Five multivariate Duchenne muscular dystrophy progression models bridging six-minute walk distance and MRI relaxometry of leg muscles.

The study aimed to provide quantitative information on the utilization of MRI transverse relaxation time constant (MRI-T2) of leg muscles in DMD clinical trials by developing multivariate disease progression models of Duchenne muscular dystrophy (DMD) using 6-min walk distance (6MWD) and MRI-T2. Clinical data were collected from the prospective and longitudinal ImagingNMD study. Disease progression models were developed by a nonlinear mixed-effect modeling approach. Univariate models of 6MWD and MRI-T2 of five muscles were developed separately. Age at assessment was the time metric. Multivariate models were developed by estimating the correlation of 6MWD and MRI-T2 model variables. Full model estimation approach for covariate analysis and five-fold cross validation were conducted. Simulations were performed to compare the models and predict the covariate effects on the trajectories of 6MWD and MRI-T2. Sigmoid Imax and Emax models best captured the profiles of 6MWD and MRI-T2 over age. Steroid use, baseline 6MWD, and baseline MRI-T2 were significant covariates. The median age at which 6MWD is half of its maximum decrease in the five models was similar, while the median age at which MRI-T2 is half of its maximum increase varied depending on the type of muscle. The models connecting 6MWD and MRI-T2 successfully quantified how individual characteristics alter disease trajectories. The models demonstrate a plausible correlation between 6MWD and MRI-T2, supporting the use of MRI-T2. The developed models will guide drug developers in using the MRI-T2 to most efficient use in DMD clinical trials.

Magnetic resonance quantification of skeletal muscle lipid infiltration in a humanized mouse model of Duchenne muscular dystrophy.

Rodent models of Duchenne muscular dystrophy (DMD) often do not recapitulate the severity of muscle wasting and resultant fibro-fatty infiltration observed in DMD patients. Having recently documented severe muscle wasting and fatty deposition in two preclinical models of muscular dystrophy (Dysferlin-null and mdx mice) through apolipoprotein E (ApoE) gene deletion without and with cholesterol-, triglyceride-rich Western diet supplementation, we sought to determine whether magnetic resonance imaging and spectroscopy (MRI and MRS, respectively) could be used to detect, characterize, and compare lipid deposition in mdx-ApoE knockout with mdx mice in a diet-dependent manner. MRI revealed that both mdx and mdx-ApoE mice exhibited elevated proton relaxation time constants (T2 ) in their lower hindlimbs irrespective of diet, indicating both chronic muscle damage and fatty tissue deposition. The mdx-ApoE mice on a Western diet (mdx-ApoEW ) presented with greatest fatty tissue infiltration in the posterior compartment of the hindlimb compared with other groups, as detected by MRI/MRS. High-resolution magic angle spinning confirmed elevated lipid deposition in the posterior compartments of mdx-ApoEW mice in vivo and ex vivo, respectively. In conclusion, the mdx-ApoEW model recapitulates some of the extreme fatty tissue deposition observed clinically in DMD muscle but typically absent in mdx mice. This preclinical model will help facilitate the development of new imaging modalities directly relevant to the image contrast generated in DMD, and help to refine MR-based biomarkers and their relationship to tissue structure and disease progression.

Effects of muscle damage on 31 phosphorus magnetic resonance spectroscopy indices of energetic status and sarcolemma integrity in young mdx mice.

