Mouse Model Of Duchenne Muscular Dystrophy Research Articles

Effects of a Ketogenic Diet on the Assessment of Biochemical and Clinical Parameters in Duchenne Muscular Dystrophy: A Preclinical Investigation.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive skeletal muscle degeneration and systemic effects, including the central nervous system (CNS). This study aimed to assess the impact of a 14-day ketogenic diet (DCet) on biochemical and clinical parameters in a DMD mouse model. Young adult mice (50 days old) were fed DCet, while control groups received a standard diet. On the 14th day, memory and behavior tests were conducted, followed by biochemical evaluations of oxidative stress, inflammatory biomarkers, body weight, feed intake, and brain-derived neurotrophic factor (BDNF) levels. mdx + DCet mice showed reduced mass (0.2 g ± 2.49) and improved memory retention (p < 0.05) compared to controls. Oxidative damage in muscle tissue and CNS decreased, along with a significant cytokine level reduction (p <0.05). The protocol led to an increase in hippocampal BDNF and mitochondrial respiratory complex activity in muscle tissue and the central nervous system (CNS), while also decreasing creatine kinase activity only in the striatum. Overall, a 14-day DCet showed protective effects by improving spatial learning and memory through reductions in oxidative stress and immune response, as well as increases in BDNF levels, consistent with our study's findings.

Expression of Dystrophin Dp71 Splice Variants Is Temporally Regulated During Rodent Brain Development.

Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.

Respiratory characterization of a humanized Duchenne muscular dystrophy mouse model

Duchenne muscular dystrophy (DMD) is the most common X-linked disease. DMD is caused by a lack of dystrophin, a critical structural protein in striated muscle. Dystrophin deficiency leads to inflammation, fibrosis, and muscle atrophy. Boys with DMD have progressive muscle weakness within the diaphragm that results in respiratory failure in the 2nd or 3rd decade of life. The most common DMD mouse model – the mdx mouse – is not sufficient for evaluating genetic medicines that specifically target the human DMD (hDMD) gene sequence. Therefore, a novel transgenic mouse carrying the hDMD gene with an exon 52 deletion was created (hDMDΔ52;mdx). We characterized the respiratory function and pathology in this model using whole body plethysmography, histology, and immunohistochemistry. At 6-months-old, hDMDΔ52;mdx mice have reduced maximal respiration, neuromuscular junction pathology, and fibrosis throughout the diaphragm, which worsens at 12-months-old. In conclusion, the hDMDΔ52;mdx exhibits moderate respiratory pathology, and serves as a relevant animal model to study the impact of novel genetic therapies, including gene editing, on respiratory function.

GDE5/Gpcpd1 activity determines phosphatidylcholine composition in skeletal muscle and regulates contractile force in mice

Glycerophosphocholine (GPC) is an important precursor for intracellular choline supply in phosphatidylcholine (PC) metabolism. GDE5/Gpcpd1 hydrolyzes GPC into choline and glycerol 3-phosphate; this study aimed to elucidate its physiological function in vivo. Heterozygous whole-body GDE5-deficient mice reveal a significant GPC accumulation across tissues, while homozygous whole-body knockout results in embryonic lethality. Skeletal muscle-specific GDE5 deletion (Gde5 skKO) exhibits reduced passive force and improved fatigue resistance in electrically stimulated gastrocnemius muscles in vivo. GDE5 deficiency also results in higher glycolytic metabolites and glycogen levels, and glycerophospholipids alteration, including reduced levels of phospholipids that bind polyunsaturated fatty acids (PUFAs), such as DHA. Interestingly, this PC fatty acid compositional change is similar to that observed in skeletal muscles of denervated and Duchenne muscular dystrophy mouse models. These are accompanied by decrease of GDE5 expression, suggesting a regulatory role of GDE5 activity for glycerophospholipid profiles. Furthermore, a DHA-rich diet enhances contractile force and lowers fatigue resistance, suggesting a functional relationship between PC fatty acid composition and muscle function. Finally, skinned fiber experiments show that GDE5 loss increases the probability of the ryanodine receptor opening and lowers the maximum Ca2+-activated force. Collectively, GDE5 activity plays roles in PC and glucose/glycogen metabolism in skeletal muscle.

