The detection of dystrophin and the proteins of the dystrophin-associated protein complex (DAPC) by immunostaining allows for the detection of the presence and localization of these proteins in slide-mounted sections of skeletal muscle or heart tissue. This technique has been used historically for the diagnosis and characterization of muscle disorders including Duchenne muscular dystrophy and many of the limb girdle and congenital muscular dystrophies. The extensive usefulness of this technique has allowed the development of numerous antibodies that can be used in immunostaining assays. As an increasing number of research and translational science studies have recently focused on the restoration of dystrophin as a therapeutic strategy, high-quality immunostaining techniques and analysis remain sought-after scientific practices. This chapter describes useful procedures to perform the immunofluorescence staining for dystrophin and several other useful DAPC proteins, while also providing guidance based on our clinical diagnostic and translational science research experience from working in the muscular dystrophy field.

SDS-PAGE Western blots allow for the identification of the presence and relative quantity of proteins of interest on the basis of their molecular weight and their recognition by specific antibodies. This technique has historically been useful in the diagnosis and study of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), as it allows the recognition of clinically relevant abnormalities in the amount or size of dystrophin. While this technique is used infrequently for diagnosis at this point (as genetic testing for pathogenic variants of the DMD gene has become standard practice), it remains an important tool in basic science research and in the evaluation of therapies that restore or replace dystrophin expression. This chapter provides a technique that is suitable for the quantitative assessment of dystrophin expression in isolates of skeletal muscle tissue, based on our experience and guidance provided by the US Food and Drug Administration (FDA) for these types of assays. Topics associated with the design of quantitative Western blot techniques, options for loading control use, and the challenges associated with variable pathology within specimens are also discussed. Performing this technique can provide reproducible and reliable quantification of full-length or shortened dystrophin protein content in frozen muscle tissue.

Adeno-associated virus (AAV)-mediated liver injury is the primary off-target toxicity observed with viral gene therapy. Understanding of the underlying mechanisms has been hindered by the fact that murine studies poorly model AAV toxicity. The X-linked myotubular myopathy (XLMTM) gene therapy program epitomizes this therapeutic discordance, because a subset of participants treated with AAV8-driven myotubularin (MTM1) gene replacement developed fatal liver injury that was not predicted by preclinical models. Here, we investigated a multihit hypothesis whereby loss of Mtm1 interacts with the postweaning environment to precipitate liver injury in XLMTM mouse models. Mtm1 knockout (KO) mice fed purified ingredient diets manifested liver dysfunction that mirrored patient clinical presentations, including elevated plasma transaminases, altered bile acid composition, and histological features of cholestasis. We further demonstrated a role for MTM1 in maintaining hepatocyte structure and localization of the bile salt export pump. Liver-specific deletion of Mtm1 recapitulated many of these features, supporting a liver-autonomous role for Mtm1. Treatment of diet-sensitized mice with AAV8-driven gene therapy increased the susceptibility of cholestasis in global KO mice and induced liver injury in wild-type mice. Last, as a proof of concept, we treated global KO mice with lipid nanoparticle MTM1 gene replacement, which prevented development of key histopathological liver abnormalities. These findings provide critical insights into the environmental precipitants and molecular mechanisms of liver dysfunction in XLMTM and the adverse events seen in viral gene therapy studies and more broadly offer a framework to model AAV-associated toxicity and identify potential therapeutic interventions.

Imaging of dystrophin and related proteins using light microscopy is a key assay in neuromuscular pathology diagnostic and research laboratories. Immunofluorescence co-staining of dystrophin alongside markers like laminin or its glycoprotein binding partners allows for quantitative analysis of these markers in relation to each other, enabling the reporting of metrics like the percentage of dystrophin-positive fibers and dystrophin staining intensity at the sarcolemma. When designed carefully, automation of image analysis approaches allows for higher throughput, greater precision, and less bias than manual counting of muscle fibers. These automated approaches are especially valuable for rigorously and objectively testing the efficacy of potential therapeutic strategies in development for restoring dystrophin expression. This chapter presents a validated method, its modifications, and some alternatives for automated quantitative analysis of immunofluorescence images of dystrophin or other muscle markers co-stained with a sarcolemmal marker for muscle fiber detection.