31 Phosphorus magnetic resonance spectroscopy (31 P-MRS) has been shown to detect altered energetic status (e.g. the ratio of inorganic phosphate to phosphocreatine: Pi/PCr), intracellular acid-base status, and free intracellular magnesium ([Mg2+ ]) in dystrophic muscle compared with unaffected muscle; however, the causes of these differences are not well understood. The purposes of this study were to examine 31 P-MRS indices of energetic status and sarcolemma integrity in young mdx mice compared with wild-type and to evaluate the effects of downhill running to induce muscle damage on 31 P-MRS indices in dystrophic muscle. In vivo 31 P-MRS spectra were acquired from the posterior hindlimb muscles in young (4-10 weeks of age) mdx (C57BL/10ScSn-DMDmdx) and wild-type (C57BL/10ScSnJ) mice using an 11.1-T MR system. The flux of phosphate from PCr to ATP was estimated by 31 P-MRS saturation transfer experiments. Relative concentrations of high-energy phosphates were measured, and intracellular pH and [Mg2+ ] were calculated. 1 H2 O-T2 was measured using single-voxel 1 H-MRS from the gastrocnemius and soleus using a 4.7-T MR system. Downhill treadmill running was performed in a subset of mice. Young mdx mice were characterized by elevated 1 H2 O-T2 (p < 0.01), Pi/PCr (p = 0.02), PCr to ATP flux (p = 0.04) and histological inflammatory markers (p < 0.05) and reduced (p < 0.01) [Mg2+ ] compared with wild-type. Furthermore, 24 h after downhill running, an increase (p = 0.02) in Pi/PCr was observed in mdx and wild-type mice compared with baseline, and a decrease (p < 0.001) in [Mg2+ ] and a lower (p = 0.048) intracellular [H+ ] in damaged muscle regions of mdx mice were observed, consistent with impaired sarcolemma integrity. Overall, our findings demonstrate that 31 P-MRS markers of energetic status and sarcolemma integrity are altered in young mdx compared with wild-type mice, and these indices are exacerbated following downhill running.

Isolated murine skeletal muscles utilize pyruvate over glucose for oxidation.

INTRODUCTION: Fuel sources for skeletal muscle tissue include carbohydrates and fatty acids, and utilization depends upon fiber type, workload, and substrate availability. The use of isotopically labeled substrate tracers combined with nuclear magnetic resonance (NMR) enables a deeper examination of not only utilization of substrates by a given tissue, but also their contribution to tricarboxylic acid (TCA) cycle intermediates. OBJECTIVES: The goal of this study was to determine the differential utilization of substrates in isolated murine skeletal muscle, and to evaluate how isopotomer anlaysis provided insight into skeletal muscle metabolism. METHODS: Isolated C57BL/6 mouse hind limb muscles were incubated in oxygenated solution containing uniformly labeled 13C6 glucose, 13C3 pyruvate, or 13C2 acetate at room temperature. Isotopomer analysis of 13C labeled glutamate was performed on pooled extracts of isolated soleus and extensor digitorum longus (EDL) muscles. RESULTS: Pyruvate and acetate were more avidly consumed than glucose with resultant increases in glutamate labeling in both muscle groups. Glucose incubation resulted in glutamate labeling, but with high anaplerotic flux in contrast to the labeling by pyruvate. Muscle fiber type distinctions were evident by differences in lactate enrichment and extent of substrate oxidation. CONCLUSION: Isotope tracing experiments in isolated muscles reveal that pyruvate and acetate are avidly oxidized by isolated soleus and EDL muscles, whereas glucose labeling of glutamate is accompanied by high anaplerotic flux. We believe our results may set the stage for future examination of metabolic signatures of skeletal muscles from pre-clinical models of aging, type-2 diabetes and neuromuscular disease.

Longitudinal changes in cardiac function in Duchenne muscular dystrophy population as measured by magnetic resonance imaging.

