Dystrophic Mice Research Articles

In vivo shear wave elasticity imaging for assessment of diaphragm function in muscular dystrophy.

Duchenne muscular dystrophy (DMD) causes patients to suffer from ambulatory disability and cardiorespiratory failure, the latter of which leads to premature death. Due to its role in respiration, the diaphragm is an important muscle for study. A common method for evaluating diaphragm function is ex vivo force testing, which only allows for an end point measurement. In contrast, ultrasound shear wave elastography imaging (US-SWEI) can assess diaphragm function over time; however, US-SWEI studies in dystrophic patients to date have focused on the limbs without preclinical studies. In this work, we used US-SWEI to estimate the shear wave speed (SWS) in diaphragm muscles of healthy (WT) mice, mdx mice, and mdx mice haploinsufficient for utrophin (mdx-utr) at 6 and 12 months of age. Diaphragms were then subjected to ex vivo force testing and histological analysis at 12 months of age. Between 6 and 12 months, a 23.8% increase in SWS was observed in WT mice and a 27.8% increase in mdx mice, although no significant difference was found in mdx-utr mice. Specific force generated by mdx-utr diaphragms was lower than that of WT diaphragms following twitch stimulus. A strong correlation between SWS and collagen deposition was observed, as well as between SWS and muscle fiber size. Together, these data demonstrate the ability of US-SWEI to evaluate dystrophic diaphragm functionality over time and predict the biochemical and morphological make-up of the diaphragm. Additionally, our results highlight the advantage of US-SWEI over ex vivo testing by obtaining longitudinal measurements in the same subject. STATEMENT OF SIGNIFICANCE: In DMD patients, muscles experience cycles of regeneration and degeneration that contribute to chronic inflammation and muscle weakness. This pathology only worsens with time and leads to muscle wasting, including in respiratory and cardiac muscles. Because respiratory failure is a major contributor to premature death in DMD patients, the diaphragm muscle is an important muscle to evaluate and treat over time. Currently, diaphragm function is assessed using ex vivo force testing, a technique that only allows measurement at sacrifice. In contrast, ultrasonography, particularly shear wave elasticity imaging (USSWEI), is a promising tool for longitudinal assessment; however, most US-SWEI in DMD patients aimed for limb muscles only with the absence of preclinical studies. This work broadens the applications of US-SWE imaging by demonstrating its ability to track properties and function of dystrophic diaphragm muscles longitudinally in multiple dystrophic mouse models.

ANT-dependent MPTP underlies necrotic myofiber death in muscular dystrophy.

Mitochondrial permeability transition pore (MPTP) formation contributes to ischemia-reperfusion injury in the heart and several degenerative diseases, including muscular dystrophy (MD). MD is a family of genetic disorders characterized by progressive muscle necrosis and premature death. It has been proposed that the MPTP has two molecular components, the adenine nucleotide translocase (ANT) family of proteins and an unknown component that requires the chaperone cyclophilin D (CypD) to activate. This model was examined in vivo by deleting the gene encoding ANT1 (Slc25a4) or CypD (Ppif) in a δ-sarcoglycan (Sgcd) gene-deleted mouse model of MD, revealing that dystrophic mice lacking Slc25a4 were partially protected from cell death and MD pathology. Dystrophic mice lacking both Slc25a4 and Ppif together were almost completely protected from necrotic cell death and MD disease. This study provides direct evidence that ANT1 and CypD are required MPTP components governing in vivo cell death, suggesting a previously unrecognized therapeutic approach in MD and other necrotic diseases.

