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Background Ischemic heart disease and the resulting heart failure continue to carry high morbidity and mortality, and a breakthrough in our understanding of this disorder is needed. The adult spiny mouse (Acomys cahirinus) has evolved the remarkable capacity to regenerate full-thickness skin tissue, including microvasculature and cartilage, without fibrosis or scarring. We hypothesized that lack of scarring and resulting functional regeneration also applies to the adult Acomys heart. Methods and results We compared responses of the Acomys heart to CD1 outbred Mus heart following acute left anterior descending coronary artery ligation to induce myocardial infarction. Both Acomys and Mus hearts showed decreased ejection fraction (EF) after ligation. However, Acomys hearts showed significant EF recovery to pre-ligation values over four weeks. Histological analysis showed comparable infarct area 24-h after ligation with a similar collateral flow in both species' hearts, but subsequently, Acomys displayed reduced infarct size, regenerated microvasculature, and increased cell proliferative activity in the infarcted area. Conclusions These observations suggest that adult Acomys displays remarkable cardiac recovery properties after acute coronary artery occlusion and may be a useful model to understand functional recovery better. Translational perspective Adult Acomys provides a novel mammalian model to further investigate the cardioprotective and regenerative signaling mechanisms in adult mammals. This opens the door to breakthrough treatment strategies for the injured myocardium and help millions of patients with heart failure secondary to tissue injury with irreversible damage.

Chronic activation of NF-κB is a key driver of muscle degeneration and suppression of muscle regeneration in Duchenne muscular dystrophy. Edasalonexent (CAT-1004) is an orally-administered novel small molecule that covalently links two bioactive compounds (salicylic acid and docosahexaenoic acid) that inhibit NF-κB. This placebo-controlled, proof-of-concept phase 2 study with open-label extension in boys ≥4-8 years old with any dystrophin mutation examined the effect of edasalonexent (67 or 100 mg/kg/day) compared to placebo or off-treatment control. Endpoints were safety/tolerability, change from baseline in MRI T

JAG2 encodes the Notch ligand Jagged2. The conserved Notch signaling pathway contributes to the development and homeostasis of multiple tissues, including skeletal muscle. We studied an international cohort of 23 individuals with genetically unsolved muscular dystrophy from 13 unrelated families. Whole-exome sequencing identified rare homozygous or compound heterozygous JAG2 variants in all 13 families. The identified bi-allelic variants include 10 missense variants that disrupt highly conserved amino acids, a nonsense variant, two frameshift variants, an in-frame deletion, and a microdeletion encompassing JAG2. Onset of muscle weakness occurred from infancy to young adulthood. Serum creatine kinase (CK) levels were normal or mildly elevated. Muscle histology was primarily dystrophic. MRI of the lower extremities revealed a distinct, slightly asymmetric pattern of muscle involvement with cores of preserved and affected muscles in quadriceps and tibialis anterior, in some cases resembling patterns seen in POGLUT1-associated muscular dystrophy. Transcriptome analysis of muscle tissue from two participants suggested misregulation of genes involved in myogenesis, including PAX7. In complementary studies, Jag2 downregulation in murine myoblasts led to downregulation of multiple components of the Notch pathway, including Megf10. Investigations in Drosophila suggested an interaction between Serrate and Drpr, the fly orthologs of JAG1/JAG2 and MEGF10, respectively. In silico analysis predicted that many Jagged2 missense variants are associated with structural changes and protein misfolding. In summary, we describe a muscular dystrophy associated with pathogenic variants in JAG2 and evidence suggests a disease mechanism related to Notch pathway dysfunction.

INTRODUCTION: 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-week in-home, remotely-supervised, mild-moderate intensity strengthening program in 8 boys. Safety measures included T(2) MRI, creatine kinase levels, and pain. Peak strength and function (time to ascend/descend 4 stairs) were also measured. RESULTS: Dose-escalation revealed no signs of muscle damage. Seven of the 8 boys completed the 12-week in-home program with a compliance of 84.9%, no signs of muscle damage, and improvements in strength (knee extensors p < 0.01; knee flexors p < 0.05) and function (descending steps p < 0.05). DISCUSSION: 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.