Use of adeno-associated virus (AAV)-mediated transfer of functional microdystrophins to address Duchenne muscular dystrophy (DMD) has been established. RGX-202, an AAV8 vector encoding a novel, optimized human microdystrophin with an extended C-terminal domain, expressed under the Spc5-12 muscle-specific promoter, was evaluated for tolerability and efficacy in dystrophin-deficient mdx mouse in 12- and 26-week studies at vector doses ranging from 3 × 1013 to 5 × 1014 gc/kg. A single intravenous administration of RGX-202 was well tolerated and led to robust, dose-dependent expression of microdystrophin in skeletal and cardiac muscles at 12 weeks, which persisted to 26 weeks post-administration. Increased microdystrophin was associated with recruitment of the dystrophin-associated protein complex to the sarcolemma, particularly at doses ≥1 × 1014 gc/kg. Histologic and magnetic resonance imaging examinations revealed marked and sustained suppression of dystrophic pathology in all RGX-202-treated mice. Functionally, gains were observed in the in vitro specific force of extensor digitorum longus muscle, in vivo grip strength, and the rescue of treadmill and gait deficits. These findings provide preclinical evidence for the therapeutic efficacy of RGX-202 at a minimum effective dose (MED) of 1 × 1014 gc/kg in the murine DMD model. This MED served as the starting dose for the RGX-202 clinical study (NCT05693142), which has currently completed phase III enrollment.

Skeletal muscle is a complex syncytial arrangement of an array of cell types and, in the case of muscle-specific cells (myofibres), subtypes. There exists extensive heterogeneity in skeletal muscle functional behaviour and molecular landscape at the cell composition, myofibre subtype and intra-myofibre subtype level. This heterogeneity highlights limitations in currently applied methodological approaches, which has stagnated our understanding of fundamental skeletal muscle biology in both healthy and myopathic contexts. Here we developed a novel approach that combines a fluorescence-based assay for the biophysical examination of the sarcomeric protein, myosin, coupled with same-myofibre high-sensitivity proteome profiling, termed single myofibre protein function-omics (SMPFO). Applying this approach as proof-of-principle we identify the integrated relationship between myofibre functionality and the underlying proteomic landscape that guides divergent, but physiologically important, behaviour in myofibre subtypes in healthy human skeletal muscle. By applying SMPFO to two forms of human nemaline myopathy (ACTA1 and TNNT1 mutations), we reveal significant reduction in the divergence of myofibre subtypes across both biophysical and proteomic behaviour. Collectively we demonstrate preliminary findings of SMPFO to support its use to study skeletal muscle with greater specificity, accuracy and resolution than currently applied methods, facilitating that advancement in understanding of skeletal muscle tissue in both healthy and diseased states. KEY POINTS: Skeletal muscle is a complex tissue made up of an array of cell and sub-cell types, with the resident muscle cell - myofibre - critical for contractile function. Although single myofibre studies have advanced, existing methods lack the precision for simultaneous multidata analysis, hindering developments in our understanding of skeletal muscle. We introduce single myofibre protein function-omics (SMPFO), a method enabling functional analysis of sarcomeric myosin alongside global protein abundance within the same myofibre. In healthy myofibres SMyoMFO reveals extensive biochemical diversity in myosin heads, correlating with the abundance of metabolic and sarcomeric proteins, including subtype-specific patterns in sarcoglycan delta (SGCD). In contrast SMyoMFO uniquely reveals a reduction in diversity of myosin function and the myofibre proteome in two forms of nemaline myopathy, highlighting disease-associated alterations. This innovative approach provides a robust framework for investigating myofibre regulation and dysfunction in skeletal muscle biology.

[This corrects the article DOI: 10.3389/fimmu.2025.1527840.].

