Jeffrey J. Widrick

Duchenne muscular dystrophy (DMD) patients suffer from skeletal and cardiopulmonary weakness, and up to one third are diagnosed on the autism spectrum. Dystrophin is an essential protein for regulating intracellular force transmission to the extracellular matrix (ECM) within the skeletal muscle, but also plays key roles in neurobehavior and cognitive function. The mouse Dmd gene is X-linked and has several isoforms with enriched tissue expression in the skeletal muscle, heart, and the brain. Constitutive and inducible deletion of muscle dystrophin resulted in a skeletal muscle myopathy, dystrophic histopathology, and functional deficits compared to the mdx mouse. Transcriptomic analysis of Dmd mKO muscles revealed dysregulation of ECM and cytokine pathways. Purkinje dystrophin knockout (Dmd:Pcp2 KO) mice displayed neurobehavioral deficits in social approach, social memory, and spatial memory. These studies reveal the essential requirement for dystrophin expression in both the skeletal muscle and brain for normal physiological and neurobehavioral function.

Background: Dystrophin-deficient zebrafish larvae are a small, genetically tractable vertebrate model of Duchenne muscular dystrophy that is well suited for early-stage therapeutic development. However, current approaches for evaluating their mobility, a physiologically relevant therapeutic outcome, yield data of low resolution and high variability that provides minimal insight into potential mechanisms responsible for their abnormal locomotion. Methods: To address these issues, we used high speed videography and deep learning-based markerless motion capture to quantify escape response (ER) swimming kinematics of two dystrophic zebrafish strains (sapje and sapje-like). Each ER was partitioned into an initiating C-start, a subsequent power stroke, and a final burst of undulatory swimming activity. Results: Markerless motion capture provided repeatable, high precision estimates of swimming kinematics. Random forest and support vector machine prediction models identified overall ER distance and peak speed, the instantaneous speed conferred by the power stroke, and the average speed and distance covered during burst swimming as the most predictive biomarkers for differentiating dystrophic from wild-type larvae. For each of these predictors, mutant and wild-type larvae differed markedly with effect sizes ranging from 2.4 to 3.7 standard deviations. To identify mechanisms underlying these performance deficits, we evaluated the amplitude and frequency of propulsive tail movements. There was little evidence that tail stroke amplitude was affected by the absence of dystrophin. Instead, temporal aspects of tail kinematics, including tail maximal angular velocity during the C-start and power stroke and tail stroke frequency during burst swimming, were slowed in mutants. In fact, tail kinematics were as effective as direct, non-survival in vitro assessments of tail muscle contractility in differentiating mutant from wild-type larvae. Conclusions: ER kinematics can be used as precise and physiologically relevant biomarkers of the dystrophic phenotype, may serve as non-lethal proxies for skeletal muscle dysfunction, and reveal new insights into why mobility is impaired in the absence of dystrophin. The approach outlined here opens new possibilities for the design and interpretation of studies using zebrafish to model movement disorders.

Therapeutic strategies for Duchenne Muscular Dystrophy (DMD) will likely require complementary approaches. One possibility is to explore genetic modifiers that improve muscle regeneration and function. The beneficial effects of the overexpression of Jagged-1 were described in escaper golden retriever muscular dystrophy (GRMD) dogs that had a near-normal life and validated in dystrophin-deficient zebrafish (1). To clarify the underlying biology of JAG1 overexpression in dystrophic muscles, we generated a transgenic mouse (mdx ^5cv - JAG1 ) model that lacks dystrophin and overexpresses human JAG1 in striated muscles. Skeletal muscles from mdx ^5cv - JAG1 and mdx ^5cv mice were studied at one, four, and twelve-month time points. JAG1 expression in mdx ^5cv - JAG1 increased by three to five times compared to mdx ^5cv . Consequently, mdx ^5cv - JAG1 muscles were significantly bigger and stronger than dystrophic controls, along with an increased number of myofibers. Proteomics data show increased dysferlin in mdx ^5cv - JAG1 muscles and an association of Nsd1 with the phenotype. Our data supports the positive effect of JAG1 overexpression in dystrophic muscles. Significance Statement: Duchenne Muscular Dystrophy (DMD) patients present a progressive decline in motor function. DMD is caused by mutations in the DMD gene that lead to the absence of dystrophin - an essential component of muscle cells. However, dystrophin-deficient dogs overexpressing JAG1 had a normal lifespan with remarkable motor function. In this study, we increased expression of human JAG1 in mouse skeletal muscles lacking dystrophin to explore mechanisms responsible for these benefits. Our observations show that overexpression of JAG1 counterbalances the lack of dystrophin by generating bigger and stronger muscles as the mouse ages. Moreover, our proteomics dataset suggests a role of dysferlin in the phenotype. Therefore, our study supports the exploration of JAG1 in pre-clinical models.

Duchenne muscular dystrophy (DMD) patients suffer from skeletal and cardiopulmonary weakness, and interestingly up to one third are diagnosed on the autism spectrum. Dystrophin is an essential protein for regulating the transmission of intracellular force to the extracellular matrix within the skeletal muscle, but also plays key roles in neurobehavior and cognitive function. The mouse dystrophin gene (also abbreviated Dmd ) is X-linked and has several isoforms with tissue-specific expression, including the large Dp427m muscle transcript found in heart and skeletal muscle, and the Dp427c transcript that encodes the brain-specific dystrophin cerebellar protein. Understanding the functional requirements and pathways that are affected by dystrophin loss will impact dystrophin replacement gene therapy and exon-skipping correction strategies. We generated conditional Dystrophin knockout mice by targeting exon 52 of the mouse Dystrophin ( Dmd ^flox52 ) locus. We generated dystrophin constitutive and inducible myofiber knockout ( Dmd mKO) mice to evaluate the tissue-specific function of the large skeletal muscle dystrophin isoform. Constitutive embryonic deletion of the Dystrophin gene exclusively in skeletal myofibers resulted in a severe skeletal muscle myopathy, dystrophic histopathology, and functional deficits compared to the mdx mouse. Transcriptomic analysis of skeletal myofibers of the Dmd mKO mice revealed the dysregulation of key extracellular matrix and cytokine signaling pathways. Separately, we generated Purkinje neuron cerebellar dystrophin knockout ( Dmd :Pcp2 KO) mice that displayed neurobehavioral deficits in social approach, social memory, and spatial navigation and working memory. These studies reveal the essential requirement for dystrophin expression in both the skeletal muscle and brain for normal physiological and neurobehavioral function. Significance Statement: Duchenne muscular dystrophy is caused by the lack of a functional dystrophin protein in muscle. The large dystrophin (Dp427m) isoform is expressed in skeletal, cardiac, and smooth muscle, but its tissue-specific requirements remain unknown. We generated and characterized a conditional skeletal muscle knockout mouse ( Dmd mKO). Constitutive embryonic genetic ablation of skeletal muscle Dystrophin resulted in muscle histopathologies similar to the mdx mouse, while postnatal muscle Dystrophin ablation resulted in milder pathologies. Ablating cerebellar Dystrophin Dp427c using a Pcp2/L7 -Cre driver resulted in sociobehavioral defects. Transcriptomic analysis of the Dmd mKO mice showed a severe reduction of extracellular matrix and cytokine signaling pathways. Our study reveals an essential role for skeletal muscle dystrophin and identifies essential pathways for modulation using dystrophin-replacement therapies.

Dystrophin-deficient zebrafish larvae are a small, genetically tractable vertebrate model of Duchenne muscular dystrophy well suited for early stage therapeutic development. However, current approaches for evaluating their impaired mobility, a physiologically relevant therapeutic target, are characterized by low resolution and high variability. To address this, we used high speed videography and deep learning-based markerless motion capture to develop linked-segment models of larval escape response (ER) swimming. Kinematic models provided repeatable, high precision estimates of larval ER performance. Effect sizes for ER peak instantaneous acceleration and speed, final displacement, and ER distance were 2 to 3.5 standard deviations less for dystrophin-deficient mutants vs. wild-types. Further analysis revealed that mutants swam slower because of a reduction in their tail stroke frequency with little change in tail stroke amplitude. Kinematic variables were highly predictive of the dystrophic phenotype with ≤ 3% of larvae misclassified by random forest and support vector machine models. Tail kinematics also performed as well as in vitro assessments of tail muscle contractility in classifying larvae as mutants or wild-type, suggesting that ER kinematics could serve as a non-lethal proxy for direct measurements of muscle function. In summary, ER kinematics can be used as precise, physiologically relevant, non-lethal biomarkers of the dystrophic phenotype. The open-source approach described here may have applications not only for studies of skeletal muscle disease but for other disciplines that use larval mobility as an experimental outcome.

DOCK (dedicator of cytokinesis) is an 11‐member family of typical guanine nucleotide exchange factors (GEFs) expressed in the brain, spinal cord, and skeletal muscle. Several DOCK proteins have been implicated in maintaining several myogenic processes such as fusion. We previously identified DOCK3 as being strongly upregulated in Duchenne muscular dystrophy (DMD), specifically in the skeletal muscles of DMD patients and dystrophic mice. Dock3 ubiquitous KO mice on the dystrophin‐deficient background exacerbated skeletal muscle and cardiac phenotypes. We generated Dock3 conditional skeletal muscle knockout mice ( Dock3 mKO) to characterize the role of DOCK3 protein exclusively in the adult muscle lineage. Dock3 mKO mice presented with significant hyperglycemia and increased fat mass, indicating a metabolic role in the maintenance of skeletal muscle health. Dock3 mKO mice had impaired muscle architecture, reduced locomotor activity, impaired myofiber regeneration, and metabolic dysfunction. We identified a novel DOCK3 interaction with SORBS1 through the C‐terminal domain of DOCK3 that may account for its metabolic dysregulation. Together, these findings demonstrate an essential role for DOCK3 in skeletal muscle independent of DOCK3 function in neuronal lineages.