Duchenne muscular dystrophy (DMD) is a muscle degenerative disorder that manifests in early childhood and results in progressive muscle weakness. Physical therapists have long been an important component of the multidisciplinary team caring for people with DMD, providing expertise in areas of disease assessment, contracture management, assistive device prescription, and exercise prescription. Over the last decade, magnetic resonance imaging of muscles in people with DMD has led to an improved understanding of the muscle pathology underlying the clinical manifestations of DMD. Findings from magnetic resonance imaging (MRI) studies in DMD, paired with the clinical expertise of physical therapists, can help guide research that leads to improved physical therapist care for this unique patient population. The 2 main goals of this perspective article are to (1) summarize muscle pathology and disease progression findings from qualitative and quantitative muscle MRI studies in DMD and (2) link MRI findings of muscle pathology to the clinical manifestations observed by physical therapists with discussion of any potential implications of MRI findings on physical therapy management.

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 ((1)H) 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 (1)H MR spectroscopy; and correlate upper extremity MRI and (1)H MR spectroscopy measures to function. MATERIALS AND METHODS: In this prospective cross-sectional study, MRI and (1)H 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 (1)H(2)O T2 in the deltoid and biceps brachii were measured from single-voxel (1)H MR spectroscopy (9000/11–243). Groups were compared by using Mann-Whitney test, and relationships between MRI and (1)H 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 (1)H 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 (1)H 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 (1)H 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.

Observational research benefits from inclusion of diverse cohorts. To characterize racial and ethnic diversity in observational and natural history research studies of Duchenne muscular dystrophy (DMD), highly cited and influential observational studies were identified. Fourteen United States-based articles were included. All studies cited >70% White participants with the majority having few racial minority participants. Enrollment of Black/African American individuals was particularly limited (

ObjectiveTo quantify disease progression in individuals with Duchenne muscular dystrophy (DMD) using magnetic resonance biomarkers of leg muscles.MethodsMRI and magnetic resonance spectroscopy (MRS) biomarkers were acquired from 104 participants with DMD and 51 healthy controls using a prospective observational study design with patients with DMD followed up yearly for up to 6 years. Fat fractions (FFs) in vastus lateralis and soleus muscles were determined with 1H MRS. MRI quantitative T2 (qT2) values were measured for 3 muscles of the upper leg and 5 muscles of the lower leg. Longitudinal changes in biomarkers were modeled with a cumulative distribution function using a nonlinear mixed-effects approach.ResultsMRS FF and MRI qT2 increased with DMD disease duration, with the progression time constants differing markedly between individuals and across muscles. The average age at half-maximal muscle involvement (μ) occurred 4.8 years earlier in vastus lateralis than soleus, and these measures were strongly associated with loss-of-ambulation age. Corticosteroid treatment was found to delay μ by 2.5 years on average across muscles, although there were marked differences between muscles with more slowly progressing muscles showing larger delay.ConclusionsMRS FF and MRI qT2 provide sensitive noninvasive measures of DMD progression. Modeling changes in these biomarkers across multiple muscles can be used to detect and monitor the therapeutic effects of corticosteroids on disease progression and to provide prognostic information on functional outcomes. This modeling approach provides a method to transform these MRI biomarkers into well-understood metrics, allowing concise summaries of DMD disease progression at individual and population levels.ClinicalTrials.gov identifier:NCT01484678.

(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 ([Mg(2+)]) 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 [Mg(2+)] were calculated. (1)H(2)O-T(2) 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)H(2)O-T(2) (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) [Mg(2+)] 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 [Mg(2+)] 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.