BackgroundInefficient transduction is a major limitation in achieving therapeutic levels of AAV-delivered microdystrophin capable of improving muscle function in patients with Duchenne muscular dystrophy. Additionally, some patients experience acute complications due to activation of innate immune pathways, such as complement. We propose that inhibiting complement receptor 1/2/3 (CR 1/2/3)-mediated phagocytosis and endosomal TLR 7/8/9 signaling pathways may decrease immune and inflammatory responses while simultaneously increasing the availability of AAV virus for muscle transduction.MethodsMdx mice were randomly assigned to the following three experimental conditions (n=8-9/group): Group 1, mdx untreated; Group 2, mdx + rAAV9-microdystrophin; Group 3, mdx + rAAV9-microdystrophin + semiweekly dosing of TLR 7/8/9 antagonist + complement receptor antibodies (combination therapy). The rAAV9-microdystrophin was administered twice to 6- and 12-week-old mice. A separate group of 6-week-old mice received a single rAAV9-microdystrophin dose and no other treatment (Group 4). We assessed several immune and inflammatory responses and dystrophin expression in the muscle.ResultsViral load was significantly increased by 77-fold in white blood cells after two rAAV9-microdystrophin doses compared to mice receiving a single dose. Repeated gene therapy resulted in a lower viral load and microdystrophin expression in muscle compared to a single rAAV dose. 63% of mice treated with two rAAV9-microdystrophin doses produced antibodies to dystrophin, which was less in mice treated with two rAAV9-microdystrophin doses and combination therapy (25%). Likewise, AAV capsid specific antibody levels were reduced in mice receiving combination therapy. Microdystrophin expression in skeletal muscle evaluated by mass spectrometry, immunofluorescence, and western blotting showed significantly higher levels in combination-treated mice compared to rAAV9-microdystrophin alone.ConclusionsOur results demonstrate that combination treatment with complement receptor 1/2/3 antibodies and a TLR 7/8/9 antagonist enhances rAAV9-microdystrophin gene therapy in mdx mice by partially reducing inflammatory and immune responses and increasing microdystrophin expression in skeletal muscle. Furthermore, repeated gene therapy is associated with greater uptake by white blood cells and less microdystrophin expression in the skeletal muscle. This suggests that blocking complement receptors and/or TLR 7/8/9 pathways would be a promising strategy to enhance AAV-microdystrophin therapy.

X-linked myotubular myopathy (XLMTM) due to MTM1 mutations is a rare and often lethal congenital myopathy. Its downstream molecular and cellular mechanisms are currently incompletely understood. The most abundant protein in muscle, myosin, has been implicated in the pathophysiology of other congenital myopathies. Hence, in the present study, we aimed to define whether myosin is also dysfunctional in XLMTM and whether it, thus, may constitute a potential drug target. To this end, we used skeletal muscle tissue from patients and canine/mouse models; we performed Mant-ATP chase experiments coupled with x-ray diffraction analyses and LC/MS-based proteomics studies. In patients with XLMTM, we found that myosin molecules are structurally disordered and preferably adopt their ATP-consuming biochemical state. This phosphorylation-related (mal)adaptation was mirrored by a striking remodeling of the myofiber energetic proteome in XLMTM dogs. In line with these, we confirmed an accrued myosin ATP consumption in mice lacking MTM1. Hence, we treated these with a myosin ATPase inhibitor, mavacamten. After a 4-week treatment period, we observed a partial restoration of the myofiber proteome, especially proteins involved in cytoskeletal, sarcomeric, and energetic pathways. Altogether, our study highlights myosin inhibition as a potentially new drug mechanism for the complex XLMTM muscle phenotype.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, resulting in dystrophin deficiency in skeletal/cardiac muscle and progressive loss of function. Although the genetic causes of DMD have been thoroughly investigated, the energetic consequences have not been well examined across animal models. Previously, the laboratory examined mitochondrial function across nemaline myopathy mouse models of varying disease severity; here, mitochondrial phenotypes in DMD are assessed through the comparison of the milder C57BL/10ScSn-Dmdmdx/J (B10-mdx) and the more severe D2.B10-Dmdmdx/J mouse (D2-mdx) mouse models. D2-mdx exhibit a significant decrease in mitochondrial respiration, undetectable ATP concentrations, increased mitochondrial membrane potential, and alterations in electron transport chain enzyme activities. In contrast, B10-mdx show only mild mitochondrial phenotypes, including decreased ATP content. The D2-mdx mouse has genetic modifiers, including latent transforming growth factor-β-binding protein 4 (LTBP4) and annexin A6, that have been shown to alter DMD severity in humans. However, these modifiers did not account for mitochondrial differences seen in mdx mice. Both models were treated with a microdystrophin adeno-associated virus gene therapy to assess whether dystrophin restoration rescued mitochondrial phenotypes. Gene therapy attenuated the ATP deficiency in the B10-mdx mice, but only improved mitochondrial membrane potentials in D2-mdx mice. The exact cause of the D2-mdx mitochondrial phenotypes remains unknown, but secondary disease processes that affect mitochondrial phenotypes should be taken into consideration when choosing an animal model for DMD studies.