Cyclo His-Pro Attenuates Muscle Degeneration in Murine Myopathy Models.

Among the inherited myopathies, a group of muscular disorders characterized by structural and metabolic impairments in skeletal muscle, Duchenne muscular dystrophy (DMD) stands out for its devastating progression. DMD pathogenesis is driven by the progressive degeneration of muscle fibers, resulting in inflammation and fibrosis that ultimately affect the overall muscle biomechanics. At the opposite end of the spectrum of muscle diseases, age-related sarcopenia is a common condition that affects an increasing proportion of the elderly. Although characterized by different pathological mechanisms, DMD and sarcopenia share the development of progressive muscle weakness and tissue inflammation. Here, the therapeutic effects of Cyclo Histidine-Proline (CHP) against DMD and sarcopenia are evaluated. In the mdx mouse model of DMD, it is shown that CHP restored muscle contractility and force production, accompanied by the reduction of fibrosis and inflammation in skeletal muscle. CHP furthermore prevented the development of cardiomyopathy and fibrosis in the diaphragm, the two leading causes of death for DMD patients. CHP also attenuated muscle atrophy and functional deterioration in a mouse model of age-related sarcopenia. These findings from two different models of muscle dysfunction hence warrant further investigation into the effects of CHP on muscle pathologies in animal models and eventually in patients.

Potential limitations of micro-dystrophin gene therapy for Duchenne muscular dystrophy.

Clinical trials delivering high doses of adeno-associated viruses (AAVs) expressing truncated dystrophin molecules (micro-dystrophins) are underway for individuals with Duchenne muscular dystrophy (DMD). We examined the efficiency and efficacy of this strategy with four micro-dystrophin constructs (three in clinical trials and a variant of the largest clinical construct), in a severe mouse model of DMD, using doses of AAV comparable to those used in the clinical trials. We achieved high levels of micro-dystrophin expression in striated muscle with cardiac expression ~10 fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of the skeletal muscle disease was observed. Surprisingly, a lethal acceleration of cardiac disease progression occurred with two of the micro-dystrophins. The detrimental impact on the heart appears to be caused by the high levels of micro-dystrophin resulting in variable competition (dependent on the design of the micro-dystrophin) between micro-dystrophin and utrophin at the cardiomyocyte membrane. There may also be a contribution from an overloading of protein degradation. The significance of these observations for patients currently being treated with AAV-micro-dystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown. However, it suggests that micro-dystrophin treatments need to avoid excessively high levels of expression in the heart and cardiac function should be carefully monitored in these patients.

Gut-muscle axis: establishing the connection between microbial signaling and skeletal muscle function in a mouse model (mdx) of Duchenne muscular dystrophy