BACKGROUND: Expiratory muscle weakness and impaired airway clearance are early signs of respiratory dysfunction in Duchenne muscular dystrophy (DMD), a degenerative muscle disorder in which muscle cells are damaged and replaced by fibrofatty tissue. Little is known about expiratory muscle pathology and its relationship to cough and airway clearance capacity; however, the level of muscle replacement by fat can be estimated using MRI and expressed as a fat fraction (FF). RESEARCH QUESTION: How does abdominal expiratory muscle fatty infiltration change over time in DMD and relate to clinical expiratory function? STUDY DESIGN AND METHODS: Individuals with DMD underwent longitudinal MRI of the abdomen to determine FF in the internal oblique, external oblique, and rectus abdominis expiratory muscles. FF data were used to estimate a model of expiratory muscle degeneration by using nonlinear mixed effects and a cumulative distribution function. FVC, maximal inspiratory and expiratory pressures, and peak cough flow were collected as clinical correlates to MRI. RESULTS: Forty individuals with DMD (aged 6-18 years at baseline) participated in up to five visits over 36 months. Modeling estimated the internal oblique progresses most quickly and reached 50% replacement by fat at a mean patient age of 13.0 years (external oblique, 14.0 years; rectus abdominis, 16.2 years). Corticosteroid-untreated individuals (n = 4) reached 50% muscle replacement by fat 3 to 4 years prior to treated individuals. Individuals with mild clinical dystrophic phenotypes (n = 3) reached 50% muscle replacement by fat 4 to 5 years later than corticosteroid-treated individuals. Internal and external oblique FFs near 50% were associated with maximal expiratory pressures < 60 cm H(2)O and peak cough flows < 270 L/min. INTERPRETATION: These data improve understanding of the early phase of respiratory compromise in DMD, which typically presents as airway clearance dysfunction prior to the onset of hypoventilation, and links expiratory muscle fatty infiltration to pulmonary function measures.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is characterized by damage to muscles including the muscles involved in respiration. Dystrophic muscles become weak and infiltrated with fatty tissue, resulting in progressive respiratory impairment. The objective of this study was to assess respiratory muscle quality and function in DMD using magnetic resonance imaging and to determine the relationship to clinical respiratory function. METHODS: Individuals with DMD (n=36) and unaffected controls (n=12) participated in this cross-sectional magnetic resonance imaging study. Participants underwent dynamic imaging of the thorax to assess diaphragm and chest wall mobility and chemical shift-encoded imaging of the chest and abdomen to determine fatty infiltration of the accessory respiratory muscles. Additionally, clinical pulmonary function measures were obtained. RESULTS: Thoracic cavity area was decreased in individuals with DMD compared to controls during tidal and maximal breathing. Individuals with DMD had reduced chest wall movement in the anterior-posterior direction during maximal inspirations and expirations, but diaphragm descent during maximal inspirations (normalized to height) was only decreased in a subset of individuals with maximal inspiratory pressures less than 60% predicted. Muscle fat fraction was elevated in all three expiratory muscles assessed (p

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.

Duchenne muscular dystrophy (DMD) is characterized by a progressive replacement of muscle by fat and fibrous tissue, muscle weakness, and loss of functional abilities. Impaired vasodilatory and blood flow responses to muscle activation have also been observed in DMD and associated with mislocalization of neuronal nitric oxide synthase mu (nNOSμ) from the sarcolemma. The objective of this study was to determine whether the postcontractile blood oxygen level-dependent (BOLD) MRI response is impaired in DMD and correlated with established markers of disease severity in DMD, including MRI muscle fat fraction (FF) and clinical functional measures. Young boys with DMD (n = 16, 5–14 yr) and unaffected controls (n = 16, 5–14 yr) were evaluated using postcontractile BOLD, FF, and functional assessments. The BOLD response was measured following five brief (2 s) maximal voluntary dorsiflexion contractions, each separated by 1 min of rest. FFs from the anterior compartment lower leg muscles were quantified via chemical shift-encoded imaging. Functional abilities were assessed using the 10 m walk/run and the 6-min walk distance (6MWD). The peak BOLD responses in the tibialis anterior and extensor digitorum longus were reduced (P < 0.001) in DMD compared with controls. Furthermore, the anterior compartment peak BOLD response correlated with function (6MWD ρ = 0.87, P < 0.0001; 10 m walk/run time ρ = −0.78, P < 0.001) and FF (ρ = −0.52, P = 0.05). The reduced postcontractile BOLD response in DMD may reflect impaired microvascular function. The relationship observed between the postcontractile peak BOLD response and functional measures and FF suggests that the BOLD response is altered with disease severity in DMD. NEW & NOTEWORTHY This study examined the postcontractile blood oxygen level-dependent (BOLD) response in boys with Duchenne muscular dystrophy (DMD) and unaffected controls, and correlated this measure to markers of disease severity. Our findings indicate that the postcontractile BOLD response is impaired in DMD after brief muscle contractions, is correlated to disease severity, and may be valuable to implement in future studies to evaluate treatments targeting microvascular function in DMD.

This case-control study uses magnetic resonance imaging and spectroscopy to evaluate the association between treatment with recombinant adeno-associated virus serotype rh74 (rAAVrh74) and muscle quality in children with Duchenne muscular dystrophy.

