been shown to be enriched in skeletal muscles, especially at the neuromuscular junction. Knockdown experiments of zbtb42 in zebrafish consistently resulted in grossly abnormal skeletal muscle development and myofibrillar disorganization at the microscopic level. This severe muscular phenotype is successfully rescued with overexpression of the human wild-type ZBTB42 gene, but not with the mutant form of ZBTB42 that models the human missense change. Our data assign a novel muscular developmental phenotype to ZBTB42 in vertebrates and establish a new LCCS6 type caused by ZBTB42 mutation.

POMK mutations disrupt muscle development leading to a spectrum of neuromuscular presentations.

Dystroglycan is a transmembrane glycoprotein whose interactions with the extracellular matrix (ECM) are necessary for normal muscle and brain development, and disruptions of its function lead to dystroglycanopathies, a group of congenital muscular dystrophies showing extreme genetic and clinical heterogeneity. Specific glycans bound to the extracellular portion of dystroglycan, α-dystroglycan, mediate ECM interactions and most known dystroglycanopathy genes encode glycosyltransferases involved in glycan synthesis. POMK, which was found mutated in two dystroglycanopathy cases, is instead involved in a glycan phosphorylation reaction critical for ECM binding, but little is known about the clinical presentation of POMK mutations or of the function of this protein in the muscle. Here, we describe two families carrying different truncating alleles, both removing the kinase domain in POMK, with different clinical manifestations ranging from Walker-Warburg syndrome, the most severe form of dystroglycanopathy, to limb-girdle muscular dystrophy with cognitive defects. We explored POMK expression in fetal and adult human muscle and identified widespread expression primarily during fetal development in myocytes and interstitial cells suggesting a role for this protein during early muscle differentiation. Analysis of loss of function in the zebrafish embryo and larva showed that pomk function is necessary for normal muscle development, leading to locomotor dysfuction in the embryo and signs of muscular dystrophy in the larva. In summary, we defined diverse clinical presentations following POMK mutations and showed that this gene is necessary for early muscle development.

Mutation of the mitochondrial carrier SLC25A42 causes a novel form of mitochondrial myopathy in humans.

Myopathies are heterogeneous disorders characterized clinically by weakness and hypotonia, usually in the absence of gross dystrophic changes. Mitochondrial dysfunction is a frequent cause of myopathy. We report a simplex case born to consanguineous parents who presented with muscle weakness, lactic acidosis, and muscle changes suggestive of mitochondrial dysfunction. Combined autozygome and exome analysis revealed a missense variant in the SLC25A42 gene, which encodes an inner mitochondrial membrane protein that imports coenzyme A into the mitochondrial matrix. Zebrafish slc25a42 knockdown morphants display severe muscle disorganization and weakness. Importantly, these features are rescued by normal human SLC25A42 RNA, but not by RNA harboring the patient's variant. Our data support a potentially causal link between SLC25A42 mutation and mitochondrial myopathy in humans.

Identification of KLHL41 Mutations Implicates BTB-Kelch-Mediated Ubiquitination as an Alternate Pathway to Myofibrillar Disruption in Nemaline Myopathy.

Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM.

Loss of catalytically inactive lipid phosphatase myotubularin-related protein 12 impairs myotubularin stability and promotes centronuclear myopathy in zebrafish.

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., "MTMRs"). Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12) binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through identification of drugs that could stabilize the myotubularin-MTMR12 complex and hence ameliorate this disorder.

Exome sequencing and functional validation in zebrafish identify GTDC2 mutations as a cause of Walker-Warburg syndrome.

Whole-exome sequencing (WES), which analyzes the coding sequence of most annotated genes in the human genome, is an ideal approach to studying fully penetrant autosomal-recessive diseases, and it has been very powerful in identifying disease-causing mutations even when enrollment of affected individuals is limited by reduced survival. In this study, we combined WES with homozygosity analysis of consanguineous pedigrees, which are informative even when a single affected individual is available, to identify genetic mutations responsible for Walker-Warburg syndrome (WWS), a genetically heterogeneous autosomal-recessive disorder that severely affects the development of the brain, eyes, and muscle. Mutations in seven genes are known to cause WWS and explain 50%-60% of cases, but multiple additional genes are expected to be mutated because unexplained cases show suggestive linkage to diverse loci. Using WES in consanguineous WWS-affected families, we found multiple deleterious mutations in GTDC2 (also known as AGO61). GTDC2's predicted role as an uncharacterized glycosyltransferase is consistent with the function of other genes that are known to be mutated in WWS and that are involved in the glycosylation of the transmembrane receptor dystroglycan. Therefore, to explore the role of GTDC2 loss of function during development, we used morpholino-mediated knockdown of its zebrafish ortholog, gtdc2. We found that gtdc2 knockdown in zebrafish replicates all WWS features (hydrocephalus, ocular defects, and muscular dystrophy), strongly suggesting that GTDC2 mutations cause WWS.

Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation.

Previously, we identified family with sequence similarity 65, member B (Fam65b), as a protein transiently up-regulated during differentiation and fusion of human myogenic cells. Silencing of Fam65b expression results in severe reduction of myogenin expression and consequent lack of myoblast fusion. The molecular function of Fam65b and whether misregulation of its expression could be causative of muscle diseases are unknown. Protein pulldowns were used to identify Fam65b-interacting proteins in differentiating human muscle cells and regenerating muscle tissue. In vitro, human muscle cells were treated with histone-deacetylase (HDAC) inhibitors, and expression of Fam65b and interacting proteins was studied. Nontreated cells were used as controls. In vivo, expression of Fam65b was down-regulated in developing zebrafish to determine the effects on muscle development. Fam65b binds to HDAC6 and dysferlin, the protein mutated in limb girdle muscular dystrophy 2B. The tricomplex Fam65b-HDAC6-dysferlin is transient, and Fam65b expression is necessary for the complex to form. Treatment of myogenic cells with pan-HDAC or HDAC6-specific inhibitors alters Fam65b expression, while dysferlin expression does not change. Inhibition of Fam65b expression in developing zebrafish results in abnormal muscle, with low birefringence, tears at the myosepta, and increased embryo lethality. Fam65b is an essential component of the HDAC6-dysferlin complex. Down-regulation of Fam65b in developing muscle causes changes consistent with muscle disease.-Balasubramanian, A., Kawahara, G., Gupta, V. A., Rozkalne, A., Beauvais, A., Kunkel, L. M., Gussoni, E. Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation.

Standardization of zebrafish drug testing parameters for muscle diseases.

Skeletal muscular diseases predominantly affect skeletal and cardiac muscle, resulting in muscle weakness, impaired respiratory function and decreased lifespan. These harmful outcomes lead to poor health-related quality of life and carry a high healthcare economic burden. The absence of promising treatments and new therapies for muscular disorders requires new methods for candidate drug identification and advancement in animal models. Consequently, the rapid screening of drug compounds in an animal model that mimics features of human muscle disease is warranted. Zebrafish are a versatile model in preclinical studies that support developmental biology and drug discovery programs for novel chemical entities and repurposing of established drugs. Due to several advantages, there is an increasing number of applications of the zebrafish model for high-throughput drug screening for human disorders and developmental studies. Consequently, standardization of key drug screening parameters, such as animal husbandry protocols, drug compound administration and outcome measures, is paramount for the continued advancement of the model and field. Here, we seek to summarize and explore critical drug treatment and drug screening parameters in the zebrafish-based modeling of human muscle diseases. Through improved standardization and harmonization of drug screening parameters and protocols, we aim to promote more effective drug discovery programs.

Intrinsic and extrinsic regulation of rhabdomyolysis susceptibility by Tango2.

Rhabdomyolysis is a clinical emergency characterized by severe muscle damage, resulting in the release of intracellular muscle components, which leads to myoglobinuria and, in severe cases, acute kidney failure. Rhabdomyolysis is caused by genetic factors linked to increased disease susceptibility in response to extrinsic triggers. Recessive mutations in TANGO2 result in episodic rhabdomyolysis, metabolic crises, encephalopathy and cardiac arrhythmia. The underlying mechanism contributing to disease onset in response to specific triggers remains unclear. To address these challenges, we created a zebrafish model of Tango2 deficiency. Here, we demonstrate that the loss of Tango2 in zebrafish results in growth defects, early lethality and increased susceptibility of skeletal muscle defects in response to extrinsic triggers, similar to TANGO2-deficient patients. Using lipidomics, we identified alterations in the glycerolipid pathway in tango2 mutants, which is critical for membrane stability and energy balance. Therefore, these studies provide insight into key disease processes in Tango2 deficiency and have increased our understanding of the impacts of specific defects on predisposition to environmental triggers in TANGO2-related disorders.