The metabolic and endocrine nature of skeletal muscle allows for systemic signaling and influence over the enteric environment influencing gut microbial populations. Concomitantly, commensal microbial signaling can alter the composition and development of skeletal muscle forming a bidirectional gut-muscle axis. Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease characterised by skeletal muscle atrophy, fibrosis, and ultimately cardiorespiratory failure. Previous studies have shown that gut microbial signaling can alter the pathways involved in muscle atrophy, enhancing atrophy when dysbiotic, or blunting the atrophic effect by restoring gut microbial populations to a healthy status; thus, we sought to characterize the metabolic signaling between the gut and the diaphragm in a mouse model of DMD ( mdx) to identify potential therapeutic targets. The study was divided into two arms and conducted following institutional ethical approval. The first arm investigated metabolomic signalling. 6-month-old male BL10 wildtype (WT; n = 12) and mdx (n = 12) mice were anaesthetized (induction by 5% isoflurane) and decapitated. Following collection of trunk blood, the diaphragm was excised, and colonic/cecal contents were collected. The samples were then processed by mass-spectrometry to isolate the metabolomic profiles of semi-polar, secondary bile acid, and short chain fatty acid (SCFA) targets in the gut, plasma and diaphragm of WT and mdx mice. The second arm of the study explored the gut microbial profile and diaphragm gene expression, structure and function. 6-month-old male WT (n = 15) and mdx (n = 15) were anesthetized (induction by 5% isoflurane) followed by cervical dislocation. The diaphragm was excised, and longitudinal strips were isolated for structural analysis (i.e., histological staining and immunohistochemistry for fibre type and size), PCR gene expression, and isometric force assessment ex vivo. Additionally, faecal pellets were collected and analysed via shotgun metagenomic sequencing to assess microbial populations. Diaphragm twitch and tetanic force in mdx were profoundly decreased compared to WT. Metabolic analysis of faecal metabolites indicated impairment in the three main tryptophan metabolic pathways in mdx mice: kynurenine, indole and serotonin. Metagenomic analysis indicated a profound genotype effect between WT and mdx gut microbial populations. Two main producers of the short chain fatty acid acetate (Lactobacillus and Bifidobacterium) were significantly decreased within the mdx group. Acetate serves as a substate in the TCA cycle contributing to ATP production and positively affects skeletal muscle via numerous pathways including the down regulation of key atrogenes associated with atrophy. Pathway enrichment analysis showed a global disruption to both purine and pyrimidine metabolism in all three tissue types. Purine/pyrimidine metabolism have been tightly linked with microbial metabolism and affect skeletal muscle health and function. Further understanding of the gut-muscle axis in dystrophic models may pave the way for novel microbiota-directed therapeutic strategies to ameliorate muscle dysfunction in DMD. Funded by Science Foundation Ireland SFI/19/6628 INSPIRE DMD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibiting Mitochondrial Fission Protein Drp1 Enhances Mitochondrial Structure, Attenuates Lipid Peroxidation, and Improves Muscle Strength in D2-mdx Mice