BACKGROUND: The lack of dystrophin in cardiomyocytes in Duchenne muscular dystrophy (DMD) is associated with progressive decline in cardiac function eventually leading to death by 20-40 years of age. The aim of this prospective study was to determine rate of progressive decline in left ventricular (LV) function in Duchenne muscular dystrophy (DMD) over 5 years. METHODS: Short axis cine and grid tagged images of the LV were acquired in individuals with DMD (n = 59; age = 5.3-18.0 years) yearly, and healthy controls at baseline (n = 16, age = 6.0-18.3 years) on a 3 T MRI scanner. Grid-tagged images were analyzed for composite circumferential strain (ℇcc%) and ℇcc% in six mid LV segments. Cine images were analyzed for left ventricular ejection fraction (LVEF), LV mass (LVM), end-diastolic volume (EDV), end-systolic volume (ESV), LV atrioventricular plane displacement (LVAPD), and circumferential uniformity ratio estimate (CURE). LVM, EDV, and ESV were normalized to body surface area for a normalized index of LVM (LVMI), EDV (EDVI) and ESV (ESVI). RESULTS: At baseline, LV ℇcc% was significantly worse in DMD compared to controls and five of the six mid LV segments demonstrated abnormal strain in DMD. Longitudinal measurements revealed that ℇcc% consistently declined in individuals with DMD with the inferior segments being more affected. LVEF progressively declined between 3 to 5 years post baseline visit. In a multivariate analysis, the use of cardioprotective drugs trended towards positively impacting cardiac measures while loss of ambulation and baseline age were associated with negative impact. Eight out of 17 cardiac parameters reached a minimal clinically important difference with a threshold of 1/3 standard deviation. CONCLUSION: The study shows a worsening of circumferential strain in dystrophic myocardium. The findings emphasize the significance of early and longitudinal assessment of cardiac function in DMD and identify early biomarkers of cardiac dysfunction to help design clinical trials to mitigate cardiac pathology. This study provides valuable non-invasive and non-contrast based natural history data of cardiac changes which can be used to design clinical trials or interpret the results of current trials aimed at mitigating the effects of decreased cardiac function in DMD.

Skeletal muscle magnetic resonance imaging in Pompe disease.

Pompe disease is characterized by a deficiency of acid alpha-glucosidase that results in muscle weakness and a variable degree of disability. There is an approved therapy based on enzymatic replacement that has modified disease progression. Several reports describing muscle magnetic resonance imaging (MRI) features of Pompe patients have been published. Most of the studies have focused on late-onset Pompe disease (LOPD) and identified a characteristic pattern of muscle involvement useful for the diagnosis. In addition, quantitative MRI studies have shown a progressive increase in fat in skeletal muscles of LOPD over time and they are increasingly considered a good tool to monitor progression of the disease. The studies performed in infantile-onset Pompe disease patients have shown less consistent changes. Other more sophisticated muscle MRI sequences, such as diffusion tensor imaging or glycogen spectroscopy, have also been used in Pompe patients and have shown promising results.

Safety, feasibility, and efficacy of strengthening exercise in Duchenne muscular dystrophy.

BACKGROUND: This two-part study explored the safety, feasibility, and efficacy of a mild-moderate resistance isometric leg exercise program in ambulatory boys with Duchenne muscular dystrophy (DMD). METHODS: First, we used a dose escalation paradigm with varying intensity and frequency of leg isometric exercise to determine the dose response and safety in 10 boys. Second, we examined safety and feasibility of a 12-wk in-home, remotely supervised, mild-moderate intensity strengthening program in eight boys. Safety measures included T2 MRI, creatine kinase levels, and pain. Peak strength and function (time to ascend/descend four stairs) were also measured. RESULTS: Dose-escalation revealed no signs of muscle damage. Seven of the eight boys completed the 12-wk in-home program with a compliance of 84.9%, no signs of muscle damage, and improvements in strength (knee extensors P < .01; knee flexors P < .05) and function (descending steps P < .05). CONCLUSIONS: An in-home, mild-moderate intensity leg exercise program is safe with potential to positively impact both strength and function in ambulatory boys with DMD.

Diaphragm weakness and proteomics (global and redox) modifications in heart failure with reduced ejection fraction in rats.

Inspiratory dysfunction occurs in patients with heart failure with reduced ejection fraction (HFrEF) in a manner that depends on disease severity and by mechanisms that are not fully understood. In the current study, we tested whether HFrEF effects on diaphragm (inspiratory muscle) depend on disease severity and examined putative mechanisms for diaphragm abnormalities via global and redox proteomics. We allocated male rats into Sham, moderate (mHFrEF), or severe HFrEF (sHFrEF) induced by myocardial infarction and examined the diaphragm muscle. Both mHFrEF and sHFrEF caused atrophy in type IIa and IIb/x fibers. Maximal and twitch specific forces (N/cm2) were decreased by 19 ± 10% and 28 ± 13%, respectively, in sHFrEF (p < .05), but not in mHFrEF. Global proteomics revealed upregulation of sarcomeric proteins and downregulation of ribosomal and glucose metabolism proteins in sHFrEF. Redox proteomics showed that sHFrEF increased reversibly oxidized cysteine in cytoskeletal and thin filament proteins and methionine in skeletal muscle α-actin (range 0.5 to 3.3-fold; p < .05). In conclusion, fiber atrophy plus contractile dysfunction caused diaphragm weakness in HFrEF. Decreased ribosomal proteins and heighted reversible oxidation of protein thiols are candidate mechanisms for atrophy or anabolic resistance as well as loss of specific force in sHFrEF.

