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.

Fiber optic Raman spectroscopy for the evaluation of disease state in Duchenne muscular dystrophy: An assessment using the mdx model and human muscle.

INTRODUCTION/AIMS: Raman spectroscopy is an emerging technique for the evaluation of muscle disease. In this study we evaluate the ability of in vivo intramuscular Raman spectroscopy to detect the effects of voluntary running in the mdx model of Duchenne muscular dystrophy (DMD). We also compare mdx data with muscle spectra from human DMD patients. METHODS: Thirty 90-day-old mdx mice were randomly allocated to an exercised group (48-hour access to a running wheel) and an unexercised group (n = 15 per group). In vivo Raman spectra were collected from both gastrocnemius muscles and histopathological assessment subsequently performed. Raman data were analyzed using principal component analysis-fed linear discriminant analysis (PCA-LDA). Exercised and unexercised mdx muscle spectra were compared with human DMD samples using cosine similarity. RESULTS: Exercised mice ran an average of 6.5 km over 48 hours, which induced a significant increase in muscle necrosis (P = .03). PCA-LDA scores were significantly different between the exercised and unexercised groups (P < .0001) and correlated significantly with distance run (P = .01). Raman spectra from exercised mice more closely resembled human spectra than those from unexercised mice. DISCUSSION: Raman spectroscopy provides a readout of the biochemical alterations in muscle in both the mdx mouse and human DMD muscle.

MRI Assessment of Motor Capabilities in Patients with Duchenne Muscular Dystrophy According to the Motor Function Measure Scale.

The research was aimed on the study of motor capabilities on the Motor Function Measure (MFM) scale in ambulant and non-ambulant patients with Duchenne muscular dystrophy, and to conduct a correlation analysis between the results of the MFM scale and Magnetic Resonance Imaging (MRI) data. A total of 46 boys who had genetically confirmed Duchenne muscular dystrophy (age from 2.1 to 16.7 years) and were in clinical rehabilitation were investigated. An assessment was performed according to the Motor Function Measure scale (subsections D1, D2, D3, and the total score), an MRI obtaining T1-VI of the muscles of the pelvic girdle was conducted, and the thighs and lower legs were further assessed in terms of the severity of fibrous-fat degeneration according to the Mercuri scale. In ambulant patients, the ability to stand up and move (D1) was 74.4%, axial and proximal motor functions (D2)­97.6%, distal motor functions (D3)­96.2%, and total score was 87.9%. In non-ambulant patients, the ability to stand up and move (D1) was 1.7%, axial and proximal motor functions (D2)­47%, distal motor functions (D3)­67.5%, and the total score­33.1%. A high inverse correlation (r = −0.7, p < 0.05) of the MRI data of the pelvic girdle and thighs with tasks D1, as well as a noticeable inverse correlation with tasks D2 (r = −0.6, p < 0.05) of the scale MFM, were revealed in the ambulant group of patients. In the non-ambulant group of patients, the MRI data of the lower legs muscles were characterized by a high inverse correlation (r = −0.7, p < 0.05) with tasks D3 and a noticeable inverse correlation (r = −0.6, p < 0.05) with tasks D1 of the MFM scale. Conclusion: The Motor Function Measure scale allows effective assessment of the motor capabilities of patients with Duchenne muscular dystrophy at different stages of the disease, which is confirmed by visualization of fibro-fatty muscle replacement.

North Star Ambulatory Assessment changes in ambulant Duchenne boys amenable to skip exons 44, 45, 51, and 53: A 3 year follow up.

INTRODUCTION: The aim of this study was to report 36-month longitudinal changes using the North Star Ambulatory Assessment (NSAA) in ambulant patients affected by Duchenne muscular dystrophy amenable to skip exons 44, 45, 51 or 53. MATERIALS AND METHODS: We included 101 patients, 34 had deletions amenable to skip exon 44, 25 exon 45, 19 exon 51, and 28 exon 53, not recruited in any ongoing clinical trials. Five patients were counted to skip exon 51 and 53 since they had a single deletion of exon 52. RESULTS: The difference between subgroups (skip 44, 45, 51 and 53) was significant at 12 (p = 0.043), 24 (p = 0.005) and 36 months (p≤0.001). DISCUSSION: Mutations amenable to skip exons 53 and 51 had lower baseline values and more negative changes than the other subgroups while those amenable to skip exon 44 had higher scores both at baseline and at follow up. CONCLUSION: Our results confirm different progression of disease in subgroups of patients with deletions amenable to skip different exons. This information is relevant as current long term clinical trials are using the NSAA in these subgroups of mutations.

A standardised framework to identify optimal animal models for efficacy assessment in drug development.