Spatial transcriptomics reveal markers of histopathological changes in Duchenne muscular dystrophy mouse models

Duchenne muscular dystrophy is caused by mutations in the DMD gene, leading to lack of dystrophin. Chronic muscle damage eventually leads to histological alterations in skeletal muscles. The identification of genes and cell types driving tissue remodeling is a key step to developing effective therapies. Here we use spatial transcriptomics in two Duchenne muscular dystrophy mouse models differing in disease severity to identify gene expression signatures underlying skeletal muscle pathology and to directly link gene expression to muscle histology. We perform deconvolution analysis to identify cell types contributing to histological alterations. We show increased expression of specific genes in areas of muscle regeneration (Myl4, Sparc, Hspg2), fibrosis (Vim, Fn1, Thbs4) and calcification (Bgn, Ctsk, Spp1). These findings are confirmed by smFISH. Finally, we use differentiation dynamic analysis in the D2-mdx muscle to identify muscle fibers in the present state that are predicted to become affected in the future state.

Gpr18 agonist dampens inflammation, enhances myogenesis, and restores muscle function in models of Duchenne muscular dystrophy.

Introduction: Muscle wasting in Duchenne Muscular Dystrophy is caused by myofiber fragility and poor regeneration that lead to chronic inflammation and muscle replacement by fibrofatty tissue. Our recent findings demonstrated that Resolvin-D2, a bioactive lipid derived from omega-3 fatty acids, has the capacity to dampen inflammation and stimulate muscle regeneration to alleviate disease progression. This therapeutic avenue has many advantages compared to glucocorticoids, the current gold-standard treatment for Duchenne Muscular Dystrophy. However, the use of bioactive lipids as therapeutic drugs also faces many technical challenges such as their instability and poor oral bioavailability. Methods: Here, we explored the potential of PSB-KD107, a synthetic agonist of the resolvin-D2 receptor Gpr18, as a therapeutic alternative for Duchenne Muscular Dystrophy. Results and discussion: We showed that PSB-KD107 can stimulate the myogenic capacity of patient iPSC-derived myoblasts in vitro. RNAseq analysis revealed an enrichment in biological processes related to fatty acid metabolism, lipid biosynthesis, small molecule biosynthesis, and steroid-related processes in PSB-KD107-treated mdx myoblasts, as well as signaling pathways such as Peroxisome proliferator-activated receptors, AMP-activated protein kinase, mammalian target of rapamycin, and sphingolipid signaling pathways. In vivo, the treatment of dystrophic mdx mice with PSB-KD107 resulted in reduced inflammation, enhanced myogenesis, and improved muscle function. The positive impact of PSB-KD107 on muscle function is similar to the one of Resolvin-D2. Overall, our findings provide a proof-of concept that synthetic analogs of bioactive lipid receptors hold therapeutic potential for the treatment of Duchenne Muscular Dystrophy.

Transgene-free direct conversion of murine fibroblasts into functional muscle stem cells

Transcription factor-based cellular reprogramming provides an attractive approach to produce desired cell types for regenerative medicine purposes. Such cellular conversions are widely dependent on viral vectors to efficiently deliver and express defined factors in target cells. However, use of viral vectors is associated with unfavorable genomic integrations that can trigger deleterious molecular consequences, rendering this method a potential impediment to clinical applications. Here, we report on a highly efficient transgene-free approach to directly convert mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by overexpression of synthetic MyoD-mRNA in concert with an enhanced small molecule cocktail. First, we performed a candidate compound screen and identified two molecules that enhance fibroblast reprogramming into iMPCs by suppression of the JNK and JAK/STAT pathways. Simultaneously, we developed an optimal transfection protocol to transiently overexpress synthetic MyoD-mRNA in fibroblasts. Combining these two techniques enabled robust and rapid reprogramming of fibroblasts into Pax7 positive iMPCs in as little as 10 days. Nascent transgene-free iMPCs proliferated extensively in vitro, expressed a suite of myogenic stem cell markers, and could differentiate into highly multinucleated and contractile myotubes. Furthermore, using global and single-cell transcriptome assays, we delineated gene expression changes associated with JNK and JAK/STAT pathway inhibition during reprogramming, and identified in iMPCs a Pax7+ stem cell subpopulation resembling satellite cells. Last, transgene-free iMPCs robustly engrafted skeletal muscles of a Duchenne muscular dystrophy mouse model, restoring dystrophin expression in hundreds of myofibers. In summary, this study reports on an improved and clinically safer approach to convert fibroblasts into myogenic stem cells that can efficiently contribute to muscle regeneration in vivo.

Prevention of early-onset cardiomyopathy in Dmd exon 52–54 deletion mice by CRISPR-Cas9-mediated exon skipping

Duchenne muscular dystrophy (DMD) is a disease with a life-threatening trajectory resulting from mutations in the dystrophin gene, leading to degeneration of skeletal muscle and fibrosis of cardiac muscle. The overwhelming majority of mutations are multiexonic deletions. We previously established a dystrophic mouse model with deletion of exons 52-54 in Dmd that develops an early-onset cardiac phenotype similar to DMD patients. Here we employed CRISPR-Cas9 delivered intravenously by adeno-associated virus (AAV) vectors to restore functional dystrophin expression via excision or skipping of exon 55. Exon skipping with a solitary guide significantly improved editing outcomes and dystrophin recovery over dual guide excision. Some improvements to genomic and transcript editing levels were observed when the guide dose was enhanced, but dystrophin restoration did not improve considerably. Editing and dystrophin recovery were restricted primarily to cardiac tissue. Remarkably, our exon skipping approach completely prevented onset of the cardiac phenotype in treated mice up to 12weeks. Thus, our results demonstrate that intravenous delivery of a single-cut CRISPR-Cas9-mediated exon skipping therapy can prevent heart dysfunction in DMD invivo.

A cellular and molecular spatial atlas of dystrophic muscle

Asynchronous skeletal muscle degeneration/regeneration is a hallmark feature of Duchenne muscular dystrophy (DMD); however, traditional -omics technologies that lack spatial context make it difficult to study the biological mechanisms of how asynchronous regeneration contributes to disease progression. Here, using the severely dystrophic D2-mdx mouse model, we generated a high-resolution cellular and molecular spatial atlas of dystrophic muscle by integrating spatial transcriptomics and single-cell RNAseq datasets. Unbiased clustering revealed nonuniform distribution of unique cell populations throughout D2-mdx muscle that were associated with multiple regenerative timepoints, demonstrating that this model faithfully recapitulates the asynchronous regeneration observed in human DMD muscle. By probing spatiotemporal gene expression signatures, we found that propagation of inflammatory and fibrotic signals from locally damaged areas contributes to widespread pathology and that querying expression signatures within discrete microenvironments can identify targetable pathways for DMD therapy. Overall, this spatial atlas of dystrophic muscle provides a valuable resource for studying DMD disease biology and therapeutic target discovery.

LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

The potential role of liver kinase B1 (LKB1) in the altered activation of the master metabolic and epigenetic regulator adenosine monophosphate-activated protein kinase (AMPK) in Duchenne muscular dystrophy has not been investigated so far. Hence, we analyzed both gene and protein levels of LKB1 and its related targets in gastrocnemius muscles of adult C57BL/10 mdx mice and D2 mdx mice, a model with a more severe dystrophic phenotype, as well as the sensitivity of the LKB1-AMPK pathway to AMPK activators, such as chronic exercise. Our data show, for the first time, a reduction in the levels of LKB1 and accessory proteins, MO25 and STRADα, in both mdx strains versus the respective wild type, which was further impaired by exercise, in parallel with a lack of further phosphorylation of AMPK. The AMPK-like kinase salt-inducible kinase (SIK) and class II histone deacetylases, along with expression of the HDAC target gene Mef2c, were also altered, supporting an impairment of LKB1-SIK-class II histone deacetylase signaling. Our results demonstrate that LKB1 may be involved in dystrophic progression, paving the way for future preclinical studies.

Ceramide compensation by ceramide synthases preserves retinal function and structure in a retinal dystrophy mouse model.

Increasing evidence has supported the role of ceramide as a mediator of photoreceptor dysfunction or cell death in ceramide accumulation and deficiency contexts. TLCD3B, a non-canonical ceramide synthase, was previously identified in addition to the six canonical ceramide synthases (CerSs), and the Tlcd3b-/- mouse model exhibited both retinal dysfunction and degeneration. As previous canonical CerS-deficient mouse models failed to display retinal degeneration, the mechanisms of how TLCD3B interacts with CerSs have not been investigated. Additionally, as the ceramide profile of each CerS is distinct, it is unclear whether the overall level or the homeostasis of different ceramide species plays a critical role in photoreceptor degeneration. Interactions between TLCD3B with canonical CerSs expressed in the retina were examined by subretinally injecting recombinant adeno-associated virus 8 vectors containing the Cers2 (rAAV8-CerS2), Cers4 (rAAV8-CerS4) and Cers5 (rAAV8-CerS5) genes. Injection of all three rAAV8-CerS vectors restored retinal functions as indicated by improved electroretinogram responses, but only rAAV8-CerS5 successfully retained retinal morphology in Tlcd3b-/- mice. CerSs and TLCD3B played partially redundant roles. Additionally, rather than acting as an integral entity, different ceramide species had different impacts on retinal cells, suggesting that the maintenance of the overall ceramide profile is critical for retinal function.

Differential histological features and myogenic protein levels in distinct muscles of d-sarcoglycan null muscular dystrophy mouse model.

Skeletal muscle (SkM) comprises slow and fast-twitch fibers, which differ in molecular composition, function, and systemic energy consumption. In addition, muscular dystrophies (DM), a group of diverse hereditary diseases, present different patterns of muscle involvement, progression, and severity, suggesting that the regeneration-degeneration process may differ depending on the muscle type. Therefore, the study aimed to explore the expression of proteins involved in the repair process in different muscles at an early stage of muscular dystrophy in the δ-sarcoglycan null mice (Sgcd-null), a limb-girdle muscular dystrophy 2F model. Hematoxylin & Eosin (H&E) Staining showed a high number of central nuclei in soleus (Sol), tibialis (Ta), gastrocnemius (Gas), and extensor digitorum longus (Edl) from four months Sgcd-null mice. However, fibrosis, determined by trichrome of Gomori modified staining, was only observed in Sgcd-null Sol. In addition, the number of Type I and II fibers variated differentially in the Sgcd-null muscles vs. wild-type muscles. Besides, the protein expression level of β-catenin, myomaker, MyoD, and myogenin also presented different expression levels in all the Sgcd-null muscles studied. In summary, our study reveals that muscles with different metabolic characteristics showed distinct expression patterns of proteins involved in the muscle regeneration process. These results could be relevant in designing therapies for genetic and acquired myopathy.

PTEN Inhibitor Treatment Lowers Muscle Plasma Membrane Damage and Enhances Muscle ECM Homeostasis after High-Intensity Eccentric Exercise in Mice.

Exercise-induced muscle damage (EIMD) is a common occurrence in athletes and can lead to delayed onset muscle soreness, reduced athletic performance, and an increased risk of secondary injury. EIMD is a complex process involving oxidative stress, inflammation, and various cellular signaling pathways. Timely and effective repair of the extracellular matrix (ECM) and plasma membrane (PM) damage is critical for recovery from EIMD. Recent studies have shown that the targeted inhibition of phosphatase and tension homolog (PTEN) in skeletal muscles can enhance the ECM environment and reduce membrane damage in Duchenne muscular dystrophy (DMD) mice. However, the effects of PTEN inhibition on EIMD are unknown. Therefore, the present study aimed to investigate the potential therapeutic effects of VO-OHpic (VO), a PTEN inhibitor, on EIMD symptoms and underlying mechanisms. Our findings indicate that VO treatment effectively enhances skeletal muscle function and reduces strength loss during EIMD by upregulating membrane repair signals related to MG53 and ECM repair signals related to the tissue inhibitor of metalloproteinases (TIMPs) and matrix metalloproteinase (MMPs). These results highlight the potential of pharmacological PTEN inhibition as a promising therapeutic approach for EIMD.

Efficacy and muscle safety assessment of fukutin-related protein gene therapy

Limb-girdle muscular dystrophy type R9 (LGMDR9) is a muscle-wasting disease that begins in the hip and shoulder regions of the body. This disease is caused by mutations in fukutin-related protein (FKRP), a glycosyltransferase critical for maintaining muscle cell integrity. Here we investigated potential gene therapies for LGMDR9 containing an FKRP expression construct with untranslated region (UTR) modifications. Initial studies treated an aged dystrophic mouse model (FKRPP448L) with adeno-associated virus vector serotype 6 (AAV6). Grip strength improved in a dose- and time-dependent manner, injected mice exhibited fewer central nuclei and serum creatine kinase levels were 3- and 5-fold lower compared to those in non-injected FKRPP448L mice. Treatment also partially stabilized the respiratory pattern during exercise and improved treadmill running, partially protecting muscle from exercise-induced damage. Western blotting of C2C12 myotubes using a novel rabbit antibody confirmed heightened translation with the UTR modifications. We further explored the question of FKRP toxicity in wild-type mice using high doses of two additional muscle-tropic capsids: AAV9 and AAVMYO1. No toxic effects were detected with either therapeutic agent. These data further support the feasibility of gene therapy to treat LGMDR9.

RANKL Inhibition Reduces Cardiac Hypertrophy in mdx Mice and Possibly in Children with Duchenne Muscular Dystrophy.

Cardiomyopathy has become one of the leading causes of death in patients with Duchenne muscular dystrophy (DMD). We recently reported that the inhibition of the interaction between the receptor activator of nuclear factor κB ligand (RANKL) and receptor activator of nuclear factor κB (RANK) significantly improves muscle and bone functions in dystrophin-deficient mdx mice. RANKL and RANK are also expressed in cardiac muscle. Here, we investigate whether anti-RANKL treatment prevents cardiac hypertrophy and dysfunction in dystrophic mdx mice. Anti-RANKL treatment significantly reduced LV hypertrophy and heart mass, and maintained cardiac function in mdx mice. Anti-RANKL treatment also inhibited NFκB and PI3K, two mediators implicated in cardiac hypertrophy. Furthermore, anti-RANKL treatment increased SERCA activity and the expression of RyR, FKBP12, and SERCA2a, leading possibly to an improved Ca2+ homeostasis in dystrophic hearts. Interestingly, preliminary post hoc analyses suggest that denosumab, a human anti-RANKL, reduced left ventricular hypertrophy in two patients with DMD. Taken together, our results indicate that anti-RANKL treatment prevents the worsening of cardiac hypertrophy in mdx mice and could potentially maintain cardiac function in teenage or adult patients with DMD.

Increasing muscle contractility through low-frequency stimulation alters tibial bone geometry and reduces bone strength in mdx and dko dystrophic mice.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by mutations or deletions in the dystrophin gene, for which there remains no cure. As DMD patients also develop bone fragility because of muscle weakness and immobilization, better understanding of the pathophysiological mechanisms of dystrophin deficiency will help develop therapies to improve musculoskeletal health. Since alterations in muscle phenotype can influence bone structure, we investigated whether modifying muscle contractile activity through low-frequency stimulation (LFS) could alter bone architecture in mouse models of DMD. We tested the hypothesis that increasing muscle contractile activity could influence bone mass and structure in dystrophin-deficient (mdx) and dystrophin- and utrophin-deficient (dko) dystrophic mice. Tibial bone structure in dko mice was significantly different from that in mdx and wild-type (C57BL/10) control mice. Effects of LFS on bone architecture differed between dystrophic and healthy mice, with LFS thinning cortical bone in both dystrophic models. Bone mass was maintained in LFS-treated healthy mice, with a reduced proportion of high-density bone and concomitant increase in low-density bone. LFS-treated dko mice exhibited a net deficit in cortical thickness and reduced high-density bone but no equivalent increase in low-density bone. These alterations in bone structure and mineral density reduced mechanical strength in mdx and dko mice. The findings reveal that muscle activity can regulate bone mass, structure, mineral accrual, and strength, especially in the context of dystrophin and/or utrophin deficiency. The results provide unique insights into the development of bone fragility in DMD and for devising interventions to improve musculoskeletal health.NEW & NOTEWORTHY Patients with Duchenne muscular dystrophy (DMD) develop bone fragility because of muscle weakness and immobilization. We investigated whether increasing muscle contractile activity through low-frequency stimulation (LFS) could alter bone architecture in dystrophin-deficient (mdx) or dystrophin- and utrophin-deficient (dko) mouse models of DMD. Chronic LFS reduced tibial diaphysis cross sections in mdx and dko mice, without affecting bone shape in healthy mice. LFS affected the distribution of bone mineral density across all phenotypes, with the magnitude of effect being dependent on disease severity.

AAV-vectored base editor trans-splicing delivers dystrophin repair

AAV-vectored base editor trans-splicing delivers dystrophin repair

Decoding the transcriptome of muscular dystrophy due to Ptrf deficiency using single-nucleus RNA sequencing.

Lacking PTRF (polymerase I and transcript release factor), an essential caveolae component, causes a secondary deficiency of caveolins resulting in muscular dystrophy. The transcriptome responses of different types of muscle fibers and mononuclear cells in skeletal muscle to muscular dystrophy caused by Ptrf deletion have not been explored. Here, we created muscular dystrophy mice by Ptrf knockout and applied single-nucleus RNA sequencing (snRNA-seq) to unveil the transcriptional changes of the skeletal muscle at single-nucleus resolution. 11 613 muscle nuclei (WT, 5838; Ptrf KO, 5775) were classified into 12 clusters corresponding to 11 nuclear types. Trajectory analysis revealed the potential transition between type IIb_1 and IIb_2 myonuclei upon muscular dystrophy. Functional enrichment analysis indicated that apoptotic signaling and enzyme-linked receptor protein signaling pathway were significantly enriched in type IIb_1 and IIb_2 myonuclei of Ptrf KO, respectively. The muscle structure development and the PI3K-AKT signaling pathway were significantly enriched in type IIa and IIx myonuclei of Ptrf KO. Meanwhile, metabolic pathway analysis showed a decrease in overall metabolic pathway activity of myonuclei subtypes upon muscular dystrophy, with the most decrease in type IIb_1 myonuclei. Gene regulatory network analysis found that the activity of Mef2c, Mef2d, Myf5, and Pax3 regulons was enhanced in type II myonuclei of Ptrf KO, especially in type IIb_2 myonuclei. In addition, we investigated the transcriptome changes in adipocytes and found that muscular dystrophy enhanced the lipid metabolic capacity of adipocytes. Our findings provide a valuable resource for exploring the molecular mechanism of muscular dystrophy due to Ptrf deficiency.

Restoration of brain dystrophin using tricyclo-DNA ASOs restores neurobehavioral deficits in DMD mice

Restoration of brain dystrophin using tricyclo-DNA ASOs restores neurobehavioral deficits in DMD mice

Modulating fast skeletal muscle contraction protects skeletal muscle in animal models of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes from mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, the relationship between how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.

Loss of the matrix metalloproteinase-10 causes premature features of aging in satellite cells.

Aged muscles accumulate satellite cells with a striking decline response to damage. Although intrinsic defects in satellite cells themselves are the major contributors to aging-associated stem cell dysfunction, increasing evidence suggests that changes in the muscle-stem cell local microenvironment also contribute to aging. Here, we demonstrate that loss of the matrix metalloproteinase-10 (MMP-10) in young mice alters the composition of the muscle extracellular matrix (ECM), and specifically disrupts the extracellular matrix of the satellite cell niche. This situation causes premature features of aging in the satellite cells, contributing to their functional decline and a predisposition to enter senescence under proliferative pressure. Similarly, reduction of MMP-10 levels in young satellite cells from wild type animals induces a senescence response, while addition of the protease delays this program. Significantly, the effect of MMP-10 on satellite cell aging can be extended to another context of muscle wasting, muscular dystrophy. Systemic treatment of mdx dystrophic mice with MMP-10 prevents the muscle deterioration phenotype and reduces cellular damage in the satellite cells, which are normally under replicative pressure. Most importantly, MMP-10 conserves its protective effect in the satellite cell-derived myoblasts isolated from a Duchenne muscular dystrophy patient by decreasing the accumulation of damaged DNA. Hence, MMP-10 provides a previously unrecognized therapeutic opportunity to delay satellite cell aging and overcome satellite cell dysfunction in dystrophic muscles.

Timed Release Ion Matrix Enhances Muscle Force in Dystrophic Mice

Background: Duchenne Muscular Dystrophy (DMD) is an x-linked, recessive chromosomal mutation of the gene dystrophin, affecting 1 in 3,500 males. DMD is associated with limb muscle dysfunction from repetitive damage to myofibers beginning at 2 years of age. In healthy muscle, dystrophin is responsible for maintaining the sarcolemma through the dystrophin-glycoprotein complex, while muscle with dystrophin deficiencies seen in DMD patients experience severe sarcolemmal disruption following muscle contraction. Despite major advances in genetic engineering, the functional outcomes of FDA-approved therapeutic strategies have failed to enhance the function of dystrophin-deficient myofibers. We have taken an alternative approach to apply a novel biomaterial, Time Release Ion Matrix (TRIM), that has the potential to release ions favorable to regeneration, restoring muscle structure and function in DMD mice. Hypothesis: We hypothesized that muscle force and myofiber cross-sectional area (CSA) of the tibialis anterior muscle (TA) in an established mouse model of muscle dystrophy [D2.B10 mdx (DBA. mdx)] would be enhanced 14 days post-injection of TRIM. Methods: The left TA of adult male DBA. mdx mice (n=3-9, age 6-7 months) was injected with 250 μg of TRIM suspended in saline or saline alone (vehicle control). At 14 days post injection, the TA muscle was isolated for in situ muscle force measurements using direct electrical stimulation. The TA was then removed and frozen in optimal cutting temperature compound (OCT) for histological analysis. Muscle cross-sections (thickness, 10 μm) were labeled with laminin to identify myofiber borders and myosin heavy chain as a marker of myofibers able to generate force. Images were analyzed using Nikon NIS-Elements software. To simulate body fluid, 300 mg of TRIM was suspended in physiological salt solution (PSS) for 120 hours. Results: The dissolution of TRIM in PSS over 120 hours was 40% (179 mg remaining). Peak isometric force of TAs injected with TRIM (60.0 ± 2.3 g) was 11% greater compared to saline alone (52.4 ± 2.4 g) (means ± SE, n=6-9 per group; P=0.048). There were fewer small diameter myofibers (&lt; 400 μm2) in TAs injected with TRIM (10 ± 7%) compared to saline controls (24 ± 5 %) (n=3/group; P=0.03). Conclusion: 14 days following injection of TRIM into the TA of dystrophic mice, muscle force and myofiber CSA were enhanced by 11 and 14% respectively. We suggest that TRIM may be an effective treatment to restore muscle function in DMD and serve as a powerful tool for discovering therapeutic targets against DMD. University of Missouri Coulter Biomedical Accelerator (SSS, ABM), Texas A&amp;M Department of Kinesiology and Sport Management (JAK, ABM) This is the full abstract presented at the American Physiology Summit 2023 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.