ObjectiveTo investigate the potential of lower extremity magnetic resonance (MR) biomarkers to serve as endpoints in clinical trials of therapeutics for Duchenne muscular dystrophy (DMD) by characterizing the longitudinal progression of MR biomarkers over 48 months and assessing their relationship to changes in ambulatory clinical function.MethodsOne hundred sixty participants with DMD were enrolled in this longitudinal, natural history study and underwent MR data acquisition of the lower extremity muscles to determine muscle fat fraction (FF) and MRI T2 biomarkers of disease progression. In addition, 4 tests of ambulatory function were performed. Participants returned for follow-up data collection at 12, 24, 36, and 48 months.ResultsLongitudinal analysis of the MR biomarkers revealed that vastus lateralis FF, vastus lateralis MRI T2, and biceps femoris long head MRI T2 biomarkers were the fastest progressing biomarkers over time in this primarily ambulatory cohort. Biomarker values tended to demonstrate a nonlinear, sigmoidal trajectory over time. The lower extremity biomarkers predicted functional performance 12 and 24 months later, and the magnitude of change in an MR biomarker over time was related to the magnitude of change in function. Vastus lateralis FF, soleus FF, vastus lateralis MRI T2, and biceps femoris long head MRI T2 were the strongest predictors of future loss of function, including loss of ambulation.ConclusionsThis study supports the strong relationship between lower extremity MR biomarkers and measures of clinical function, as well as the ability of MR biomarkers, particularly those from proximal muscles, to predict future ambulatory function and important clinical milestones.ClinicalTrials.gov identifierNCT01484678.

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.

Objective Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway that produces nicotinamide adenine dinucleotide (NAD+), an essential co-substrate regulating a myriad of signaling pathways. We produced a mouse that overexpressed NAMPT in skeletal muscle (NamptTg) and hypothesized that NamptTg mice would have increased oxidative capacity, endurance performance, and mitochondrial gene expression, and would be rescued from metabolic abnormalities that developed with high fat diet (HFD) feeding. Methods Insulin sensitivity (hyperinsulinemic-euglycemic clamp) was assessed in NamptTg and WT mice fed very high fat diet (VHFD, 60% by kcal) or chow diet (CD). The aerobic capacity (VO2max) and endurance performance of NamptTg and WT mice before and after 7 weeks of voluntary exercise training (running wheel in home cage) or sedentary conditions (no running wheel) were measured. Skeletal muscle mitochondrial gene expression was also measured in exercised and sedentary mice and in mice fed HFD (45% by kcal) or low fat diet (LFD, 10% by kcal). Results NAMPT enzyme activity in skeletal muscle was 7-fold higher in NamptTg mice versus WT mice. There was a concomitant 1.6-fold elevation of skeletal muscle NAD+. NamptTg mice fed VHFD were partially protected against body weight gain, but not against insulin resistance. Notably, voluntary exercise training elicited a 3-fold higher exercise endurance in NamptTg versus WT mice. Mitochondrial gene expression was higher in NamptTg mice compared to WT mice, especially when fed HFD. Mitochondrial gene expression was higher in exercised NamptTg mice than in sedentary WT mice. Conclusions Our studies have unveiled a fascinating interaction between elevated NAMPT activity in skeletal muscle and voluntary exercise that was manifest as a striking improvement in exercise endurance., Highlights • Skeletal muscle NAMPT overexpression increases NAD+ via elevated NAMPT activity. • Elevated NAMPT partially protects against very-high-fat-diet-induced weight gain. • Elevated NAMPT amplifies exercise-induced improvements in exercise endurance. • Fascinating interaction between elevated NAMPT activity in muscle and exercise.

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/cm(2)) were decreased by 19±10% and 28±13%, respectively, in sHFrEF (p < 0.05), 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 < 0.05). 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.

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.

Skeletal muscle wasting is a devastating consequence of cancer that contributes to increased complications and poor survival, but is not well understood at the molecular level. Herein, we investigated the role of Myocilin (Myoc), a skeletal muscle hypertrophy-promoting protein that we showed is downregulated in multiple mouse models of cancer cachexia. Loss of Myoc alone was sufficient to induce phenotypes identified in mouse models of cancer cachexia, including muscle fiber atrophy, sarcolemmal fragility, and impaired muscle regeneration. By 18 months of age, mice deficient in Myoc showed significant skeletal muscle remodeling, characterized by increased fat and collagen deposition compared with wild-type mice, thus also supporting Myoc as a regulator of muscle quality. In cancer cachexia models, maintaining skeletal muscle expression of Myoc significantly attenuated muscle loss, while mice lacking Myoc showed enhanced muscle wasting. Furthermore, we identified the myocyte enhancer factor 2 C (MEF2C) transcription factor as a key upstream activator of Myoc whose gain of function significantly deterred cancer-induced muscle wasting and dysfunction in a preclinical model of pancreatic ductal adenocarcinoma (PDAC). Finally, compared with noncancer control patients, MYOC was significantly reduced in skeletal muscle of patients with PDAC defined as cachectic and correlated with MEF2c. These data therefore identify disruptions in MEF2c-dependent transcription of Myoc as a novel mechanism of cancer-associated muscle wasting that is similarly disrupted in muscle of patients with cachectic cancer. Significance: This work identifies a novel transcriptional mechanism that mediates skeletal muscle wasting in murine models of cancer cachexia that is disrupted in skeletal muscle of patients with cancer exhibiting cachexia.