Duchenne Muscular Dystrophy (DMD) is the most severe muscular dystrophy manifested by progressive muscle wasting and weakness. Mitochondrial dysfunction and reactive oxygen species (ROS) accumulation are early pathological features of DMD and precede myopathy. A fine balance of mitochondrial dynamics (fission and fusion) is crucial to maintain mitochondrial function and skeletal muscle health. We recently reported excessive activation of Dynamin-Related Protein 1 (Drp1)-mediated mitochondrial fission in two animal models of DMD. Mitochondrial fission inhibitor‐1 (Mdivi‐1) is an effective pharmacological inhibitor of Drp1-mediated mitochondrial fission and has been shown benefits in numerous diseases. The objective of the study was to test whether Mdivi-1 could improve skeletal muscle mitochondrial structure, function and muscle strength in D2- mdx mouse model of DMD. Methods: 9-10-week-old male D2- mdx mice were treated with either Mdivi-1 (I.P., 40 mg/kg BW, mdx/Mdivi-1) or vehicle (2%DMSO in PBS, mdx/VEH) for 5 weeks. D2-wildtype mice treated with vehicle (WT/VEH) were used as the control (n=8/group). Muscle strength was measured using a grip strength meter. Mitochondria were isolated from quad muscles collected 24 hours after the last injection to measure mitochondrial respiration. Transmission Electron Microscopy was used to image mitochondrial structure. Gastrocnemius muscles were collected for immunoblot analysis. Results: Grip strength was significantly lower in muscles from D2- mdx mice compared to WT controls (P = 0.01) but was improved in mdx+Mdivi-1 mice (P = 0.05). Mitochondrial fission protein markers, Drp1(Ser616) phosphorylation and Fis1 were markedly higher in skeletal muscles from mdx/VEH than WT/VEH (P= 0.009 and 0.001) but were attenuated in mdx/Mdivi-1 mice (P = 0.041 and 0.1), indicating a rebalance of mitochondrial dynamics. This rebalance subsequently led to improvement in skeletal muscle mitochondrial structure in mdx/Mdivi-1 mice, with evidence of lower numbers of damaged mitochondria and reduced circularity (P = 0.005 and 0.038). Although not statistically significant, ADP and FCCP-stimulated mitochondrial respiration were enhanced by 93.8% and 92.4% in mdx+Mdivi-1 mice compared to mdx/VEH. In addition, lipid peroxidation marker, 4-HNE was significantly higher in mdx/VEH group in comparison to WT/VEH group (P = &lt;0.0001), but was attenuated in mdx/Mdivi-1 mice (P = 0.08). Conclusion: These data demonstrate that inhibition of Drp1-mediated mitochondrial fission is effective in improving muscular strength in D2- mdx mice, which is at least partly due to enhanced mitochondrial dynamics, structure and attenuated lipid peroxidation. Mdivi-1 may be a viable treatment for DMD. National Institute of Health (R15DK131512). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Leucyl-tRNA Synthetase Contributes to Muscle Weakness through Mammalian Target of Rapamycin Complex 1 Activation and Autophagy Suppression in a Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD), caused by loss-of-function mutations in the dystrophin gene, results in progressive muscle weakness and early fatality. Impaired autophagy is one of the cellular hallmarks of DMD, contributing to the disease progression. Molecular mechanisms underlying the inhibition of autophagy in DMD are not well understood. In the current study the DMD mouse model mdx is used for the investigation of signaling pathways leading to suppression of autophagy. Mammalian target of rapamycin complex 1 (mTORC1) is found to be hyperactive in the DMD muscles, accompanying muscle weakness and autophagy impairment. Surprisingly, Akt, a well-known upstream regulator of mTORC1, is not responsible for mTORC1 activation or the dystrophic muscle phenotypes. Instead, leucyl-tRNA synthetase (LeuRS) is found to be overexpressed in mdx muscles compared to the wild-type. LeuRS is known to activate mTORC1 in a non-canonical mechanism that involves interaction with RagD, an activator of mTORC1. Disrupting LeuRS interaction with RagD by the small-molecule inhibitor BC-LI-0186 reduces mTORC1 activity, restores autophagy, and ameliorates myofiber damage in the mdx muscles. Furthermore, inhibition of LeuRS by BC-LI-0186 improves dystrophic muscle strength in an autophagy-dependent manner. Taken together, our findings uncover a non-canonical function of the house-keeping protein LeuRS as a potential therapeutic target in the treatment of DMD.

Myospreader improves gene editing in skeletal muscle by myonuclear propagation

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

The BALB/c.mdx62 mouse exhibits a dystrophic muscle pathology and is a model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating monogenic skeletal muscle-wasting disorder. Although many pharmacological and genetic interventions have been reported in preclinical studies, few have progressed to clinical trials with meaningful benefit. Identifying therapeutic potential can be limited by availability of suitable preclinical mouse models. More rigorous testing across models with varied background strains and mutations can identify treatments for clinical success. Here, we report the generation of a DMD mouse model with a CRISPR-induced deletion within exon 62 of the dystrophin gene (Dmd) and the first generated in BALB/c mice. Analysis of mice at 3, 6 and 12 months of age confirmed loss of expression of the dystrophin protein isoform Dp427 and resultant dystrophic pathology in limb muscles and the diaphragm, with evidence of centrally nucleated fibers, increased inflammatory markers and fibrosis, progressive decline in muscle function, and compromised trabecular bone development. The BALB/c.mdx62 mouse is a novel model of DMD with associated variations in the immune response and muscle phenotype, compared with those of existing models. It represents an important addition to the preclinical model toolbox for developing therapeutic strategies.