Upper and Lower Extremities in Duchenne Muscular Dystrophy Evaluated with Quantitative MRI and Proton MR Spectroscopy in a Multicenter Cohort.

Background Upper extremity MRI and proton MR spectroscopy are increasingly considered to be outcome measures in Duchenne muscular dystrophy (DMD) clinical trials. Purpose To demonstrate the feasibility of acquiring upper extremity MRI and proton (1H) MR spectroscopy measures of T2 and fat fraction in a large, multicenter cohort (ImagingDMD) of ambulatory and nonambulatory individuals with DMD; compare upper and lower extremity muscles by using MRI and 1H MR spectroscopy; and correlate upper extremity MRI and 1H MR spectroscopy measures to function. Materials and Methods In this prospective cross-sectional study, MRI and 1H MR spectroscopy and functional assessment data were acquired from participants with DMD and unaffected control participants at three centers (from January 28, 2016, to April 24, 2018). T2 maps of the shoulder, upper arm, forearm, thigh, and calf were generated from a spin-echo sequence (repetition time msec/echo time msec, 3000/20-320). Fat fraction maps were generated from chemical shift-encoded imaging (eight echo times). Fat fraction and 1H2O T2 in the deltoid and biceps brachii were measured from single-voxel 1H MR spectroscopy (9000/11-243). Groups were compared by using Mann-Whitney test, and relationships between MRI and 1H MR spectroscopy and arm function were assessed by using Spearman correlation. Results This study evaluated 119 male participants with DMD (mean age, 12 years ± 3 [standard deviation]) and 38 unaffected male control participants (mean age, 12 years ± 3). Deltoid and biceps brachii muscles were different in participants with DMD versus control participants in all age groups by using quantitative T2 MRI (P < .001) and 1H MR spectroscopy fat fraction (P < .05). The deltoid, biceps brachii, and triceps brachii were affected to the same extent (P > .05) as the soleus and medial gastrocnemius. Negative correlations were observed between arm function and MRI (T2: range among muscles, ρ = -0.53 to -0.73 [P < .01]; fat fraction, ρ = -0.49 to -0.70 [P < .01]) and 1H MR spectroscopy fat fraction (ρ = -0.64 to -0.71; P < .01). Conclusion This multicenter study demonstrated early and progressive involvement of upper extremity muscles in Duchenne muscular dystrophy (DMD) and showed the feasibility of MRI and 1H MR spectroscopy to track disease progression over a wide range of ages in participants with DMD. © RSNA, 2020 Online supplemental material is available for this article.

Conference report on contractures in musculoskeletal and neurological conditions.

Limb contractures are debilitating complications associated with various muscle and nervous system disorders. This report summarizes presentations at a conference at the Shirley Ryan AbilityLab in Chicago, Illinois, on April 19-20, 2018, involving researchers and physicians from diverse disciplines who convened to discuss current clinical and preclinical understanding of contractures in Duchenne muscular dystrophy, stroke, cerebral palsy, and other conditions. Presenters described changes in muscle architecture, activation, extracellular matrix, satellite cells, and muscle fiber sarcomeric structure that accompany or predispose muscles to contracture. Participants identified ongoing and future research directions that may lead to understanding of the intersecting factors that trigger contractures. These include additional studies of changes in muscle, tendon, joint, and neuronal tissues during contracture development with imaging, molecular, and physiologic approaches. Participants identified the requirement for improved biomarkers and outcome measures to identify patients likely to develop contractures and to accurately measure efficacy of treatments currently available and under development.

Age-dependent changes in metabolite profile and lipid saturation in dystrophic mice.