Lack of sarcolemma-localized neuronal nitric oxide synthase mu (nNOSμ) contributes to muscle damage and fatigue in dystrophic muscle. In this study, we examined the effects of compensating for lack of nNOSμ with a phosphodiesterase type 5 (PDE5) inhibitor in mdx mice following downhill running and endurance training. Dystrophic mice ( mdx) were treated with sildenafil citrate and compared with untreated mdx and wild-type mice after an acute bout of downhill running and during a progressive low-intensity treadmill running program (5 days/wk, 4 wk). Magnetic resonance imaging (MRI) and spectroscopy (MRS) transverse relaxation time constant (T2) of hindlimb and forelimb muscles were measured as a marker of muscle damage after downhill running and throughout training. The MRI blood oxygenation level dependence (BOLD) response and 31phosphorus MRS (31P-MRS) data were acquired after stimulated muscle contractions. After downhill running, the increase in T2 was attenuated ( P < 0.05) in treated mdx and wild-type mice compared with untreated mdx. During training, resting T2 values did not change in wild-type and mdx mice from baseline values; however, the running distance completed during training was greater ( P < 0.05) in treated mdx (>90% of target distance) and wild-type (100%) than untreated mdx (60%). The post-contractile BOLD response was greater ( P < 0.05) in treated mdx that trained than untreated mdx, with no differences in muscle oxidative capacity, as measured by 31P-MRS. Our findings indicate that PDE5 inhibition reduces muscle damage after a single bout of downhill running and improves performance during endurance training in dystrophic mice, possibly because of enhanced microvascular function. NEW & NOTEWORTHY This study examined the combined effects of PDE5 inhibition and exercise in dystrophic muscle using high-resolution magnetic resonance imaging and spectroscopy. Our findings demonstrated that sildenafil citrate reduces muscle damage after a single bout of downhill running, improves endurance-training performance, and enhances microvascular function in dystrophic muscle. Collectively, the results support the combination of exercise and PDE5 inhibition as a therapeutic approach in muscular dystrophies lacking nNOSμ.

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-T(2), 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.

Aims Under pressure overload, initial adaptive hypertrophy of the heart is followed by cardiomyocyte elongation, reduced contractile force, and failure. The mechanisms governing the transition to failure are not fully understood. Pressure overload reduced cardiac myosin light chain kinase (cMLCK) by ∼80% within 1 week and persists. Knockdown of cMLCK in cardiomyocytes resulted in reduced cardiac contractility and sarcomere disorganization. Thus, we hypothesized that acute reduction of cMLCK may be causative for reduced contractility and cardiomyocyte remodelling during the transition from compensated to decompensated cardiac hypertrophy. Methods and results To mimic acute cMLCK reduction in adult hearts, the floxed- Mylk3 gene that encodes cMLCK was inducibly ablated in Mylk3flox/flox / merCremer mice ( Mylk3-KO ), and compared with two control mice ( Mylk3flox/flox and Mylk3+/+ / merCremer ) following tamoxifen injection (50 mg/kg/day, 2 consecutive days). In Mylk3-KO mice, reduction of cMLCK protein was evident by 4 days, with a decline to below the level of detection by 6 days. By 7 days, these mice exhibited heart failure, with reduction of fractional shortening compared with those in two control groups (19.8 vs. 28.0% and 27.7%). Severely convoluted cardiomyocytes with sarcomeric disorganization, wavy fibres, and cell death were demonstrated in Mylk3-KO mice. The cardiomyocytes were also unable to thicken adaptively to pressure overload. Conclusion Our results, using a new mouse model mimicking an acute reduction of cMLCK, suggest that cMLCK plays a pivotal role in the transition from compensated to decompensated hypertrophy via sarcomeric disorganization.

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 1H2O 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. Ann Neurol 2016;79:535–547

OBJECTIVE The main objective of this study was to examine the effect of disease on strength in two functionally important lower limb muscles for a period of 2 yrs in children with Duchene muscular dystrophy. DESIGN Seventy-seven Duchene muscular dystrophy children participated in this study. Plantar flexors, knee extensors, strength, and performance on timed tests (6-min walk, 4-stairs, 10-m walk, supine-up) were assessed yearly for 2 yrs. Multivariate normal regression was used to assess changes in strength over time in the Duchene muscular dystrophy group. Spearman correlations were computed to examine relationship between strength and function. RESULTS Normalized plantar flexor and knee extensor strength showed a significant decrease (P < 0.05) over 2 yrs, with larger declines in knee extensor. At baseline, knee extensor strongly correlated with performance on timed tests. However, plantar flexor strength was found to be a stronger predictor of loss in ambulatory function. Modest correlations (r = 0.19-0.34) were found between the decline in strength and functional performance over 2 yrs. CONCLUSIONS This study describes the loss of lower limb strength in a large cohort of Duchene muscular dystrophy children for 2 yrs. The findings support that lower limb strength alone cannot account for the decline in performance on functional tests, and the role of other contributing factors, such as compensatory strategies, should be considered.