MCU-independent Ca2+ uptake mediates mitochondrial Ca2+ overload and necrotic cell death in a mouse model of Duchenne muscular dystrophy

Mitochondrial Ca2+ overload can mediate mitochondria-dependent cell death, a major contributor to several human diseases. Indeed, Duchenne muscular dystrophy (MD) is driven by dysfunctional Ca2+ influx across the sarcolemma that causes mitochondrial Ca2+ overload, organelle rupture, and muscle necrosis. The mitochondrial Ca2+ uniporter (MCU) complex is the primary characterized mechanism for acute mitochondrial Ca2+ uptake. One strategy for preventing mitochondrial Ca2+ overload is deletion of the Mcu gene, the pore forming subunit of the MCU-complex. Conversely, enhanced MCU-complex Ca2+ uptake is achieved by deleting the inhibitory Mcub gene. Here we show that myofiber-specific Mcu deletion was not protective in a mouse model of Duchenne MD. Specifically, Mcu gene deletion did not reduce muscle histopathology, did not improve muscle function, and did not prevent mitochondrial Ca2+ overload. Moreover, myofiber specific Mcub gene deletion did not augment Duchenne MD muscle pathology. Interestingly, we observed MCU-independent Ca2+ uptake in dystrophic mitochondria that was sufficient to drive mitochondrial permeability transition pore (MPTP) activation and skeletal muscle necrosis, and this same type of activity was observed in heart, liver, and brain mitochondria. These results demonstrate that mitochondria possess an uncharacterized MCU-independent Ca2+ uptake mechanism that is sufficient to drive MPTP-dependent necrosis in MD in vivo.

Apelin stimulation of the vascular skeletal muscle stem cell niche enhances endogenous repair in dystrophic mice.

Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here, we showed that defects in the endothelial cell (EC) compartment of the vascular stem cell niche in mouse models of Duchenne MD, laminin α2-related MD, and collagen VI-related myopathy were associated with inefficient mobilization of MuSCs after tissue damage. Using chemoinformatic analysis, we identified the 13-amino acid form of the peptide hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. Systemic administration of AP-13 using osmotic pumps generated a pro-proliferative EC-rich niche that supported MuSC function through angiocrine factors and markedly improved tissue regeneration and muscle strength in all three dystrophic mouse models. Moreover, EC-specific knockout of the apelin receptor led to regenerative defects that phenocopied key pathological features of MD, including vascular defects, fibrosis, muscle fiber necrosis, impaired MuSC function, and reduced force generation. Together, these studies provide in vivo proof of concept that enhancing endogenous skeletal muscle repair by targeting the vascular niche is a viable therapeutic avenue for MD and characterized AP-13 as a candidate for further study for the systemic treatment of MuSC dysfunction.

Correction of human nonsense mutation via adenine base editing for Duchenne muscular dystrophy treatment in mouse

Duchenne muscular dystrophy (DMD) is the most prevalent herediatry disease in men, characterized by dystrophin deficiency, progressive muscle wasting, cardiac insufficiency, and premature mortality, with no effective therapeutic options. Here, we investigated whether adenine base editing can correct pathological non-sense point mutations leading to premature stop codons in the dystrophin gene. We identified 27 causative nonsense mutations in our DMD patient cohort. Treatment with adenine base editor (ABE) could restore dystrophin expression by direct A-to-G editing of pathological nonsense mutations in cardiomyocytes generated from DMD patient-derived induced pluripotent stem cells. We also generated two humanized mouse models of DMD expressing mutation-bearing exons 23 or 30 of human dystrophin gene. Intramuscular administration of ABE, driven by ubiquitous or muscle-specific promoters could correct these non-sense mutations in vivo, albeit with higher efficiency in exon 30, restoring dystrophin expression in skeletal fibers of humanized DMD mice. Moreover, a single systemic delivery of ABE with human sgRNA could induce body-wide dystrophin expression and improve muscle function in rotarod tests of humanized DMD mice. These findings demonstrate that ABE with human sgRNAs can confer therapeutic alleviation of DMD in mice, providing a basis for development of adenine base editing therapies in monogenic diseases.

Amelioration of Morphological Pathology in Cardiac, Respiratory, and Skeletal Muscles Following Intraosseous Administration of Human Dystrophin Expressing Chimeric (DEC) Cells in Duchenne Muscular Dystrophy Model.

Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutation in the dystrophin gene. Currently there is no cure for DMD. We introduced a novel human Dystrophin Expressing Chimeric (DEC) cell therapy of myoblast origin and confirmed the safety and efficacy of DEC in the mdx mouse models of DMD. In this study, we assessed histological and morphological changes in the cardiac, diaphragm, and gastrocnemius muscles of the mdx/scid mice after the transplantation of human DEC therapy via the systemic-intraosseous route. The efficacy of different DEC doses was evaluated at 90 days (0.5 × 106 and 1 × 106 DEC cells) and 180 days (1 × 106 and 5 × 106 DEC cells) after administration. The evaluation of Hematoxylin & Eosin (H&E)-stained sectional slices of cardiac, diaphragm, and gastrocnemius muscles included assessment of muscle fiber size by minimal Feret's diameter method using ImageJ software. The overall improvement in muscle morphology was observed in DMD-affected target muscles in both studies, as evidenced by a shift in fiber size distribution toward the wild type (WT) phenotype and by an increase in the mean Feret's diameter compared to the vehicle-injected controls. These findings confirm the long-term efficacy of human DEC therapy in the improvement of overall morphological pathology in the muscles affected by DMD and introduce DEC as a novel therapeutic approach for DMD patients.

Cell-mediated exon skipping normalizes dystrophin expression and muscle function in a new mouse model ofDuchenne Muscular Dystrophy.

Cell therapy for muscular dystrophy has met with limited success, mainly due to the poor engraftment of donor cells, especially in fibrotic muscle at an advanced stage of the disease. We developed a cell-mediated exon skipping that exploits the multinucleated nature of myofibers to achieve cross-correction of resident, dystrophic nuclei by the U7 small nuclear RNA engineered to skip exon 51 of the dystrophin gene. We observed that co-culture of genetically corrected human DMD myogenic cells (but not of WT cells) with their dystrophic counterparts at a ratio of either 1:10 or 1:30 leads to dystrophin production at a level several folds higher than what predicted by simple dilution. This is due to diffusion of U7 snRNA to neighbouring dystrophic resident nuclei. When transplanted into NSG-mdx-Δ51mice carrying a mutation of exon 51, genetically corrected human myogenic cells produce dystrophin at much higher level than WT cells, well in the therapeutic range, and lead to force recovery even with an engraftment of only 3-5%. This level of dystrophin production is an important step towards clinical efficacy for cell therapy.

Retention of stress susceptibility in the mdx mouse model of Duchenne muscular dystrophy after PGC-1α overexpression or ablation of IDO1 or CD38.

Duchenne muscular dystrophy (DMD) is a lethal degenerative muscle wasting disease caused by the loss of the structural protein dystrophin with secondary pathological manifestations including metabolic dysfunction, mood and behavioral disorders. In the mildly affected mdx mouse model of DMD, brief scruff stress causes inactivity, while more severe subordination stress results in lethality. Here, we investigated the kynurenine pathway of tryptophan degradation and the nicotinamide adenine dinucleotide (NAD+) metabolic pathway in mdx mice and their involvement as possible mediators of mdx stress-related pathology. We identified downregulation of the kynurenic acid shunt, a neuroprotective branch of the kynurenine pathway, in mdx skeletal muscle associated with attenuated peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) transcriptional regulatory activity. Restoring the kynurenic acid shunt by skeletal muscle-specific PGC-1α overexpression in mdx mice did not prevent scruff -induced inactivity, nor did abrogating extrahepatic kynurenine pathway activity by genetic deletion of the pathway rate-limiting enzyme, indoleamine oxygenase 1. We further show that reduced NAD+ production in mdx skeletal muscle after subordination stress exposure corresponded with elevated levels of NAD+ catabolites produced by ectoenzyme cluster of differentiation 38 (CD38) that have been implicated in lethal mdx response to pharmacological β-adrenergic receptor agonism. However, genetic CD38 ablation did not prevent mdx scruff-induced inactivity. Our data do not support a direct contribution by the kynurenine pathway or CD38 metabolic dysfunction to the exaggerated stress response of mdx mice.

The slow-release adiponectin analog ALY688-SR modifies early-stage disease development in the D2.mdx mouse model of Duchenne muscular dystrophy.

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-β1 (TGF-β1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.