What is this summary about? This summary describes the results of a research study (clinical trial) called ASPIRO that was published in the Lancet Neurology in 2023. This study looked at an investigational gene therapy called resamirigene bilparvovec (also known as AT132) as a possible treatment for children with a disease called X-linked myotubular myopathy (abbreviated as XLMTM).

In skeletal muscle, troponin T (TnT) exists in two isoforms, slow skeletal TnT (ssTnT) and fast skeletal TnT (fsTnT), encoded by the TNNT1 and TNNT3 genes, respectively. Nonsense or missense TNNT1 variants have been associated with skeletal muscle weakness and contractures and a histopathological appearance of nemaline myopathy (NM) on muscle biopsy. Little is known about how TNNT1 mutations ultimately lead to muscle dysfunction, preventing the development of targeted therapeutic interventions. Here, we aimed to identify the underlying molecular biophysical mechanisms, by investigating isolated skeletal myofibres from patients with TNNT1-related NM as well as from controls through a combination of structural and functional assays. Our studies revealed variable and unusual ssTnT and fsTnT expression patterns and post-translational modifications. We also observed that, in the presence of TNNT1 variants, the thin filament was more compliant, and this was associated with a higher myofibre Ca2+ sensitivity. Altogether, our findings suggest TnT remodelling as the key mechanism ultimately leading to molecular and cellular hyper-contractility, and then inhibitors of altered contractility as potential therapeutic modalities for TNNT1-associated NM. KEY POINTS: No therapeutic treatment exists for patients with genetic TNNT1 mutations and skeletal muscle weakness/contractures. In these patients, expression and post-translational modifications of troponin T are severely disrupted. These are associated with changes in thin filament compliance where troponin T is located. All these induce muscle fibre hyper-contractility that can be reversed by mavacamten, a myosin ATPase inhibitor.

Background: Slow skeletal troponin T (ssTnT, TNNT1 ) is the tropomyosin-binding subunit of the troponin complex in the slow-twitch fibers of skeletal muscle. Exon 5 of TNNT1 is alternatively spliced, and retention of the 3’ region of intron 11 (exon 12’) has also been described. Variants in TNNT1 are known to cause nemaline myopathy (NM). Objective: To identify and further investigate the disease-causing variant in a patient with lethal NM. Methods: The genetic analyses included a gene panel, Sanger sequencing, whole-exome sequencing, and targeted array-CGH. Muscle biopsy was analyzed using routine histopathological methods. The alternative splicing of TNNT1 exon 12 in patient muscle was quantified from RNA sequencing data, and the protein expression was confirmed by western blot. Expression of ssTnT in patient muscle was studied by immunohistology. Results: The patient presented with arthrogryposis, stiffness, respiratory insufficiency, and minimal spontaneous movements. Histopathology showed hypotrophy and predominance of type II fibers, perimysial connective tissue accumulation, and nemaline bodies. The patient was homozygous for the TNNT1 missense variant (NM_003283.6:c.653C > G, p.(Pro218Arg), NM_ 001126132.3:c.612-7C > G), predicted to disrupt splicing. RNA-seq revealed inclusion of exon 12’ in 49.85% of transcripts, whereas in controls exon 12’ was not expressed. Exon 12’ expression on the protein level was confirmed by western blot. Immunohistology showed strong ssTnT expression in remaining type I fibers, and low expression in type IIA fibers. Conclusions: The c.653C > G variant was shown to alter TNNT1 splicing. The results suggest a novel pathogenetic mechanism involving abnormal expression of a troponin T isoform.