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. Muscle Nerve 53: 84–90, 2016

Gene therapy strategies have the potential to advance treatment options for pediatric neuromuscular disorders. The capacity to restore or preserve integrity and functionality of the neuromuscular junction (NMJ) is presumably limited by multiple factors, including bioavailability of tissue composing the NMJ at the time of intervention and efficacy of transgene replacement to both pre- and postsynaptic components. For these reasons, evaluating adeno-associated virus (AAV) serotypes with favorable tissue transduction characteristics and establishing early markers of NMJ deterioration to define an optimal therapeutic window are essential to preclinical development of gene therapy strategies to treat neuromuscular disorders. Maximum therapeutic benefit is presumably conferred by intervention at a prepathological stage; however, in the clinical setting, presymptomatic intervention is not always possible without newborn screening or clinical suspicion owing to an affected sibling. In the advanced stage of disease, the reversibility of pathology or plasticity of NMJ components may be limited. Examples from studies in spinal muscular atrophy have defined a narrow therapeutic window for scAAV9 therapy to impart functional benefit by restoration of survival motor neuron expression. Loss of therapeutic efficacy is related to rapid degeneration of the NMJ.1 Therefore, understanding the influence of NMJ pathology on the optimal therapeutic window is critical in evaluating a therapeutic strategy for patients challenged with a variety of neuromuscular disorders. Pompe disease is a neuromuscular disorder characterized by a deficiency in degradation of lysosomal glycogen resulting from reduced or absent acid alpha-glucosidase (GAA).2 Severe glycogen accumulation causes muscle atrophy and weakness and leads to loss of muscle function and cardiorespiratory failure before 12 months of age in early-onset patients.2,3 We have previously characterized contractile dysfunction and weakness of the diaphragm in the murine model of Pompe disease4–6 that mirrors the progressive phenotype of human patients.7 More recently, we demonstrated NMJ dysfunction in both the diaphragm and tibialis anterior (TA) muscles in Gaa-deficient mice, confirming that both muscle and nerve pathophysiology are likely contributors to disease progression.8–10 These observations justify the development of AAV-based gene therapy as a viable therapeutic candidate for Pompe, based on the intrinsic properties of AAV9 to target transgene replacement in both muscle and motor neurons.4–6,9,11,12 Supporting this hypothesis, correction of the Pompe phenotype, as demonstrated by previous studies, is likely attributable to both muscle and neuronal targeting of AAV9 expressing GAA (AAV9-hGAA).1,4,5 In this study, we used the Pompe mouse model to evaluate the therapeutic benefit of AAV9 vectors by direct intramuscular injection at early, mid, and advanced stages of NMJ pathology. We demonstrate that direct intramuscular administration of AAV9-hGAA is sufficient to promote glycogen clearance in gene-corrected muscle at all disease stages; however, normalizing muscle glycogen is ineffective in restoring motor endplate gene expression or neuromuscular functional profiles in the setting of end-stage disease. Overall, these data have important implications to guide the preclinical development of AAV9 vectors for treatment of Pompe disease and also provide potential markers of NMJ dysfunction that may be used to interpret therapeutic outcomes.

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.

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.

Duchenne muscular dystrophy (DMD) is a devastating muscle disease characterized by progressive muscle deterioration and replacement with an aberrant fatty, fibrous matrix. Chronic upregulation of nuclear factor κB (NF-κB) is implicated as a driver of the dystrophic pathogenesis. Herein, 2 members of a novel class of NF-κB inhibitors, edasalonexent (formerly CAT-1004) and CAT-1041, were evaluated in both mdx mouse and golden retriever muscular dystrophy (GRMD) dog models of DMD. These orally bioavailable compounds consist of a polyunsaturated fatty acid conjugated to salicylic acid and potently suppress the pathogenic NF-κB subunit p65/RelA in vitro. In vivo, CAT-1041 effectively improved the phenotype of mdx mice undergoing voluntary wheel running, in terms of activity, muscle mass and function, damage, inflammation, fibrosis, and cardiac pathology. We identified significant increases in dysferlin as a possible contributor to the protective effect of CAT-1041 to sarcolemmal damage. Furthermore, CAT-1041 improved the more severe GRMD phenotype in a canine case study, where muscle mass and diaphragm function were maintained in a treated GRMD dog. These results demonstrate that NF-κB modulation by edasalonexent and CAT-1041 is effective in ameliorating the dystrophic process and these compounds are candidates for new treatments for DMD patients.