Empagliflozin treatment rescues abnormally reduced Na+ currents in ventricular cardiomyocytes from dystrophin-deficient mdx mice.

Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a severe muscle illness caused by mutations in the gene encoding for the intracellular protein dystrophin. A major source for arrhythmia vulnerability in patients with DMD is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, decreased cardiac output, and the development of reentrant circuits. Using the dystrophin-deficient mdx mouse model for human DMD, we previously reported that the lack of dystrophin causes a significant loss of peak Na+ current (INa) in ventricular cardiomyocytes. This finding provided a mechanistic explanation for ventricular conduction defects and concomitant arrhythmias in the dystrophic heart. In the present study, we explored the hypothesis that empagliflozin (EMPA), an inhibitor of sodium/glucose cotransporter 2 in clinical use to treat type II diabetes and nondiabetic heart failure, rescues peak INa loss in dystrophin-deficient ventricular cardiomyocytes. We found that INa of cardiomyocytes derived from mdx mice, which had received clinically relevant doses of EMPA for 4 wk, was restored to wild-type level. Moreover, incubation of isolated mdx ventricular cardiomyocytes with 1 µM EMPA for 24 h significantly increased their peak INa. This effect was independent of Na+-H+ exchanger 1 inhibition by the drug. Our findings imply that EMPA treatment can rescue abnormally reduced peak INa of dystrophin-deficient ventricular cardiomyocytes. Long-term EMPA administration may diminish arrhythmia vulnerability in patients with DMD.NEW & NOTEWORTHY Dystrophin deficiency in cardiomyocytes leads to abnormally reduced Na+ currents. These can be rescued by long-term empagliflozin treatment.

Electrical impedance myography detects dystrophin-related muscle changes in mdx mice

BackgroundThe lack of functional dystrophin protein in Duchenne muscular dystrophy (DMD) causes chronic skeletal muscle inflammation and degeneration. Therefore, the restoration of functional dystrophin levels is a fundamental approach for DMD therapy. Electrical impedance myography (EIM) is an emerging tool that provides noninvasive monitoring of muscle conditions and has been suggested as a treatment response biomarker in diverse indications. Although magnetic resonance imaging (MRI) of skeletal muscles has become a standard measurement in clinical trials for DMD, EIM offers distinct advantages, such as portability, user-friendliness, and reduced cost, allowing for remote monitoring of disease progression or response to therapy. To investigate the potential of EIM as a biomarker for DMD, we compared longitudinal EIM data with MRI/histopathological data from an X-linked muscular dystrophy (mdx) mouse model of DMD. In addition, we investigated whether EIM could detect dystrophin-related changes in muscles using antisense-mediated exon skipping in mdx mice.MethodsThe MRI data for muscle T2, the magnetic resonance spectroscopy (MRS) data for fat fraction, and three EIM parameters with histopathology were longitudinally obtained from the hindlimb muscles of wild-type (WT) and mdx mice. In the EIM study, a cell-penetrating peptide (Pip9b2) conjugated antisense phosphorodiamidate morpholino oligomer (PPMO), designed to induce exon-skipping and restore functional dystrophin production, was administered intravenously to mdx mice.ResultsMRI imaging in mdx mice showed higher T2 intensity at 6 weeks of age in hindlimb muscles compared to WT mice, which decreased at ≥ 9 weeks of age. In contrast, EIM reactance began to decline at 12 weeks of age, with peak reduction at 18 weeks of age in mdx mice. This decline was associated with myofiber atrophy and connective tissue infiltration in the skeletal muscles. Repeated dosing of PPMO (10 mg/kg, 4 times every 2 weeks) in mdx mice led to an increase in muscular dystrophin protein and reversed the decrease in EIM reactance.ConclusionsThese findings suggest that muscle T2 MRI is sensitive to the early inflammatory response associated with dystrophin deficiency, whereas EIM provides a valuable biomarker for the noninvasive monitoring of subsequent changes in skeletal muscle composition. Furthermore, EIM reactance has the potential to monitor dystrophin-deficient muscle abnormalities and their recovery in response to antisense-mediated exon skipping.