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Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

Zebrafish are a preferred vertebrate model for delineating genotype-phenotype relationships. One of the most studied features of zebrafish is their exceptional swimming ability. By 7 days postfertilization (dpf), zebrafish spend over two-thirds of their time engaged in spontaneous swimming activity and several months later they are capable of attaining some of the fastest swimming velocities relative to body length ever recorded in the laboratory. However, laboratory-assembled flumes capable of achieving the slow flow velocities characteristics of larvae as well as the relatively fast maximal velocities of adults have not been described in sufficient detail to allow easy replication. Here we describe an easily assembled, open-source zebrafish-scaled flume for assessing swimming performance. The flume uses two independent spherical-impeller pumps modulated by a microcontroller to achieve flow velocities ranging from 1 to 70 cm s-1. The microcontroller also monitors water temperature and flow velocity and sends these data to a personal computer for real-time display and storage. Incremental protocols for assessing maximal swimming speed (Umax) were developed, stored in custom software, and then uploaded to the microcontroller in order to assess performance of larval (14, 21, 28 dpf), juvenile (35, 42 dpf), and adult (8, 22 month) zebrafish. The flume had sufficient range and sensitivity to detect developmental changes in Umax of larvae and juveniles, an 18-24% faster Umax of adult males vs. females, and a 14-20% age-related reduction in Umax for the oldest zebrafish. Detailed information is provided to assemble and operate this low-cost, versatile, and reliable tool for assessing zebrafish swimming performance.

PurposeChanges in stiffness or extensibility of the muscle or muscle–tendon unit with aging could lead to impaired function and an increased vulnerability to injury. We aimed to investigate the passive force and viscoelastic properties of single muscle fibers in older adults.MethodsSeven older adults (mean age 79.0 ± 3.8 years) and 10 young control (mean age 25.6 ± 4.5 years) were recruited. Biopsy specimens were obtained percutaneously from m. vastus lateralis and skinned single fibers were used for the experiments. Slack tests were performed to determine maximal force and maximal unloaded shortening velocity. Passive force was measured in pCa 9.0 solution using a stepwise stretch technique with increment of sarcomere length from 2.4 to 4.2 µm. Myosin heavy chain (MHC) isoform was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Specific force was calculated as maximal force divided by cross-sectional area. Passive force, peak passive force, time to half stress relaxation (T1/2) and force decay index (a force time integral under a stress relaxation curve) were measured.ResultsNo difference between the groups were found in specific force and shortening velocity. Passive force and peak passive force were greater in both MHC I and IIa fibers of older adults (p < 0.001, p = 0.012, respectively, at 4.2 mm SL). Force decay index was higher in older adults. (p = 0.001 at 4.2 µm SL). There were no significant differences in passive force and viscoelastic properties between fiber types.ConclusionWe demonstrated greater passive force and viscoelastic properties at the level of single fibers in older adults.

Non-invasive and effort independent biomarkers are needed to better assess the effects of drug therapy on healthy muscle and that affected by muscular dystrophy (mdx). Here we evaluated the use of multi-frequency electrical impedance for this purpose with comparison to force and histological parameters. Eight wild-type (wt) and 10 mdx mice were treated weekly with RAP-031 activin type IIB receptor at a dose of 10 mg kg-1 twice weekly for 16 weeks; the investigators were blinded to treatment and disease status. At the completion of treatment, impedance measurements, in situ force measurements, and histology analyses were performed. As compared to untreated animals, RAP-031 wt and mdx treated mice had greater body mass (18% and 17%, p < 0.001 respectively) and muscle mass (25% p < 0.05 and 22% p < 0.001, respectively). The Cole impedance parameters in treated wt mice, showed a 24% lower central frequency (p < 0.05) and 19% higher resistance ratio (p < 0.05); no significant differences were observed in the mdx mice. These differences were consistent with those seen in maximum isometric force, which was greater in the wt animals (p < 0.05 at > 70 Hz), but not in the mdx animals. In contrast, maximum force normalized by muscle mass was unchanged in the wt animals and lower in the mdx animals by 21% (p < 0.01). Similarly, myofiber size was only non-significantly higher in treated versus untreated animals (8% p = 0.44 and 12% p = 0.31 for wt and mdx animals, respectively). Our findings demonstrate electrical impedance of muscle reproduce the functional and histological changes associated with myostatin pathway inhibition and do not reflect differences in muscle size or volume. This technique deserves further study in both animal and human therapeutic trials.

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmdmdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmdmdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle-specific miR-486 overexpression in Dmdmdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.

Age-related deficits in skeletal muscle function, termed sarcopenia, are due to loss of muscle mass and changes in the intrinsic mechanisms underlying contraction. Sarcopenia is associated with falls, functional decline, and mortality. Electrical impedance myography (EIM)—a minimally invasive, rapid electrophysiological tool—can be applied to animals and humans to monitor muscle health, thereby serving as a biomarker in both preclinical and clinical studies. EIM has been successfully employed in several species; however, the application of EIM to the assessment of zebrafish—a model organism amenable to high-throughput experimentation—has not been reported. Here, we demonstrated differences in EIM measures between the skeletal muscles of young (6 months of age) and aged (33 months of age) zebrafish. For example, EIM phase angle and reactance at 2 kHz showed significantly decreased phase angle (5.3 ± 2.1 versus 10.7 ± 1.5°; p = 0.001) and reactance (89.0 ± 3.9 versus 172.2 ± 54.8 ohms; p = 0.007) in aged versus young animals. Total muscle area, in addition to other morphometric features, was also strongly correlated to EIM 2 kHz phase angle across both groups (r = 0.7133, p = 0.01). Moreover, there was a strong correlation between 2 kHz phase angle and established metrics of zebrafish swimming performance, including turn angle, angular velocity, and lateral motion (r = 0.7253, r = 0.7308, r = 0.7857, respectively, p < 0.01 for all). In addition, the technique was shown to have high reproducibility between repeated measurements with a mean percentage difference of 5.34 ± 1.17% for phase angle. These relationships were also confirmed in a separate replication cohort. Together, these findings establish EIM as a fast, sensitive method for quantifying zebrafish muscle function and quality. Moreover, identifying the abnormalities in the bioelectrical properties of sarcopenic zebrafish provides new opportunities to evaluate potential therapeutics for age-related neuromuscular disorders and to interrogate the disease mechanisms of muscle degeneration.

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.

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.

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.

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. Generation and characterization of a conditional dystrophin ( Dmd ) knockout mouse reveals myofiber-specific requirements for muscle homeostasis, and an essential role for Purkinje neuron Dmd in neurobehavior and learning function.