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked genetic disorder. In DMD, the absence of the dystrophin protein causes decreased sarcolemmal integrity resulting in progressive replacement of muscle with fibrofatty tissue. The effects of lacking dystrophin on muscle and systemic metabolism are still unclear. Therefore, to determine the impact of the absence of dystrophin on metabolism, we investigated the metabolic and lipid profile at two different, well-defined stages of muscle damage and stabilization in mdx mice. We measured NMR-detectable metabolite and lipid profiles in the serum and muscles of mdx mice at 6 and 24 weeks of age. Metabolites were determined in muscle in vivo using 1 H MRI/MRS, in isolated muscles using 1 H-HR-MAS NMR, and in serum using high resolution 1 H/13 C NMR. Dystrophic mice were found to have a unique lipid saturation profile compared with control mice, revealing an age-related metabolic change. In the 6-week-old mdx mice, serum lipids were increased and the degree of lipid saturation changed between 6 and 24 weeks. The serum taurine-creatine ratio increased over the life span of mdx, but not in control mice. Furthermore, the saturation index of lipids increased in the serum but decreased in the tissue over time. Finally, we demonstrated associations between MRI-T2 , a strong indicator of inflammation/edema, with tissue and serum lipid profiles. These results indicate the complex temporal changes of metabolites in the tissue and serum during repetitive bouts of muscle damage and regeneration that occur in dystrophic muscle.

Texture Analysis for Muscular Dystrophy Classification in MRI with Improved Class Activation Mapping.

The muscular dystrophies are made up of a diverse group of rare genetic diseases characterized by progressive loss of muscle strength and muscle damage. Since there is no cure for muscular dystrophy and clinical outcome measures are limited, it is critical to assess the progression of MD objectively. Imaging muscle replacement by fibrofatty tissue has been shown to be a robust biomarker to monitor disease progression in DMD. In magnetic resonance imaging (MRI) data, specific texture patterns are found to correlate to certain MD subtypes and thus present a potential way for automatic assessment. In this paper, we first apply state-of-the-art convolutional neural networks (CNNs) to perform accurate MD image classification and then propose an effective visualization method to highlight the important image textures. With a dystrophic MRI dataset, we found that the best CNN model delivers an 91.7% classification accuracy, which significantly outperforms non-deep learning methods, e.g., >40% improvement has been found over the traditional mean fat fraction (MFF) criterion for DMD and CMD classification. After investigating every single neuron at the top layer of CNN model, we found the superior classification ability of CNN can be explained by its 91 and 118 neurons were performing better than the MFF criterion under the measurements of Euclidean and Chi-square distance, respectively. In order to further interpret CNNs predictions, we tested an improved class activation mapping (ICAM) method to visualize the important regions in the MRI images. With this ICAM, CNNs are able to locate the most discriminative texture patterns of DMD in soleus, lateral gastrocnemius, and medial gastrocnemius; for CMD, the critical texture patterns are highlighted in soleus, tibialis posterior, and peroneus.

Magnetic Resonance Monitoring of Disease Progression in mdx Mice on Different Genetic Backgrounds.

Genetic modifiers alter disease progression in both preclinical models and subjects with Duchenne muscular dystrophy (DMD). Using multiparametric magnetic resonance (MR) techniques, we compared the skeletal and cardiac muscles of two different dystrophic mouse models of DMD, which are on different genetic backgrounds, the C57BL/10ScSn-Dmdmdx (B10-mdx) and D2.B10-Dmdmdx (D2-mdx). The proton transverse relaxation constant (T2) using both MR imaging and spectroscopy revealed significant age-related differences in dystrophic skeletal and cardiac muscles as compared with their age-matched controls. D2-mdx muscles demonstrated an earlier and accelerated decrease in muscle T2 compared with age-matched B10-mdx muscles. Diffusion-weighted MR imaging indicated differences in the underlying muscle structure between the mouse strains. The fractional anisotropy, mean diffusion, and radial diffusion of water varied significantly between the two dystrophic strains. Muscle structural differences were confirmed by histological analyses of the gastrocnemius, revealing a decreased muscle fiber size and increased fibrosis in skeletal muscle fibers of D2-mdx mice compared with B10-mdx and control. Cardiac involvement was also detected in D2-mdx myocardium based on both decreased function and myocardial T2. These data indicate that MR parameters may be used as sensitive biomarkers to detect fibrotic tissue deposition and fiber atrophy in dystrophic strains.

