Dystrophin Deficiency Research Articles

Long-Term Biodistribution and Safety of Human Dystrophin Expressing Chimeric Cell Therapy After Systemic-Intraosseous Administration to Duchenne Muscular Dystrophy Model

Duchenne muscular dystrophy (DMD) is a lethal disease caused by X-linked mutations in the dystrophin gene. Dystrophin deficiency results in progressive degeneration of cardiac, respiratory and skeletal muscles leading to premature death due to cardiopulmonary complications. Currently, no cure exists for DMD. Based on our previous reports confirming a protective effect of human dystrophin expressing chimeric (DEC) cell therapy on cardiac, respiratory, and skeletal muscle function after intraosseous administration, now we assessed long-term safety and biodistribution of human DEC therapy for potential clinical applications in DMD patients. Safety of different DEC doses (1 × 106 and 5 × 106) was assessed at 180 days after systemic-intraosseous administration to mdx/scid mice, a model of DMD. Assessments included: single cell gel electrophoresis assay (COMET assay) to confirm lack of genetic toxicology, magnetic resonance imaging (MRI) for tumorigenicity, and body, muscle and organ weights. Human DEC biodistribution to the target (heart, diaphragm, gastrocnemius muscle) and non-target (blood, bone marrow, lung, liver, spleen) organs was detected by flow cytometry assessment of HLA-ABC markers. Human origin of dystrophin was verified by co-localization of dystrophin and human spectrin by immunofluorescence. No complications were observed after intraosseous transplant of human DEC. COMET assay of donors and fused DEC cells confirmed lack of DNA damage. Biodistribution analysis of HLA-ABC expression revealed dose-dependent presence of human DEC cells in target organs, whereas negligible presence was detected in non-target organs. Human origin of dystrophin in the heart, diaphragm and gastrocnemius muscle was confirmed by co-localization of dystrophin expression with human spectrin. MRI revealed no evidence of tumor formation. Body mass and muscle and organ weights were stable and comparable to vehicle controls, further confirming DEC safety at 180 days post- transplant. This preclinical study confirmed long-term local and systemic safety of human DEC therapy at 180 days after intraosseous administration. Thus, DEC can be considered as a novel myoblast based advanced therapy medicinal product for DMD patients.

Abstract P2038: Synthetic Membrane Stabilizers Reduce Serum Ck And Immune Cell Infiltrate In Neonatal Mdx Skeletal Muscle Prior To Overt Muscle Necrosis Onset In Vivo

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease caused by a mutation in the dystrophin gene resulting in a complete loss of dystrophin from the striated muscle. This leads to the disruption of the dystrophin-associated glycoprotein complex and altered mechanical and signaling functions subsequently causing membrane instability, necrosis, and progressive muscle wasting. Immune cell infiltration is a prominent feature of the DMD pathophysiology and is strongly associated with disease severity. To date, no curative treatment is available for DMD. A logical therapeutic approach for DMD is to directly target the primary defect of DMD, sarcolemma instability, and thus prevent contraction-induced membrane damage. First in class synthetic triblock copolymer, poloxamer 188 (P188), is a muscle membrane interfacing and stabilizing molecule that has been shown to protect dystrophic myocardium and skeletal muscle from acute stress induced injury in vivo . Creatine kinase (CK) is a well-established biomarker of DMD that reflects sarcolemma damage, and serum levels have been shown to dramatically increase in mdx mice, but this has not been fully characterized during the early postnatal growth period prior to overt disease onset at 3 weeks. Under standard laboratory housing conditions for adult mdx mice (6-9 month old), we found that daily administration of P188 for two weeks did not significantly change serum CK levels or Evan’s blue dye uptake by diaphragm, heart or tibialis anterior muscles. In order to evaluate P188’s ability to protect dystrophin-deficient striated muscle prior to the overt disease, serum CK levels need to be characterized during this time period to identify the earliest muscle membrane damage event in mdx striated muscle and thus the earliest time point for therapeutic intervention. Results show that mdx serum CK levels are significantly higher than control serum levels 15 days after birth and continue to increase dramatically to 20 days after birth, and then CK remains high for the lifespan. Systemic delivery of P188 to neonatal mdx mice (12 days old) prior to CK peak significantly reduced serum CK levels and blunted immune cell infiltrate as compared to saline control mdx mice. These findings support the hypothesis that early intervention with membrane stabilizers has the potential to mitigate muscle damage and blunt the inflammatory response characteristic of DMD disease pathology. More work must be done to fully understand the early pathophysiological changes associated with dystrophin deficiency in order to implement novel therapeutic strategies including membrane stabilizers to enhance the standard of care for these patients and their families.