Several studies provide compelling support for the use of MRI as a sensitive non-invasive method to assess skeletal muscle disease progression in various neuromuscular diseases, including Duchenne muscular dystrophy1,2 and limb girdle muscular dystrophy type 2I.3 In The Lancet Neurology, Jasper Morrow and colleagues4 now report the sensitivity of MRI to track disease progression in 20 patients with Charcot-Marie-Tooth disease 1A and 20 patients with inclusion body myositis. The investigators used a comprehensive study design that included magnetic resonance measures of muscle fat fraction, transverse relaxation time constant (T2), and magnetisation transfer ratio (MTR), along with relevant clinical functional tests (lower limb myometry, Medical Research Council score, Short-Form 36 Quality of Life Score, and Charcot-Marie-Tooth examination score or inclusion body myositis functional rating scale). In this study, the validity of the magnetic resonance measures was supported by strong correlations with clinical functional measures and the responsiveness to disease progression over 1 year was shown to be better with MRI than with the clinical functional tests. Notably, standardised response mean values were greater than 1 in inclusion body myositis and greater than 0·8 in Charcot-Marie-Tooth disease 1A, indicating that magnetic resonance measures are highly sensitive to disease progression and more responsive than established clinical measures. Even though Charcot-Marie-Tooth disease 1A progresses slowly, magnetic resonance measures detected substantial increases in disease pathological changes in 1 year. Thus, the encouraging results of Morrow and colleagues’ study might have a profound effect on clinical trials, potentially leading to a need for fewer participants to show efficacy or futility, shorter trials, and ultimately more rapid approval of treatments. An additional advantage of magnetic resonance measures compared with timed functional and strength measures, which are highly relevant to paediatric neuromuscular diseases, is that they are not dependent on participant motivation, an issue that has been of concern in Duchenne muscular dystrophy trials.5 Along with showing that MRI is a sensitive measure of disease progression in Charcot-Marie-Tooth disease 1A and inclusion body myositis, Morrow and colleagues also report that MRI-measured T2 and MTR are abnormal in these diseases even when fat fraction values are within normal limits. The authors interpret this to suggest that tissue water distribution changes before fat infiltration. Because MRI-measured T2 and MTR are affected by both fat and water content, the interpretation of these measures can be difficult in the context of neuromuscular diseases, and several analysis approaches are being developed to address this concern.6–8 Alternatively, magnetic resonance spectroscopy (MRS) could be used to more directly measure 1H2O T2 (figure) and MTR, and this would avoid the influence of fat.9 This method has already been applied to neuromuscular diseases10,11 1H2O T2 has been shown to differentiate boys with Duchenne muscular dystrophy and healthy individuals, even at a young age, when muscle fat fraction levels are normal.10 Furthermore, 1H2O T2 measured with MRS decreases with corticosteroid treatment in Duchenne muscular dystrophy, presumably because of reduced inflammation.11 Therefore, although lacking in spatial resolution, MRS is a high-fidelity approach to calculate 1H2O T2 and MTR measurements in neuromuscular diseases. Figure MRI and magnetic resonance spectroscopy of skeletal muscle in Duchenne muscular dystrophy In view of the low prevalence of neuromuscular diseases, standardisation of methods over several sites will be crucial to the successful implementation of magnetic resonance biomarkers in clinical trials. Although challenging, when standardised protocols are carefully implemented across sites, several important magnetic resonance measures can be reproducibly obtained, including MRI-measured T2 and MRS measures of fat fraction and 1H2O T2.12 Similar to neuroimaging and musculoskeletal studies, for larger trials, incorporation of an infrastructure that enables automated or semi-automated processing, analysis, and quality control procedures that can detect and correct for system deviations, including instrument modifications and upgrades, will be particularly important. Importantly, Morrow and colleagues showed the effectiveness of monitoring disease progression using magnetic resonance sequences commonly available on clinical scanners. Although the measures used were highly effective in tracking progression, future studies might include some developments. For example, the accuracy of fat fraction from the Dixon fat-water imaging sequence might be further improved by correcting for T2*, accounting for noise bias, and using a multipeak Dixon model specific to skeletal muscle.13,14 As suggested by Morrow and colleagues, the optimum outcome measures will depend on the pathophysiology, stage of disease, muscles affected, and intervention to be tested. This could include different types of magnetic resonance measures and analysis procedures. For example, targeting of specific muscles might be optimum for certain stages of a particular disease. Also, use of large regions of interest that encompass the entire muscle might be needed to detect pathological changes and improve responsiveness in some diseases, by contrast with small portions of the muscle, as used by Morrow and colleagues to measure T2 and MTR. Overall, this study by Morrow and colleagues clearly shows the value of MRI to monitor disease progression and sets the stage for its potential use in clinical trials for Charcot-Marie-Tooth disease 1A and inclusion body myositis. With increased evidence of the validity and sensitivity of magnetic resonance biomarkers in neuromuscular diseases, the path for biomarker qualification (eg, their approval by the Food and Drug Administration) should be carefully explored, with the ultimate goal of using magnetic resonance measures as surrogate endpoints in clinical trials.