Abnormalities of myofiber size are often of diagnostic significance on skeletal muscle biopsies, mainly when myofibers are excessively small. The establishment of standards for myofiber size in children has been hampered until recently by the lack of tools to assess large numbers of fibers across a sizeable number of samples. This study describes the assessment of myofiber size in 349 histologically normal patient biopsy specimens obtained between 4 weeks and 25 years of life and corresponding primarily to locations in the thigh/quadriceps/vastus lateralis region. Biopsy specimens were selected for inclusion based on histologically normal light microscopic findings and minimal technical artifacts. H&E-stained slides were scanned and evaluated for minFeret diameter using a Visiopharm software app (APP #10164). MinFeret diameter fiber size data were then grouped into 18 age cohorts to establish normal ranges for males and females within each age cohort. A pilot study to compare known abnormal cases to these normal ranges was then performed to demonstrate how cases with abnormal fiber size might compare to these standards. This dataset provides a user-friendly and applicable set of standard fiber size ranges to assist in diagnostic and scientific work in children and young adults.

The occurrence of severe adverse events (SAEs) in patients with Duchenne muscular dystrophy (DMD), X-linked myotubular myopathy (XLMTM), and other neuromuscular diseases treated with adeno-associated virus (AAV) constructs has prompted studies to improve the safety and efficacy of gene therapy. Physicians have weighed the medical tenet of “first, do no harm” against the perspective of patients with progressive life-threatening conditions who may accept greater risk. Regarding SAE pathogenesis, discussion has focused on total AAV exposure and patient mutations more likely to induce immunity, while stressing the limitations of animal models in predicting adverse events. Therapeutic strategies for reducing side effects have employed more myotropic AAV serotypes and efficient transgenes. Other recommendations include excluding certain DMD gene mutations associated with SAEs and substituting less immunogenic transgenes such as utrophin (DMD) and myotubularin-related protein (XLMTM). For the sake of preclinical studies, emphasis has been placed on outbred rodents and larger animals that better predict immunity. Here, the absence of side effects in canine DMD and XLMTM models might be explained partly by phenotypic differences between affected humans and dogs. Specifically, dystrophin- and myotubularin-deficient dogs exhibit milder lesions, including less muscle fat deposition and the absence of hepatopathy, respectively, which could lead to reduced immune responses to AAV constructs. To better predict future problems, thought should be given to tracking early subclinical markers of the innate immune response, especially complement activation. Regardless of steps taken to improve the predictive value of animal models for SAEs, some questions will only be answered through human clinical trials after carefully considering the risk-benefit ratio.

BACKGROUND:X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital muscle disease caused by mutations in the MTM1 gene that result in profound muscle weakness, significant respiratory insufficiency, and high infant mortality. There is no approved disease-modifying therapy for XLMTM. Resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) is an investigational adeno-associated virus (AAV8)-mediated gene replacement therapy designed to deliver MTM1 to skeletal muscle cells and achieve long-term correction of XLMTM-related muscle pathology. The clinical trial ASPIRO (NCT03199469) investigating resamirigene bilparvovec in XLMTM is currently paused while the risk:benefit balance associated with this gene therapy is further investigated.METHODS:Muscle biopsies were taken before treatment and 24 and 48 weeks after treatment from ten boys with XLMTM in a clinical trial of resamirigene bilparvovec (ASPIRO; NCT03199469). Comprehensive histopathological analysis was performed.FINDINGS:Baseline biopsies uniformly showed findings characteristic of XLMTM, including small myofibres, increased internal or central nucleation, and central aggregates of organelles. Biopsies taken at 24 weeks post-treatment showed marked improvement of organelle localisation, without apparent increases in myofibre size in most participants. Biopsies taken at 48 weeks, however, did show statistically significant increases in myofibre size in all nine biopsies evaluated at this timepoint. Histopathological endpoints that did not demonstrate statistically significant changes with treatment included the degree of internal/central nucleation, numbers of triad structures, fibre type distributions, and numbers of satellite cells. Limited (predominantly mild) treatment-associated inflammatory changes were seen in biopsy specimens from five participants.INTERPRETATION:Muscle biopsies from individuals with XLMTM treated with resamirigene bilparvovec display statistically significant improvement in organelle localisation and myofibre size during a period of substantial improvements in muscle strength and respiratory function. This study identifies valuable histological endpoints for tracking treatment-related gains with resamirigene bilparvovec, as well as endpoints that did not show strong correlation with clinical improvement in this human study.FUNDING:Astellas Gene Therapies (formerly Audentes Therapeutics, Inc.).

X-linked myotubular myopathy (XL-MTM) is a rare life-threatening congenital myopathy caused by mutations in the MTM1 gene that encodes myotubularin, a ubiquitous enzyme required for normal development and function of skeletal muscle. The incidence of XLMTM in newborn males is 1:50,000. Recent evidence has revealed a previously unrecognized cholestatic tendency in patients with XL-MTM. EXCEL is a 48-week, prospective, observational, multicenter study to evaluate hepatobiliary health in patients with XLMTM. Approximately 50 male participants <18 years of age (up to half <5 years of age) with genetically confirmed XLMTM will be enrolled at 15–25 specialist sites in Canada, UK, and USA. The primary objective is to assess hepatobiliary health by estimating the incidence and prevalence of cholestatic complications in participants with XL-MTM. Secondary objectives are to evaluate the: (1) association between genetic variants of MTM1 and cholestasis; (2) association between environmental modifiers and cholestasis; and (3) healthcare utilization related to hepatobiliary and cholestatic complications. Participant assessments will be performed at the investigator's discretion based on standard of care at the site. A recommended schedule of assessments includes liver function tests (including serum bile acids), blood clotting parameters, creatinine, triglycerides and total cholesterol, and vitamin panel. Further clinical evaluation including liver ultrasound and Fibroscan will be used where appropriate. To reduce patient and caregiver burden, home healthcare services will collect laboratory tests when possible. Remote data collection may be used for healthcare resource utilization and patient medical history. Findings are expected to improve our understanding of cholestatic tendencies in participants with XL-MTM and provide critical information to improve the current management and treatment of XL-MTM, as well as future clinical trial designs. *Co-first authors.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder affecting 1:3500 male births and is associated with myofiber degeneration, regeneration, and inflammation. Glucocorticoid treatments have been the standard of care due to immunomodulatory/ immunosuppressive properties but novel genetic approaches, including exon skipping and gene replacement therapy, are currently being developed. The identification of additional biomarkers to assess DMDrelated inflammatory responses and the potential efficacy of these therapeutic approaches are thus of critical importance. The current study uses RNA sequencing of skeletal muscle from two mdx mouse models to identify high mobility group box 1 (HMGB1) as a candidate biomarker potentially contributing to DMD-related inflammation. HMGB1 protein content was increased in a human iPSC-derived skeletal myocyte model of DMD and microdystrophin treatment decreased HMGB1 back to control levels. In vivo, , HMGB1 protein levels were increased in vehicle treated B10-mdx mdx skeletal muscle compared to B10-WT and significantly decreased in B10-mdx mdx animals treated with adeno-associated virus (AAV)-microdystrophin. However, HMGB1 protein levels were not increased in D2-mdx mdx skeletal muscle compared to D2-WT, demonstrating a strain-specific difference in DMDrelated immunopathology.