Abstract P2069: In Vivo Investigation Of The Mechanisms Regulating Truncated Dystrophin Protein Turnover In The Heart

Numerous inherited and acquired cardiac diseases are directly linked to a deficiency in the protein dystrophin, such as the development of severe cardiomyopathy in Duchenne Muscular Dystrophy (DMD) patients. Dilated cardiomyopathy, arrhythmias, and congestive heart failure represent the most important life-limiting condition in DMD. Currently, there is great excitement in the muscular dystrophy community as multiple ongoing gene-based therapies, are advancing through clinical trials. These therapies utilize miniaturized dystrophin constructs, and these modified dystrophins are the focus of this study. We implemented an innovative and valuable system to determine the direct physiological significance of dystrophin protein deficiency including determining the half-life of truncated dystrophin constructs. This system provides a novel means to investigate mechanistically how deletions in dystrophin affect therapeutically shortened dystrophin turnover in cardiac muscle in vivo. We used a floxed allele approach together with a cardiac directed (αMHC Mer-Cre-Mer) inducible Cre for precise control of full-length dystrophin or therapeutic micro-dystrophin gene excision. We examined the time course of full-length and micro-dystrophin mRNA as a biologically relevant surrogate for intact gene excision efficiency. Our data showed evidence of significant full-length dystrophin cDNA and micro-dystrophin gene excision, with a complete loss of micro-dystrophin mRNA and a gene excision efficiency of 80% for full-length dystrophin. Heart tissues were then extracted for protein quantitative analysis of micro-dystrophin (5, 10 and 30 days) and of full-length dystrophin (1, 3 and 6 months) post tamoxifen administration. Results reported a fast turnover rate for micro-dystrophin in the heart, with calculated half-life of between 5-7 days. In marked contrast, full-length dystrophin was highly stable with dystrophin protein content (~ 40%) at 6 months, after dystrophin gene excision. Studies of in vivo full-length and micro-dystrophin protein half-life in skeletal muscle, are ongoing. This work will provide key information required for long-term success of ongoing and future DMD therapies featuring gene therapy with shortened dystrophins.

Drug development progress in duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive, and incurable X-linked disorder caused by mutations in the dystrophin gene. Patients with DMD have an absence of functional dystrophin protein, which results in chronic damage of muscle fibers during contraction, thus leading to deterioration of muscle quality and loss of muscle mass over time. Although there is currently no cure for DMD, improvements in treatment care and management could delay disease progression and improve quality of life, thereby prolonging life expectancy for these patients. Furthermore, active research efforts are ongoing to develop therapeutic strategies that target dystrophin deficiency, such as gene replacement therapies, exon skipping, and readthrough therapy, as well as strategies that target secondary pathology of DMD, such as novel anti-inflammatory compounds, myostatin inhibitors, and cardioprotective compounds. Furthermore, longitudinal modeling approaches have been used to characterize the progression of MRI and functional endpoints for predictive purposes to inform Go/No Go decisions in drug development. This review showcases approved drugs or drug candidates along their development paths and also provides information on primary endpoints and enrollment size of Ph2/3 and Ph3 trials in the DMD space.

DMD, RIPK3, and MLKL gene editing by CRISPR Cas9 as myofiber protection against dystrophin deficiency and necroptosis in Duchenne muscular dystrophy: A literature review


 BACKGROUND: Duchenne muscular dystrophy is a neuromuscular disease caused by a deficiency of dystrophin, which causes the skeletal and cardiac muscles to degenerate. Targeted deletion of DMD, RIPK3, and MLKL has been shown in several studies to prevent dystrophin deficiency and necroptosis, a critical hypothesis in the etiology of Duchenne muscular dystrophy. AIM: This research aimed to see if using CRISPR/Cas9 to target DMD, RIPK3, and MLKL is an effective therapeutic and if it has a long-term effect on Duchenne muscular dystrophy. METHODS: Abstracts and titles of articles were searched for specific keywords to summarize them using the method used in this study. The researcher will look over the entire article to see if it is valuable and relevant to the topic. RESULTS: CRISPR/Cas9-mediated genome editing in MDX mice can improve the primary genetic lesions that cause muscular dystrophy (DMD) and prevent disease development. Furthermore, Ripk3/Mlk1 double knockout completely blocked necroptosis susceptibility in necroptosis-sensitive cell lines, each to an indistinguishable degree. CONCLUSION: DMD, RIPK3, and MLKL gene editing by CRISPR/Cas9 is effective dystrophin insufficiency, sarcolemma fragility, poor intracellular signaling, myocyte death, inflammatory infiltration, muscle replacement, and necroptosis. However, more research is needed to determine its side effects and safety.

Breathing in Duchenne muscular dystrophy: translation to therapy.

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease caused by a deficiency in dystrophin - a structural protein which stabilises muscle during contraction. Dystrophin deficiency adversely affects the respiratory system leading to sleep-disordered breathing, hypoventilation, and weakness of the expiratory and inspiratory musculature, which culminate in severe respiratory dysfunction. Muscle degeneration-associated respiratory impairment in neuromuscular disease is a result of disruptions at multiple sites of the respiratory control network, including sensory and motor pathways. As a result of this pathology, respiratory failure is a leading cause of premature death in DMD patients. Currently available treatments for DMD respiratory insufficiency attenuate respiratory symptoms without completely reversing the underlying pathophysiology. This underscores the need to develop curative therapies to improve quality of life and longevity of DMD patients. This review summarises research findings on the pathophysiology of respiratory insufficiencies in DMD disease in humans and animal models, the clinical interventions available to ameliorate symptoms, and gene-based therapeutic strategies uncovered by preclinical animal studies.

Forced activation of dystrophin transcription by CRISPR/dCas9 reduced arrhythmia susceptibility via restoring membrane Nav1.5 distribution.

Dystrophin deficiency due to genetic mutations causes cardiac abnormalities in Duchenne's muscular dystrophy. Dystrophin is also shown to be downregulated in conventional failing hearts. Whether restoration of dystrophin expression possesses any therapeutic potential for conventional heart failure (HF) remains to be examined. HF mouse model was generated by transverse aortic constriction (TAC). In vivo activation of dystrophin transcription was achieved by tail-vein injection of adeno-associated virus 9 carrying CRISPR/dCas system for dystrophin. We found that activation of dystrophin expression in TAC mice significantly reduced the susceptibility to arrhythmia of TAC mice and the mortality rate. We further demonstrated that over-expression of dystrophin increased cardiac conduction of hearts in TAC mice by optical mapping evaluation. Activation of dystrophin expression also increased peak sodium current in isolated ventricular myocytes from hearts of TAC mice as recorded by the patch-clamp technique. Immunoblotting and immunofluorescence showed that increased dystrophin transcription restored the membrane distribution of Nav1.5 in the hearts of TAC mice. In summary, correction of dystrophin downregulation by the CRISPR-dCas9 system reduced the susceptibility to arrhythmia of conventional HF mice through restoring Nav1.5 membrane distribution. This study paved the way to develop a new therapeutic strategy for HF-related ventricular arrhythmia.

Sarcoplasmic reticulum calcium handling in unbranched, immediately post-necrotic fast-twitch mdx fibres is similar to wild-type littermates.

What is the central question of this study? What are the early effects of dystrophin deficiency on sarcoplasmic reticulum Ca2+ handling in the mdx mouse? What is the main finding and its importance? In the mdx mouse, Ca2+ handling by the sarcoplasmic reticulum is little affected by the absence of dystrophin when looking at fibres without branches that have recently regenerated after massive myonecrosis. This has important implications for our understanding of Ca2+ pathology in the mdx mouse. There is a variety of results in the literature regarding the effects of dystrophin deficiency on the Ca2+ handling properties of the sarcoplasmic reticulum (SR) in the mdx mouse, an animal model of Duchenne muscular dystrophy. One possible source of variation is the presence of branched fibres. Fibre branching, a consequence of degenerative-regenerative processes such as muscular dystrophy, has in itself a significant influence on the function of the SR. In this study, we attempted to detect early effects of dystrophin deficiency on SR Ca2+ handling by using unbranched fibres from the immediate post-necrotic stage in mdx mice (recently regenerated after massive necrosis). Using kinetically corrected fura-2 fluorescence signals measured during twitch and tetanus, we analysed the amplitude, rise time and decay time of Δ[Ca2+ ]i in unfatigued and fatigued fibres. Decay was also resolved into SR pump and SR leak components. Fibres from mdx mice were similar in all respects to fibres from wild-type littermates apart from: (1) a smaller amplitude of the initial spike of Δ[Ca2+ ]i during a tetanus; and (2) a mitigation of the fall in Δ[Ca2+ ]i amplitude during the course of fatigue. Our findings suggest that the early effects of a loss of dystrophin on SR Ca2+ handling in mdx mice are subtle, and we emphasize the importance of distinguishing between Ca2+ pathology that is attributable to lack of dystrophin and Ca2+ pathology that is attributable to muscle degeneration.

Altered Activation of Inflammatory Signaling with Diet‐induced Insulin Resistance in the Dystrophic Diaphragm

Duchenne muscular dystrophy (DMD) affects 1 in 3500 to 5000 boys born worldwide. It is caused by the absence of functional dystrophin, a sarcolemmal protein. Dystrophin deficiency causes progressive muscle degeneration, leading to numerous cellular dysfunctions, ultimately resulting in death due to respiratory or cardiac failure. Although insulin resistance (IR) is a frequently reported comorbidity in boys with DMD, how IR impacts DMD is less studied. Additionally, the compounded effects of IR and dystropathology on cellular pathways, including inflammatory signaling, have not been previously considered. We hypothesized that a high‐fat, high sucrose diet (HF/HSD) would induce IR and cause activation of inflammatory signaling in dystrophic muscles. To address this hypothesis, 7‐week‐old mdx (diseased) and C57 (healthy) mice were fed a control diet (CD) or HF/HSD for 15 weeks, after which tissues were collected and analyzed. Data were compared using two‐way ANOVAs with Tukey adjustments and repeated measures ANOVA, with significance established at p<0.05. Insulin resistance was confirmed in the HF/HSD groups via increased area under the curve following a glucose tolerance test (p<0.05) and decreased rate of glucose disappearance following an insulin tolerance test (ITT; p<0.05). Further, these outcomes also suggest IR in mdx‐CD compared to C57‐CD as glucose clearance was impaired following an ITT. Visceral adipose (peri‐renal and epididymal) was impacted by disease and diet such that it was lower in mdx mice than C57 and increased in HF/HSD compared to CD, with a significant interaction (p<0.05). Of note, the perirenal adipose weights were increased almost 2‐fold in mdx‐HF/HSD compared to mdx‐CD. To probe for changes associated with both IR and DMD in skeletal muscle, we measured markers of inflammatory signaling using western blot. In whole muscle protein from the diaphragm, TLR4, a pattern recognition receptor, was increased approximately 70% by disease (p<0.05) and 40% by HF/HSD (p<0.05). TNFα, a pro‐inflammatory cytokine, had a significant interaction effect, was increased approximately 50% by disease (p<0.05), decreased 28% by diet (p<0.05), and mdx‐CD was higher (p<0.05) than the other groups. NFκB, a transcription factor that regulates inflammatory responses, increased (p<0.05) as a main effect of disease in whole muscle homogenate and nuclear fraction, but was not changed by diet. IκBα, an inhibitor of NFκB activation, and IKKα, an inhibitor of IκBα and promoter of NFκB, were increased by 1.6‐fold and 2‐fold, respectively (p<0.05), as a function of disease but were resistant to changes caused by diet. AP1 was increased approximately 3‐fold (p<0.05) with dystrophin deficiency in whole muscle homogenate but decreased in the nuclear fraction (45%, p<0.05). In summary, we discovered a HF/HSD induced IR in mdx mice, and our data indicate IR may be a fundamental consequence of dystrophin deficiency. Further, counter to our hypothesis, we discovered that a HF/HSD did not promote inflammatory signaling in dystrophin‐deficient skeletal muscle.

Analysis of Brain Structure and Neural Organization in Dystrophin-Deficient Model Mice with Magnetic Resonance Imaging at 7 T

Background: Dystrophin strengthens muscle cells; however, in muscular dystrophy, dystrophin is deficient due to an abnormal sugar chain. This abnormality occurs in skeletal muscle and in brain tissue. Objective: This study aimed to non-invasively analyze the neural organization of the brain in muscular dystrophy. We used a mouse model of muscular dystrophy to study whether changes in brain structure and neurodegeneration following dystrophin deficiency can be assessed by 7T magnetic resonance imaging. Methods: C57BL/10-mdx (X chromosome-linked muscular dystrophy) mice were used as the dystrophic mouse model and healthy mice were used as controls. Ventricular enlargement is one of the most common brain malformations in dystrophin-deficient patients. Therefore, we examined whether ventricular enlargement was observed in C57BL/10-mdx using transverse-relaxation weighted images. Brain parenchyma analysis was performed using diffusion MRI with diffusion tensor images and neurite orientation dispersion and density imaging. Parenchymal degeneration was assessed in terms of directional diffusion, nerve fiber diffusion, and dendritic scattering density. Results: For the volume of brain ventricles analyzed by T2WI, the average size was 1.5 times larger in mdx mice compared to control mice. In the brain parenchyma, a significant difference (p < 0.05) was observed in parameters indicating disturbances in the direction of nerve fibers and dendritic scattering density in the white matter region. Conclusion: Our results show that changes in brain structure due to dystrophin deficiency can be assessed in detail without tissue destruction by combining diffusion tensor images and neurite orientation dispersion and density imaging analyses.

Generation of a Dystrophin Mutant in Dog by Nuclear Transfer Using CRISPR/Cas9-Mediated Somatic Cells: A Preliminary Study.

Dystrophinopathy is caused by mutations in the dystrophin gene, which lead to progressive muscle degeneration, necrosis, and finally, death. Recently, golden retrievers have been suggested as a useful animal model for studying human dystrophinopathy, but the model has limitations due to difficulty in maintaining the genetic background using conventional breeding. In this study, we successfully generated a dystrophin mutant dog using the CRISPR/Cas9 system and somatic cell nuclear transfer. The dystrophin mutant dog displayed phenotypes such as elevated serum creatine kinase, dystrophin deficiency, skeletal muscle defects, an abnormal electrocardiogram, and avoidance of ambulation. These results indicate that donor cells with CRISPR/Cas9 for a specific gene combined with the somatic cell nuclear transfer technique can efficiently produce a dystrophin mutant dog, which will help in the successful development of gene therapy drugs for dogs and humans.

Passive-Stretch Induced Skeletal Muscle Injury Platform for Duchenne Muscular Dystrophy Modeling

<h3>Research Objectives</h3> Inactivity following skeletal muscle dysfunction in DMD usually causes compromised soft tissue and decreased joint range of motion. Passive stretch techniques in combination with an exercise program are used as interventions to prevent musculoskeletal complications in children with DMD. However, the exact role of stretch-based rehabilitation methods is not well established in children with DMD. In fact, the underlying molecular and cellular mechanisms of how stretch-based rehabilitation methods in dystrophin-deficient muscle fibers might worsen the disease phenotype have not been fully explained. Therefore, the purpose was to establish an in vitro stretch-induced injury model in normal and dystrophic rat skeletal muscle fibers. <h3>Design</h3> Randomized experimental. <h3>Setting</h3> Laboratory Setting. <h3>Participants</h3> Normal and dystrophic primary rat myoblasts (N=8) were platted on two (control and experimental) nanopatterned plates. The experimental setting was replicated three times. <h3>Interventions</h3> Myofibers were subjected to passive stretch on day three differentiation using the Cytostretcher device for 10 hours. <h3>Main Outcome Measures</h3> A electrochemiluminescence assay was used to quantify concentrations of skeletal muscle-specific injury biomarkers, FABP3, Myl3, and STnI. Two-way analysis of variance (ANOVA) with repeated measure test was used. <h3>Results</h3> STnI concentration was significantly higher on day four post differentiation compared to day three in the DMD-stretched group; whereas STnI concentration in all three groups did not show significant changes between the two-time points. FABP3 concentrations showed a significant increase on day four of differentiation compared to pre-stretch in both the DMD-stretched and the WT-stretched groups. However, MyL3 concentration decreased across all groups on day four of differentiation compared to day three. <h3>Conclusions</h3> The initial findings of this study supported the hypothesis that the passive-stretch protocol caused a greater magnitude of injury in dystrophic myofibers compared to normal. However, the preliminary findings did not support the hypothesis that dystrophin deficiency could worsen the response to passive-stretch-induced injury. These findings will guide the development of future in vitro studies for disease modeling, and to better understand the molecular mechanism(s) responsible for adaptation to exercise-induced muscle injury. <h3>Author(s) Disclosures</h3> The authors declare no conflict of interest.

Validation of DE50-MD dogs as a model for the brain phenotype of Duchenne muscular dystrophy.

ABSTRACTDuchenne muscular dystrophy (DMD), a fatal musculoskeletal disease, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin deficiency. Dog models of DMD represent key translational tools to study dystrophin biology and to develop novel therapeutics. However, characterisation of dystrophin expression and function in the canine brain is lacking. We studied the DE50-MD canine model of DMD that has a missense mutation in the donor splice site of exon 50. Using a battery of cognitive tests, we detected a neurocognitive phenotype in DE50-MD dogs, including reduced attention, problem solving and exploration of novel objects. Through a combination of capillary immunoelectrophoresis, immunolabelling, quantitative PCR and RNAScope in situ hybridisation, we show that regional dystrophin expression in the adult canine brain reflects that of humans, and that the DE50-MD dog lacks full-length dystrophin (Dp427) protein expression but retains expression of the two shorter brain-expressed isoforms, Dp140 and Dp71. Thus, the DE50-MD dog is a translationally relevant pre-clinical model to study the consequences of Dp427 deficiency in the brain and to develop therapeutic strategies for the neurological sequelae of DMD.

A sandwich ELISA kit reveals marked elevation of titin N-terminal fragment levels in the urine of mdx mice.

The mdx mouse is a model of Duchenne muscular dystrophy (DMD), a fatal progressive muscle wasting disease caused by dystrophin deficiency, and is used most widely in preclinical studies. Mice with dystrophin deficiency, however, show milder muscle strength phenotypes than humans. In human, the introduction of a sandwich enzyme‐linked immunosorbent assay (ELISA) kit revealed a more than 700‐fold increase in titin N‐terminal fragment levels in the urine of pediatric patients with DMD. Notably, the urinary titin level declines with aging, reflecting progression of muscle wasting. In mouse, development of a highly sensitive ELISA kit has been awaited. Here, a sandwich ELISA kit to measure titin N‐terminal fragment levels in mouse urine was developed. The developed kit showed good linearity, recovery, and repeatability in measuring recombinant or natural mouse titin N‐terminal fragment levels. The titin N‐terminal fragment concentration in the urine of mdx mice was more than 500‐fold higher than that of normal mice. Urinary titin was further analyzed by extending the collection of urine samples to both young (3–11 weeks old) and aged (56–58 weeks old) mdx mice. The concentration in the young group was significantly higher than that in the aged group. It was concluded that muscle protein breakdown is active and persistent in mdx mice even though the muscle phenotype is mild. Our results provide an opportunity to develop DMD treatments that aim to alleviate muscle protein breakdown by monitoring urinary titin levels.

Preload-induced cardiomyocyte calcium overload and ventricular damage in isolated hearts from young Duchenne muscular dystrophy (Dmdmdx-4CV) mice

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, which leads to structural and functional defects of the muscle cell sarcolemma. Patients with DMD progress to dilated cardiomyopathy leading to sudden cardiac death. Dystrophin deficiency associates with stretch-induced membrane instability and/or stretch-activated ion channel activation leading to cellular calcium overload. In this study we test the hypothesis that dystrophin deficiency leads to calcium handling abnormalities and cardiomyocyte damage following ventricular preload challenge in ex vivo perfused hearts.

Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues

Cardiomyopathy is currently the leading cause of death for patients with Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys. Animal models have provided insight into the mechanisms by which dystrophin protein deficiency causes cardiomyopathy, but there remains a need to develop human models of DMD to validate pathogenic mechanisms and identify therapeutic targets. Here, we have developed human engineered heart tissues (EHTs) from genetically edited, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing a truncated dystrophin protein.

Mechanisms and Consequences of Cerebellar Purkinje Cell Disinhibition in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), the most common form of childhood muscular dystrophy, is caused by mutations in the dystrophin gene. In addition to debilitating muscle degeneration, patients display a range of cognitive deficits thought to result from the loss of dystrophin normally expressed in the brain. While the function of dystrophin in muscle tissue is well characterized, its role in the brain is still poorly understood. The highest expression of dystrophin in the mouse brain is in cerebellar Purkinje cells (PCs), where it colocalizes with GABAA receptor clusters. Using ex vivo electrophysiological recordings from connected molecular layer interneuron (MLI)-PC pairs, we investigated changes in inhibitory synaptic transmission caused by dystrophin deficiency. In male mdx mice (which lack long-form dystrophin), we found that responses at MLI-PC pairs were reduced by ∼60% because of both decreased quantal response amplitude and a reduced number of functional vesicle release sites. Using electron microscopy, we found significantly fewer and smaller anatomically defined inhibitory synapses contacting the soma of PCs in mdx mice, suggesting that dystrophin may play a critical role in synapse formation and/or maintenance. Functionally, we found reduced MLI-evoked pauses in PC firing in acute slices. In vivo recordings from awake mdx mice showed increased sensory-evoked simple spike firing in positively modulating PCs, consistent with reduced feedforward inhibition, but no change in negatively modulating PCs. These data suggest that dystrophin deficiency in PCs disrupts inhibitory signaling in the cerebellar circuit and PC firing patterns, potentially contributing to cognitive and motor deficits observed in mdx mice and DMD patients.SIGNIFICANCE STATEMENT Duchenne muscular dystrophy (DMD) is primarily characterized by progressive muscle weakening caused by genetic mutations in the gene for dystrophin. Dystrophin is also normally expressed in the CNS, and DMD patients experience a range of nonprogressive cognitive deficits. The pathophysiology of CNS neurons resulting from loss of dystrophin and the function of dystrophin in neurons are still poorly understood. Using cerebellar PCs as a model, we found that the loss of dystrophin specifically disrupts the number and strength of inhibitory synaptic connections, suggesting that dystrophin participates in formation and/or maintenance of these synapses. This work provides insight into the function of dystrophin in the CNS and establishes neuronal and synaptic dysfunction, which may underlie cognitive deficits in DMD.

Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues

Cardiomyopathy is currently the leading cause of death for patients with Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys. Animal models have provided insight into the mechanisms by which dystrophin protein deficiency causes cardiomyopathy, but there remains a need to develop human models of DMD to validate pathogenic mechanisms and identify therapeutic targets. Here, we have developed human engineered heart tissues (EHTs) from CRISPR-edited, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing a truncated dystrophin protein lacking part of the actin-binding domain. The 3D EHT platform enables direct measurement of contractile force, simultaneous monitoring of Ca2+ transients, and assessment of myofibril structure. Dystrophin-mutant EHTs produced less contractile force as well as delayed kinetics of force generation and relaxation, as compared to isogenic controls. Contractile dysfunction was accompanied by reduced sarcomere length, increased resting cytosolic Ca2+ levels, delayed Ca2+ release and reuptake, and increased beat rate irregularity. Transcriptomic analysis revealed clear differences between dystrophin-deficient and control EHTs, including downregulation of genes related to Ca2+ homeostasis and extracellular matrix organization, and upregulation of genes related to regulation of membrane potential, cardiac muscle development, and heart contraction. These findings indicate that the EHT platform provides the cues necessary to expose the clinically-relevant, functional phenotype of force production as well as mechanistic insights into the role of Ca2+ handling and transcriptomic dysregulation in dystrophic cardiac function, ultimately providing a powerful platform for further studies in disease modeling and drug discovery.

Full-Length Dystrophin Deficiency Leads to Contractile and Calcium Transient Defects in Human Engineered Heart Tissues

Full-Length Dystrophin Deficiency Leads to Contractile and Calcium Transient Defects in Human Engineered Heart Tissues

Clinical aspects, food management, and clinical evolution of a cat with hypertrophic muscular dystrophy

ABSTRACT: Hypertrophic feline muscular dystrophy (HFMD), rarely reported in the literature, is a disease caused by a hereditary recessive dystrophin deficiency linked to the X chromosome, mainly affecting young male cats. Here, we presented the clinical aspects, food management, and clinical evolution of a seven-year-old mixed-breed cat diagnosed with HFMD, having a primary history of progressive tongue protrusion.