INTRODUCTION: Poor translation of efficacy data derived from animal models can lead to clinical trials unlikely to benefit patients-or even put them at risk-and is a potential contributor to costly and unnecessary attrition in drug development. OBJECTIVES: To develop a tool to assess, validate and compare the clinical translatability of animal models used for the preliminary assessment of efficacy. DESIGN AND RESULTS: We performed a scoping review to identify the key aspects used to validate animal models. Eight domains (Epidemiology, Symptomatology and Natural History-SNH, Genetic, Biochemistry, Aetiology, Histology, Pharmacology and Endpoints) were identified. We drafted questions to evaluate the different facets of human disease simulation. We designed the Framework to Identify Models of Disease (FIMD) to include standardised instructions, a weighting and scoring system to compare models as well as factors to help interpret model similarity and evidence uncertainty. We also added a reporting quality and risk of bias assessment of drug intervention studies in the Pharmacological Validation domain. A web-based survey was conducted with experts from different stakeholders to gather input on the framework. We conducted a pilot study of the validation in two models for Type 2 Diabetes (T2D)-the ZDF rat and db/db mouse. Finally, we present a full validation and comparison of two animal models for Duchenne Muscular Dystrophy (DMD): the mdx mouse and GRMD dog. We show that there are significant differences between the mdx mouse and the GRMD dog, the latter mimicking the human epidemiological, SNH, and histological aspects to a greater extent than the mouse despite the overall lack of published data. CONCLUSIONS: FIMD facilitates drug development by serving as the basis to select the most relevant model that can provide meaningful data and is more likely to generate translatable results to progress drug candidates to the clinic.

Quantitative Ultrasound Assessment of Duchenne Muscular Dystrophy Using Edge Detection Analysis.

OBJECTIVES: The purpose of this study was to investigate the ability of quantitative ultrasound (US) using edge detection analysis to assess patients with Duchenne muscular dystrophy (DMD). METHODS: After Institutional Review Board approval, US examinations with fixed technical parameters were performed unilaterally in 6 muscles (biceps, deltoid, wrist flexors, quadriceps, medial gastrocnemius, and tibialis anterior) in 19 boys with DMD and 21 age-matched control participants. The muscles of interest were outlined by a tracing tool, and the upper third of the muscle was used for analysis. Edge detection values for each muscle were quantified by the Canny edge detection algorithm and then normalized to the number of edge pixels in the muscle region. The edge detection values were extracted at multiple sensitivity thresholds (0.01-0.99) to determine the optimal threshold for distinguishing DMD from normal. Area under the receiver operating curve values were generated for each muscle and averaged across the 6 muscles. RESULTS: The average age in the DMD group was 8.8 years (range, 3.0-14.3 years), and the average age in the control group was 8.7 years (range, 3.4-13.5 years). For edge detection, a Canny threshold of 0.05 provided the best discrimination between DMD and normal (area under the curve, 0.96; 95% confidence interval, 0.84-1.00). According to a Mann-Whitney test, edge detection values were significantly different between DMD and controls (P < .0001). CONCLUSIONS: Quantitative US imaging using edge detection can distinguish patients with DMD from healthy controls at low Canny thresholds, at which discrimination of small structures is best. Edge detection by itself or in combination with other tests can potentially serve as a useful biomarker of disease progression and effectiveness of therapy in muscle disorders.

Non-invasive assessment of muscle stiffness in patients with Duchenne muscular dystrophy.

INTRODUCTION: Assessment of muscle mechanical properties may provide clinically valuable information for follow-up of patients with Duchenne muscular dystrophy (DMD) through the course of their disease. In this study we aimed to assess the effect of DMD on stiffness of relaxed muscles using elastography (supersonic shear imaging). METHODS: Fourteen DMD patients and 13 control subjects were studied. Six muscles were measured at 2 muscle lengths (shortened and stretched): gastrocnemius medialis (GM); tibialis anterior (TA); vastus lateralis (VL); biceps brachii (BB); triceps brachii (TB); and abductor digiti minimi (ADM). RESULTS: Stiffness was significantly higher in DMD patients compared with controls for all the muscles (main effect for population, P < 0.033 in all cases), except for ADM. The effect size was small (d = 0.33 for ADM at both muscle lengths) to large (d = 0.86 for BB/stretched). CONCLUSIONS: Supersonic shear imaging is a sensitive non-invasive technique to assess the increase in muscle stiffness associated with DMD.

Quantitative MRI and strength measurements in the assessment of muscle quality in Duchenne muscular dystrophy.

The purpose of this study was to assess leg muscle quality and give a detailed description of leg muscle involvement in a series of Duchenne muscular dystrophy patients using quantitative MRI and strength measurements. Fatty infiltration, as well as total and contractile (not fatty infiltrated) cross sectional areas of various leg muscles were determined in 16 Duchenne patients and 11 controls (aged 8-15). To determine specific muscle strength, four leg muscle groups (quadriceps femoris, hamstrings, anterior tibialis and triceps surae) were measured and related to the amount of contractile tissue. In patients, the quadriceps femoris showed decreased total and contractile cross sectional area, attributable to muscle atrophy. The total, but not the contractile, cross sectional area of the triceps surae was increased in patients, corresponding to hypertrophy. Specific strength decreased in all four muscle groups of Duchenne patients, indicating reduced muscle quality. This suggests that muscle hypertrophy and fatty infiltration are two distinct pathological processes, differing between muscle groups. Additionally, the quality of remaining muscle fibers is severely reduced in the legs of Duchenne patients. The combination of quantitative MRI and quantitative muscle testing could be a valuable outcome parameter in longitudinal studies and in the follow-up of therapeutic effects.

Quantitative assessment of muscle damage in the mdx mouse model of Duchenne muscular dystrophy using polarization-sensitive optical coherence tomography.

Minimally invasive, high-resolution imaging of muscle necrosis has the potential to aid in the assessment of diseases such as Duchenne muscular dystrophy. Undamaged muscle tissue possesses high levels of optical birefringence due to its anisotropic ultrastructure, and this birefringence decreases when the tissue undergoes necrosis. In this study, we present a novel technique to image muscle necrosis using polarization-sensitive optical coherence tomography (PS-OCT). From PS-OCT scans, our technique is able to quantify the birefringence in muscle tissue, generating an image indicative of the tissue ultrastructure, with areas of abnormally low birefringence indicating necrosis. The technique is demonstrated on excised skeletal muscles from exercised dystrophic mdx mice and control C57BL/10ScSn mice with the resulting images validated against colocated histological sections. The technique additionally gives a measure of the proportion (volume fraction) of necrotic tissue within the three-dimensional imaging field of view. The percentage necrosis assessed by this technique is compared against the percentage necrosis obtained from manual assessment of histological sections, and the difference between the two methods is found to be comparable to the interobserver variability of the histological assessment. This is the first published demonstration of PS-OCT to provide automated assessment of muscle necrosis.

Assessment of the structural and functional impact of in-frame mutations of the DMD gene, using the tools included in the eDystrophin online database.

BACKGROUND: Dystrophin is a large essential protein of skeletal and heart muscle. It is a filamentous scaffolding protein with numerous binding domains. Mutations in the DMD gene, which encodes dystrophin, mostly result in the deletion of one or several exons and cause Duchenne (DMD) and Becker (BMD) muscular dystrophies. The most common DMD mutations are frameshift mutations resulting in an absence of dystrophin from tissues. In-frame DMD mutations are less frequent and result in a protein with partial wild-type dystrophin function. The aim of this study was to highlight structural and functional modifications of dystrophin caused by in-frame mutations. METHODS AND RESULTS: We developed a dedicated database for dystrophin, the eDystrophin database. It contains 209 different non frame-shifting mutations found in 945 patients from a French cohort and previous studies. Bioinformatics tools provide models of the three-dimensional structure of the protein at deletion sites, making it possible to determine whether the mutated protein retains the typical filamentous structure of dystrophin. An analysis of the structure of mutated dystrophin molecules showed that hybrid repeats were reconstituted at the deletion site in some cases. These hybrid repeats harbored the typical triple coiled-coil structure of native repeats, which may be correlated with better function in muscle cells. CONCLUSION: This new database focuses on the dystrophin protein and its modification due to in-frame deletions in BMD patients. The observation of hybrid repeat reconstitution in some cases provides insight into phenotype-genotype correlations in dystrophin diseases and possible strategies for gene therapy. The eDystrophin database is freely available: http://edystrophin.genouest.org/.

Assessment of cardiac abnormalities in Duchenne's muscular dystrophy by (99m)Tc-MIBI gated myocardial perfusion imaging.