Longitudinal timed function tests in Duchenne muscular dystrophy: ImagingDMD cohort natural history.

INTRODUCTION: Tests of ambulatory function are common clinical trial endpoints in Duchenne muscular dystrophy (DMD). Using these tests, the ImagingDMD study has generated a large data set that can describe the contemporary natural history of DMD in 5-12.9-year-olds. METHODS: Ninety-two corticosteroid-treated boys with DMD and 45 controls participated in this longitudinal study. Participants performed the 6-minute walk test (6MWT) and timed function tests (TFT: 10-m walk/run, climbing 4 stairs, supine to stand). RESULTS: Boys with DMD had impaired functional performance even at 5-6.9 years old. Boys older than 7 had significant declines in function over 1 year for 10-m walk/run and 6MWT. Eighty percent of participants could perform all functional tests at 9 years old. TFTs appear to be slightly more responsive and predictive of disease progression than the 6MWT in 7-12.9 year olds. DISCUSSION: This study provides insight into the contemporary natural history of key functional endpoints in DMD. Muscle Nerve 58: 631-638, 2018.

Contrast-Enhanced Near-Infrared Optical Imaging Detects Exacerbation and Amelioration of Murine Muscular Dystrophy.

Assessment of muscle pathology is a key outcome measure to measure the success of clinical trials studying muscular dystrophies; however, few robust minimally invasive measures exist. Indocyanine green (ICG)-enhanced near-infrared (NIR) optical imaging offers an objective, minimally invasive, and longitudinal modality that can quantify pathology within muscle by imaging uptake of ICG into the damaged muscles. Dystrophic mice lacking dystrophin (mdx) or gamma-sarcoglycan (Sgcg-/-) were compared to control mice by NIR optical imaging and magnetic resonance imaging (MRI). We determined that optical imaging could be used to differentiate control and dystrophic mice, visualize eccentric muscle induced by downhill treadmill running, and restore the membrane integrity in Sgcg-/- mice following adeno-associated virus (AAV) delivery of recombinant human SGCG (desAAV8hSGCG). We conclude that NIR optical imaging is comparable to MRI and can be used to detect muscle damage in dystrophic muscle as compared to unaffected controls, monitor worsening of muscle pathology in muscular dystrophy, and assess regression of pathology following therapeutic intervention in muscular dystrophies.

Near-Infrared Optical Imaging Noninvasively Detects Acutely Damaged Muscle.

Muscle damage is currently assessed through methods such as muscle biopsy, serum biomarkers, functional testing, and imaging procedures, each with its own inherent limitations, and a pressing need for a safe, repeatable, inexpensive, and noninvasive modality to assess the state of muscle health remains. Our aim was to develop and assess near-infrared (NIR) optical imaging as a novel noninvasive method of detecting and quantifying muscle damage. An immobilization-reambulation model was used for inducing muscle damage and recovery in the lower hindlimbs in mice. Confirmation of muscle damage was obtained using in vivo indocyanine green-enhanced NIR optical imaging, magnetic resonance imaging, and ex vivo tissue analysis. The soleus of the immobilized-reambulated hindlimb was found to have a greater amount of muscle damage compared to that in the contralateral nonimmobilized limb, confirmed by in vivo indocyanine green-enhanced NIR optical imaging (3.86-fold increase in radiant efficiency), magnetic resonance imaging (1.41-fold increase in T2), and an ex vivo spectrophotometric assay of indocyanine green uptake (1.87-fold increase in normalized absorbance). Contrast-enhanced NIR optical imaging provides a sensitive, rapid, and noninvasive screening method that can be used for imaging and quantifying muscle damage and recovery in vivo.

Multicenter prospective longitudinal study of magnetic resonance biomarkers in a large duchenne muscular dystrophy cohort.