Zebrafish larvae are one of the few vertebrates amenable to large-scale drug discovery screens. Larval swimming behavior is often used as an outcome variable and many fields of study have developed assays for evaluating swimming performance. An unintended consequence of this wide interest is that details related to assay methodology and interpretation become scattered across the literature. The aim of this review is to consolidate this information, particularly as it relates to high-throughput approaches. The authors describe larval swimming behaviors as this forms the basis for understanding their experimentally evoked swimming or spontaneous activity. Next, they detail how swimming activity can serve as an outcome variable, particularly in the multi-well formats used in large-scale screening studies. They also highlight biological and technical factors that can impact the sensitivity and variability of these measurements. Careful attention to animal husbandry, experimental design, data acquisition, and interpretation of results can improve screen outcomes by maximizing swimming activity while minimizing intra- and inter-larval variability. The development of more sensitive, quantitative methods of assessing swimming performance that can be incorporated into high-throughput workflows will be important in order to take full advantage of the zebrafish model.

DOCK (dedicator of cytokinesis) is an 11-member family of typical guanine nucleotide exchange factors (GEFs) expressed in the brain, spinal cord, and skeletal muscle. Several DOCK proteins have been implicated in maintaining several myogenic processes such as fusion. We previously identified DOCK3 as being strongly upregulated in Duchenne muscular dystrophy (DMD), specifically in the skeletal muscles of DMD patients and dystrophic mice. Dock3 ubiquitous KO mice on the dystrophin-deficient background exacerbated skeletal muscle and cardiac phenotypes. We generated Dock3 conditional skeletal muscle knockout mice ( Dock3 mKO) to characterize the role of DOCK3 protein exclusively in the adult muscle lineage. Dock3 mKO mice presented with significant hyperglycemia and increased fat mass, indicating a metabolic role in the maintenance of skeletal muscle health. Dock3 mKO mice had impaired muscle architecture, reduced locomotor activity, impaired myofiber regeneration, and metabolic dysfunction. We identified a novel DOCK3 interaction with SORBS1 through the C-terminal domain of DOCK3 that may account for its metabolic dysregulation. Together, these findings demonstrate an essential role for DOCK3 in skeletal muscle independent of DOCK3 function in neuronal lineages.

Striated preferentially expressed protein kinase ( SPEG ), a myosin light chain kinase, is mutated in centronuclear myopathy (CNM) and/or dilated cardiomyopathy. No precise therapies are available for this disorder, and gene replacement therapy is not a feasible option due to the large size of SPEG . We evaluated the potential of dynamin-2 (DNM2) reduction as a potential therapeutic strategy because it has been shown to revert muscle phenotypes in mouse models of CNM caused by MTM1 , DNM2 , and BIN1 mutations. We determined that SPEG-β interacted with DNM2, and SPEG deficiency caused an increase in DNM2 levels. The DNM2 reduction strategy in Speg -KO mice was associated with an increase in life span, body weight, and motor performance. Additionally, it normalized the distribution of triadic proteins, triad ultrastructure, and triad number and restored phosphatidylinositol-3-phosphate levels in SPEG-deficient skeletal muscles. Although DNM2 reduction rescued the myopathy phenotype, it did not improve cardiac dysfunction, indicating a differential tissue-specific function. Combining DNM2 reduction with other strategies may be needed to target both the cardiac and skeletal defects associated with SPEG deficiency. DNM2 reduction should be explored as a therapeutic strategy against other genetic myopathies (and dystrophies) associated with a high level of DNM2.

MiR-486 is a muscle-enriched microRNA, or “myomiR,” that has reduced expression correlated with Duchenne muscular dystrophy (DMD). To determine the function of miR-486 in normal and dystrophin-deficient muscles and elucidate miR-486 target transcripts in skeletal muscle, we characterized mir-486 knockout mice (mir-486 KO). mir-486 KO mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects were exacerbated in mir-486 KO:mdx^5cv (DKO) mice. To identify direct in vivo miR-486 muscle target transcripts, we integrated RNA sequencing and chimeric miRNA eCLIP sequencing to identify key transcripts and pathways that contribute towards mir-486 KO and dystrophic disease pathologies. These targets included known and novel muscle metabolic and dystrophic structural remodeling factors of muscle and skeletal muscle contractile transcript targets. Together, our studies identify miR-486 as essential for normal muscle function, a driver of pathological remodeling in dystrophin-deficient muscle, a useful biomarker for dystrophic disease progression, and highlight the use of multiple omic platforms to identify in vivo microRNA target transcripts.