Cardiac abnormalities in Duchenne's muscular dystrophy (DMD) are often detected in adult patients and their early detection is warranted. Studies have suggested that myocardial damage may be detected by cardiovascular magnetic resonance imaging or by echocardiography at the early stage of DMD. We aimed to identify early changes of cardiac abnormalities in children with DMD by technetium 99m-methoxyisobutylisonitrile ((99m)Tc-MIBI) gated myocardial perfusion imaging (GMPI). Forty-three boys aged 3 to 14 years (mean age 8.2±3.6 years) with DMD and 12 age-matched normal boys as control were studied by G-MPI. These patients were at early stage according to previous studies on DMD. Uptake of (99m)Tc-MIBI in 7 regional walls and 17 segments of the left ventricle were visually analyzed. Quantitative gated single photon emission tomography (QGSSPET) analysis of myocardium was performed to evaluate left ventricular function (LVEF). Gated myocardial perfusion imaging revealed cardiac abnormalities in 81.4% of all patients. Regional perfusion decrease involving multiple walls of LV was present. Four of the patients demonstrated mild abnormalities (11.4%), 7 moderate (20.0%) and 24 severe abnormalities (68.6%). Evident LV ejection fraction (EF) decrease (42.1±6.4%) and dilation with globally poor perfusion were found in three patients, aged 10 to 14, which had significant difference compared with the control group (EF=58.4±4.7%, P=0.001). The rest cases, aged 3 to 9 years, had normal LVF. In conclusion, from the 35 cases of DMD patients (aged 3 to 14 years), regional myocardial perfusion decrease was detected in multiple walls by (99m)Tc-MIBI G-MPI at an early stage, while left ventricular function decrease (3/35, 8.6%) appeared late at about 10 years of age or older as compared with the control group in this study.

Proteomic assessment of the acute phase of dystrophin deficiency in mdx mice.

Duchenne muscular dystrophy (DMD) is caused by the absence of a functional dystrophin protein and is modeled by the mdx mouse. The mdx mouse suffers an early necrotic bout in the hind limb muscles lasting from approximately 4 to 7 weeks. The purpose of this investigation was to determine the extent to which dystrophin deficiency changed the proteome very early in the disease process. In order to accomplish this, proteins from gastrocnemius from 6-week-old C57 (n = 6) and mdx (n = 6) mice were labeled with fluorescent dye and subjected to two-dimensional differential in-gel electrophoresis (2D-DIGE). Resulting differentially expressed spots were excised and protein identity determined via MALDI-TOF followed by database searching using MASCOT. Proteins of the immediate energy system and glycolysis were generally down-regulated in mdx mice compared to C57 mice. Conversely, expression of proteins involved in the Kreb's cycle and electron transport chain were increased in dystrophin-deficient muscle compared to control. Expression of cytoskeletal components, including tubulins, vimentin, and collagen, were increased in mdx mice compared to C57 mice. Importantly, these changes are occurring at only 6 weeks of age and are caused by acute dystrophin deficiency rather than more chronic injury. These data may provide insight regarding early pathologic changes occurring in dystrophin-deficient skeletal muscle.

Assessment of regional body composition with dual-energy X-ray absorptiometry in Duchenne muscular dystrophy: correlation of regional lean mass and quantitative strength.

The purpose of this study was to assess regional body composition and its correlation with regional strength in Duchenne muscular dystrophy (DMD) subjects and able-bodied controls. Regional dual-energy X-ray absorptiometry (DEXA) measurements and isometric strength were obtained for 23 DMD subjects and 23 control subjects. DMD subjects showed a decreased regional lean mass (P < 0.001). The correlation between regional strength and regional lean mass was stronger for controls than for DMD subjects. DMD subjects had decreased regional lean mass, increased regional fat mass, and decreased strength. Muscle Nerve 39: 647-651, 2009.

Preclinical drug trials in the mdx mouse: assessment of reliable and sensitive outcome measures.

The availability of animal models for Duchenne muscular dystrophy has led to extensive preclinical research on potential therapeutics. Few studies have focused on reliability and sensitivity of endpoints for mdx mouse drug trials. Therefore, we sought to compare a wide variety of reported and novel endpoint measures in exercised mdx and normal control mice at 10, 20, and 40 weeks of age. Statistical analysis as well as power calculations for expected effect sizes in mdx preclinical drug trials across different ages showed that body weight, normalized grip strength, horizontal activity, rest time, cardiac function measurements, blood pressure, total central/peripheral nuclei per fiber, and serum creatine kinase are the most effective measurements for detecting drug-induced changes. These data provide an experimental basis upon which standardization of preclinical drug testing can be developed. Muscle Nerve, 2008.

Assessment of the feasibility of exon 45-55 multiexon skipping for Duchenne muscular dystrophy.

BACKGROUND: The specific skipping of an exon, induced by antisense oligonucleotides (AON) during splicing, has shown to be a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients. As different mutations require skipping of different exons, this approach is mutation dependent. The skipping of an entire stretch of exons (e.g. exons 45 to 55) has recently been suggested as an approach applicable to larger groups of patients. However, this multiexon skipping approach is technically challenging. The levels of intended multiexon skips are typically low and highly variable, and may be dependent on the order of intron removal. We hypothesized that the splicing order might favor the induction of multiexon 45-55 skipping. METHODS: We here tested the feasibility of inducing multiexon 45-55 in control and patient muscle cell cultures using various AON cocktails. RESULTS: In all experiments, the exon 45-55 skip frequencies were minimal and comparable to those observed in untreated cells. CONCLUSION: We conclude that current state of the art does not sufficiently support clinical development of multiexon skipping for DMD.

Body composition and resting energy expenditure of individuals with Duchenne and Becker muscular dystrophy.

PURPOSE: The relationship between body composition and resting energy expenditure (REE) was investigated in two boys and two men with Duchenne muscular dystrophy (DMD) (ages 11 to 22.4 years) and two boys with Becker muscular dystrophy (BMD) (ages 7.75 and 13.75 years). METHODS: The REE was assessed by indirect calorimetry; body composition indices (weight, height, skinfolds, and mid-arm circumference) were measured using standardized techniques and compared with healthy reference data. RESULTS: Those with DMD had reduced corrected mid-upper-arm muscle area (C-MUMA) in comparison with healthy peers, and approximately twice the subcutaneous fat levels of subjects with BMD and healthy peers. Boys with BMD had remarkably lower muscle status than did boys with DMD and healthy peers. In both groups, REE was lower than in healthy peers; REE was associated with body weight (r=0.85), height (r=0.92), mid-upper arm fat area (MUFA) (r=0.97), and C-MUMA (r=0.65). CONCLUSIONS: Individuals with muscular dystrophy (MD) exhibit considerable disease-specific alterations in body composition. The REE had a stronger relationship with growth (weight and height) and subcutaneous body fat composition and a weaker association with C-MUMA. Understanding the effect of MD on body composition and REE will allow dietitians to individualize energy recommendations.

Measurement in Duchenne muscular dystrophy: considerations in the development of a neuromuscular assessment tool.

A review of the measures relating to the monitoring of disease progression in Duchenne muscular dystrophy was undertaken as part of the work of the North Star Clinical Network for Paediatric Neuromuscular Disease Management developing a standardized assessment protocol for ambulant children in the UK. This article outlines the process of identifying possible measures. Detailed consideration has been given to key measures of muscle strength and function. As well as the usual assessment of the validity and reliability of the measures, three key characteristics were identified as necessary to the assessment of scales used in health care: (1) the type of scale used; (2) the clinical significance of the attribute being measured; and (3) the mathematical properties of the data provided. Consideration of such aspects in the early stage of a study facilitates the choice of measures, and the analysis and interpretation of data in the longer term.

Body composition and energy expenditure in Duchenne muscular dystrophy.

OBJECTIVE: To investigate the relationship between resting energy expenditure (REE) and body composition in Duchenne Muscular Dystrophy (DMD). DESIGN: An observational study. SETTING: University Research Centre. SUBJECTS: Nine Duchenne children (age range 6-12 y), mean relative weight 128%, agreed to undergo the investigation and all of them completed the study; INTERVENTIONS: Assessment of body composition (total body fat and skeletal muscle mass) by magnetic resonance imaging and resting energy expenditure by indirect calorimetry. MAIN OUTCOME MEASURES: Fat mass (FM; kg and percentage weight), fat-free mass (FFM; kg and percentage weight), muscle mass (kg and percentage weight), resting energy expenditure (kJ/kg body weight and kJ/kg fat-free mass). RESULTS: : In Duchenne children fat mass averages 32% and total skeletal muscle mass 20% of body weight. Resting energy expenditure per kg of body weight falls within the normal range for children of the same age range, while when expressed per kg of FFM is significantly higher than reference values. No relationship was found between REE and total skeletal muscle mass. CONCLUSIONS: Our results do not demonstrate a low REE in DMD boys; on the contrary REE per kg of FFM is higher than normal, probably due to the altered FFM composition. We suggest that the development of obesity in DMD children is not primarily due to a low REE but to other causes such as a reduction in physical activity and or overfeeding.

Quantitative assessment of calf circumference in Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy is clinically characterised by progressive muscle weakness and a gradual increase in the size of some affected muscles, especially calf muscles. The extent of calf enlargement is usually determined by subjective visual assessment. The purpose of this study was to determine the extent of calf muscle enlargement in Duchenne muscular dystrophy (DMD) patients compared with healthy age matched boys by quantifying calf circumference. Calf circumference in the group of DMD patients is significantly increased. However, in individual patients calf enlargement can be feigned by a discrepancy between calf circumference and circumference of the upper leg and arm muscles as part of a general muscle atrophy.