OBJECTIVE: The aim of this study was to describe Duchenne muscular dystrophy (DMD) disease progression in the lower extremity muscles over 12 months using quantitative magnetic resonance (MR) biomarkers, collected across three sites in a large cohort. METHODS: A total of 109 ambulatory boys with DMD (8.7 ± 2.0 years; range, 5.0-12.9) completed baseline and 1-year follow-up quantitative MR imaging (transverse relaxation time constant; MRI-T2 ), MR spectroscopy (fat fraction and (1) H2 O T2 ), and 6-minute walk test (6MWT) measurements. A subset of boys completed additional measurements after 3 or 6 months. RESULTS: MRI-T2 and fat fraction increased significantly over 12 months in all age groups, including in 5- to 6.9-year-old boys. Significant increases in vastus lateralis (VL) fat fraction were observed in 3 and 6 months. Even in boys whose 6MWT performance improved or remained stable over 1 year, significant increases in MRI-T2 and fat fraction were found. Of all the muscles examined, the VL and biceps femoris long head were the most responsive to disease progression in boys with DMD. INTERPRETATION: MR biomarkers are responsive to disease progression in 5- to 12.9-year-old boys with DMD and able to detect subclinical disease progression in DMD, even within short (3-6 months) time periods. The measured sensitivity of MR biomarkers in this multicenter study may be critically important to future clinical trials, allowing for smaller sample sizes and/or shorter study windows in this fatal rare disease.

Correcting Neuromuscular Deficits With Gene Therapy in Pompe Disease.

OBJECTIVE: We have recently reported on the pathology of the neuromuscular junction (NMJ) in Pompe disease, reflecting disruption of neuronal and muscle homeostasis as a result of glycogen accumulation. The aim of this study was to examine how the alteration of NMJ physiology contributes to Pompe disease pathology; we performed molecular, physiological, and histochemical analyses of NMJ-related measures of the tibialis anterior muscles of young-, mid-, and late-stage alpha-glucosidase (GAA)-deficient mice. METHODS: We performed intramuscular injection of an adeno-associated virus (AAV)9 vector expressing GAA (AAV9-hGAA) into the tibialis anterior muscle of Gaa(-/-) mice at early, mid, and severe pathological time points. We analyzed expression of NMJ-related genes, in situ muscle force production, and clearance of glycogen in conjunction with histological assessment of the NMJ. RESULTS: Our data demonstrate that AAV9-hGAA is able to replace GAA to the affected tissue and modify AChR mRNA expression, muscle force production, motor endplate area, and innervation status. Importantly, the degree of restoration for these outcomes is limited by severity of disease. Early restoration of GAA activity was most effective, whereas late correction of GAA expression was not effective in modifying parameters reflecting NMJ structure and function nor in force restoration despite resolution of glycogen storage in muscle. INTERPRETATION: Our data provide new mechanistic insight into the pathology of Pompe disease and suggest that early systemic correction to both neural and muscle tissues may be essential for successful correction of neuromuscular function in Pompe disease. Ann Neurol 2015;78:222-234.

Age-related T2 changes in hindlimb muscles of mdx mice.

INTRODUCTION: Magnetic resonance imaging (MRI) was used to monitor changes in the transverse relaxation time constant (T2) in lower hindlimb muscles of mdx mice at different ages. METHODS: Young (5 weeks), adult (44 weeks), and old mdx (96 weeks), and age-matched control mice were studied. Young mdx mice were imaged longitudinally, whereas adult and old mdx mice were imaged at a single time-point. RESULTS: Mean muscle T2 and percent of pixels with elevated T2 were significantly different between mdx and control mice at all ages. In young mdx mice, mean muscle T2 peaked at 7-8 weeks and declined at 9-11 weeks. In old mdx mice, mean muscle T2 was decreased compared with young and adult mice, which could be attributed to fibrosis. CONCLUSIONS: MRI captured longitudinal changes in skeletal muscle integrity of mdx mice. This information will be valuable for pre-clinical testing of potential therapeutic interventions for muscular dystrophy.

Dysferlin and myoferlin regulate transverse tubule formation and glycerol sensitivity.

Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy.