ResearchPad - dystrophin https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Serum glutamate dehydrogenase activity enables early detection of liver injury in subjects with underlying muscle impairments]]> https://www.researchpad.co/article/elastic_article_14604 Serum activities of alanine and aspartate aminotransferases (ALT and AST) are used as gold standard biomarkers for the diagnosis of hepatocellular injury. Since ALT and AST lack liver specificity, the diagnosis of the onset of hepatocellular injury in patients with underlying muscle impairments is severely limited. Thus, we evaluated the potential of glutamate dehydrogenase (GLDH) as a liver specific alternative biomarker of hepatocellular injury. In our study, serum GLDH in subjects with Duchene muscular dystrophy (DMD) was equivalent to serum GLDH in age matched healthy subjects, while serum ALT was increased 20-fold in DMD subjects. Furthermore, serum GLDH in 131 subjects with variety of muscle impairments was similar to serum GLDH of healthy subjects while serum ALT corelated with serum creatine kinase, a widely accepted biomarker of muscle impairment. In addition, significant elevations of ALT, AST, and CK were observed in a case of a patient with rhabdomyolysis, while serum GLDH stayed within the normal range until the onset of hypoxia-induced liver injury. In a mouse model of DMD (DMDmdx), serum GLDH but not serum ALT clearly correlated with the degree of acetaminophen-induced liver injury. Taken together, our data support the utility of serum GLDH as a liver-specific biomarker of liver injury that has a potential to improve diagnosis of hepatocellular injury in patients with underlying muscle impairments. In drug development, GLDH may have utility as a biomarker of drug induced liver injury in clinical trials of new therapies to treat muscle diseases such as DMD.

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<![CDATA[Activation of Both the Calpain and Ubiquitin-Proteasome Systems Contributes to Septic Cardiomyopathy through Dystrophin Loss/Disruption and mTOR Inhibition]]> https://www.researchpad.co/article/5989da43ab0ee8fa60b8ad90

Cardiac dysfunction caused by the impairment of myocardial contractility has been recognized as an important factor contributing to the high mortality in sepsis. Calpain activation in the heart takes place in response to increased intracellular calcium influx resulting in proteolysis of structural and contractile proteins with subsequent myocardial dysfunction. The purpose of the present study was to test the hypothesis that increased levels of calpain in the septic heart leads to disruption of structural and contractile proteins and that administration of calpain inhibitor-1 (N-acetyl-leucinyl-leucinyl-norleucinal (ALLN)) after sepsis induced by cecal ligation and puncture prevents cardiac protein degradation. We also tested the hypothesis that calpain plays a role in the modulation of protein synthesis/degradation through the activation of proteasome-dependent proteolysis and inhibition of the mTOR pathway. Severe sepsis significantly increased heart calpain-1 levels and promoted ubiquitin and Pa28β over-expression with a reduction in the mTOR levels. In addition, sepsis reduced the expression of structural proteins dystrophin and β-dystroglycan as well as the contractile proteins actin and myosin. ALLN administration prevented sepsis-induced increases in calpain and ubiquitin levels in the heart, which resulted in decreased of structural and contractile proteins degradation and basal mTOR expression levels were re-established. Our results support the concept that increased calpain concentrations may be part of an important mechanism of sepsis-induced cardiac muscle proteolysis.

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<![CDATA[Long-Term Efficacy, Safety, and Pharmacokinetics of Drisapersen in Duchenne Muscular Dystrophy: Results from an Open-Label Extension Study]]> https://www.researchpad.co/article/5989da05ab0ee8fa60b75811

Background

Drisapersen induces exon 51 skipping during dystrophin pre-mRNA splicing and allows synthesis of partially functional dystrophin in Duchenne muscular dystrophy (DMD) patients with amenable mutations.

Methods

This 188-week open-label extension of the dose-escalation study assessed the long-term efficacy, safety, and pharmacokinetics of drisapersen (PRO051/GSK2402968), 6 mg/kg subcutaneously, in 12 DMD subjects. Dosing was once weekly for 72 weeks. All subjects had a planned treatment interruption (weeks 73–80), followed by intermittent dosing (weeks 81–188).

Results

Subjects received a median (range) total dose of 5.93 (5.10 to 6.02) mg/kg drisapersen. After 177 weeks (last efficacy assessment), median (mean [SD]) six-minute walk distance (6MWD) improved by 8 (-24.5 [161]) meters for the 10 subjects able to complete the 6MWD at baseline (mean age [SD]: 9.5 [1.9] years). These statistics include 2 subjects unable to complete the test at later visits and who scored “zero”. When only the 8 ambulant subjects at week 177 were taken into account, a median (mean [SD]) increase of 64 (33 [121]) meters in 6MWD was observed. Of 7 subjects walking ≥330 m at extension baseline, 5 walked farther at week 177. Of 3 subjects walking <330 m, 2 lost ambulation, while 1 declined overall but walked farther at some visits. Over the 188 weeks, the most common adverse events were injection-site reactions, raised urinary α1-microglobulin and proteinuria. Dystrophin expression was detected in all muscle biopsies obtained at week 68 or 72.

Conclusion

Drisapersen was generally well tolerated over 188 weeks. Possible renal effects, thrombocytopenia and injection-site reactions warrant continued monitoring. Improvements in the 6MWD at 12 weeks were sustained after 3.4 years of dosing for most patients. For a small, uncontrolled study, the outcomes are encouraging, as natural history studies would anticipate a decline of over 100 meters over a 3-year period in a comparable cohort.

Trial Registration

ClinicalTrials.gov NCT01910649

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<![CDATA[Long-Term Quercetin Dietary Enrichment Partially Protects Dystrophic Skeletal Muscle]]> https://www.researchpad.co/article/5989daffab0ee8fa60bc6081

Duchenne muscular dystrophy (DMD) results from a genetic lesion in the dystrophin gene and leads to progressive muscle damage. PGC-1α pathway activation improves muscle function and decreases histopathological injury. We hypothesized that mild disease found in the limb muscles of mdx mice may be responsive to quercetin-mediated protection of dystrophic muscle via PGC-1α pathway activation. To test this hypothesis muscle function was measured in the soleus and EDL from 14 month old C57, mdx, and mdx mice treated with quercetin (mdxQ; 0.2% dietary enrichment) for 12 months. Quercetin reversed 50% of disease-related losses in specific tension and partially preserved fatigue resistance in the soleus. Specific tension and resistance to contraction-induced injury in the EDL were not protected by quercetin. Given some functional gain in the soleus it was probed with histological and biochemical approaches, however, in dystrophic muscle histopathological outcomes were not improved by quercetin and suppressed PGC-1α pathway activation was not increased. Similar to results in the diaphragm from these mice, these data suggest that the benefits conferred to dystrophic muscle following 12 months of quercetin enrichment were underwhelming. Spontaneous activity at the end of the treatment period was greater in mdxQ compared to mdx indicating that quercetin fed mice were more active in addition to engaging in more vigorous activity. Hence, modest preservation of muscle function (specific tension) and elevated spontaneous physical activity largely in the absence of tissue damage in mdxQ suggests dietary quercetin may mediate protection.

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<![CDATA[Increased Echogenicity and Radiodense Foci on Echocardiogram and MicroCT in Murine Myocarditis]]> https://www.researchpad.co/article/5989daacab0ee8fa60ba9d99

Objectives

To address the question as to whether echocardiographic and/or microcomputed tomography (microCT) analysis can be utilized to assess the extent of Coxsackie B virus (CVB) induced myocarditis in the absence of left ventricular dysfunction in the mouse.

Background

Viral myocarditis is a significant clinical problem with associated inflammation of the myocardium and myocardial injury. Murine models of myocarditis are commonly used to study the pathophysiology of the disease, but methods for imaging the mouse myocardium have been limited to echocardiographic assessment of ventricular dysfunction and, to a lesser extent, MRI imaging.

Methods

Using a murine model of myocarditis, we used both echocardiography and microCT to assess the extent of myocardial involvement in murine myocarditis using both wild-type mice and CVB cleavage-resistant dystrophin knock-in mice.

Results

Areas of increased echogenicity were only observed in the myocardium of Coxsackie B virus infected mice. These echocardiographic abnormalities correlated with the extent of von Kossa staining (a marker of membrane permeability), inflammation, and fibrosis. Given that calcium phosphate uptake as imaged by von Kossa staining might also be visualized using microCT, we utilized microCT imaging which allowed for high-resolution, 3-dimensional images of radiodensities that likely represent calcium phosphate uptake. As with echocardiography, only mice infected with Coxsackie B virus displayed abnormal accumulation of calcium within individual myocytes indicating increased membrane permeability only upon exposure to virus.

Conclusions

These studies demonstrate new, quantitative, and semi-quantitative imaging approaches for the assessment of myocardial involvement in the setting of viral myocarditis in the commonly utilized mouse model of viral myocarditis.

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<![CDATA[Individualized Prediction of Changes in 6-Minute Walk Distance for Patients with Duchenne Muscular Dystrophy]]> https://www.researchpad.co/article/5989db05ab0ee8fa60bc816d

Background

Deficits in ambulatory function progress at heterogeneous rates among individuals with Duchenne muscular dystrophy (DMD). The resulting inherent variability in ambulatory outcomes has complicated the design of drug efficacy trials and clouded the interpretation of trial results. We developed a prediction model for 1-year change in the six minute walk distance (6MWD) among DMD patients, and compared its predictive value to that of commonly used prognostic factors (age, baseline 6MWD, and steroid use).

Methods

Natural history data were collected from DMD patients at routine follow up visits approximately every 6 months over the course of 2–5 years. Assessments included ambulatory function and steroid use. The annualized change in 6MWD (Δ6MWD) was studied between all pairs of visits separated by 8–16 months. Prediction models were developed using multivariable regression for repeated measures, and evaluated using cross-validation.

Results

Among n = 191 follow-up intervals (n = 39 boys), mean starting age was 9.4 years, mean starting 6MWD was 351.8 meters, and 75% had received steroids for at least one year. Over the subsequent 8–16 months, mean Δ6MWD was -37.0 meters with a standard deviation (SD) of 93.7 meters. Predictions based on a composite of age, baseline 6MWD, and steroid use explained 28% of variation in Δ6MWD (R2 = 0.28, residual SD = 79.4 meters). A broadened prognostic model, adding timed 10-meter walk/run, 4-stair climb, and rise from supine, as well as height and weight, significantly improved prediction, explaining 59% of variation in Δ6MWD after cross-validation (R2 = 0.59, residual SD = 59.7 meters).

Conclusions

A prognostic model incorporating timed function tests significantly improved prediction of 1-year changes in 6MWD. Explained variation was more than doubled compared to predictions based only on age, baseline 6MWD, and steroid use. There is significant potential for composite prognostic models to inform DMD clinical trials and clinical practice.

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<![CDATA[Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy]]> https://www.researchpad.co/article/5989db03ab0ee8fa60bc750a

Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required. With the recent introduction of 3rd generation digital droplet PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of transcript copy numbers with and without specific exon skip with high precision, sensitivity and reproducibility. Using Taqman assays with probes targeting specific exon-exon junctions, we here demonstrate that ddPCR reproducibly quantified cDNA fragments with and without exon 51 of the DMD gene over a 4-log dynamic range. In a comparison of conventional nested PCR, qPCR and ddPCR using cDNA constructs with and without exon 51 mixed in different molar ratios using, ddPCR quantification came closest to the expected outcome over the full range of ratios (0–100%), while qPCR and in particular nested PCR overestimated the relative percentage of the construct lacking exon 51. Highest accuracy was similarly obtained with ddPCR in DMD patient-derived muscle cells treated with an AON inducing exon 51 skipping. We therefore recommend implementation of ddPCR for quantification of exon skip efficiencies of AONs in (pre)clinical development for DMD.

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<![CDATA[The mdx Mutation in the 129/Sv Background Results in a Milder Phenotype: Transcriptome Comparative Analysis Searching for the Protective Factors]]> https://www.researchpad.co/article/5989da13ab0ee8fa60b7a648

The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process.

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<![CDATA[Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies]]> https://www.researchpad.co/article/5989daa8ab0ee8fa60ba8324

Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.

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