To validate a multicenter protocol that examines lower extremity skeletal muscles of children with Duchenne muscular dystrophy (DMD) by using magnetic resonance (MR) imaging and MR spectroscopy in terms of reproducibility of these measurements within and across centers.This HIPAA-compliant study was approved by the institutional review boards of all participating centers, and informed consent was obtained from each participant or a guardian. Standardized procedures with MR operator training and quality assurance assessments were implemented, and data were acquired at three centers by using different 3-T MR imaging instruments. Measures of maximal cross-sectional area (CSAmax), transverse relaxation time constant (T2), and lipid fraction were compared among centers in two-compartment coaxial phantoms and in two unaffected adult subjects who visited each center. Also, repeat MR measures were acquired twice on separate days in 30 boys with DMD (10 per center) and 10 unaffected boys. Coefficients of variation (CVs) were computed to examine the repeated-measure variabilities within and across centers.CSAmax, T2 from MR imaging and MR spectroscopy, and lipid fraction were consistent across centers in the phantom (CV,3%) and in the adult subjects who traveled to each site (CV, 2%-7%). High day-to-day reproducibility in MR measures was observed in boys with DMD (CSAmax, CV = 3.7% [25th percentile, 1.3%; 75th percentile, 5.1%]; contractile area, CV = 4.2% [25th percentile, 0.8%; 75th percentile, 4.9%]; MR imaging T2, CV = 3.1% [25th percentile, 1.2%; 75th percentile, 4.7%]; MR spectroscopy T2, CV = 3.9% [25th percentile, 1.5%; 75th percentile, 5.1%]; and lipid fraction, CV = 4.7% [25th percentile, 1.0%; 75th percentile, 5.3%]).The MR protocol implemented in this multicenter study achieved highly reproducible measures of lower extremity muscles across centers and from day to day in ambulatory boys with DMD.

Purpose The relationship between fat fractions (FFs) determined based on multiple TE, unipolar gradient echo images and 1H magnetic resonance spectroscopy (MRS) was evaluated using different models for fat-water decomposition, signal-to-noise ratios, and excitation flip angles. Methods A combination of single-voxel proton spectroscopy (1H-MRS) and gradient echo imaging was used to determine muscle FFs in both normal and dystrophic muscles. In order to cover a large range of FFs, the soleus and vastus lateralis muscles of 22 unaffected control subjects, 16 subjects with collagen VI deficiency (COL6), and 71 subjects with Duchenne muscular dystrophy (DMD) were studied. 1H-MRS–based FF were corrected for the increased muscle 1H2O T1 and T2 values observed in dystrophic muscles. Results Excellent agreement was found between coregistered FFs derived from gradient echo images fit to a multipeak model with noise bias correction and the relaxation-corrected 1H-MRS FFs (y = 0.93x + 0.003; R2 = 0.96) across the full range of FFs. Relaxation-corrected 1H-MRS FFs and imaging-based FFs were significantly elevated (P < 0.01) in the muscles of COL6 and DMD subjects. Conclusion FFs, T2, and T1 were all sensitive to muscle involvement in dystrophic muscle. MRI offered an additional advantage over single-voxel spectroscopy in that the tissue heterogeneity in FFs could be readily determined. Magn Reson Med 72:8–19, 2014. © 2013 Wiley Periodicals, Inc.

Purpose Golden retriever muscular dystrophy (GRMD) is a widely used canine model of Duchenne muscular dystrophy (DMD). Recent studies have shown that magnetic resonance imaging (MRI) can be used to non-invasively detect consistent changes in both DMD and GRMD. In this paper, we propose a semiautomated system to quantify MRI biomarkers of GRMD. Methods Our system was applied to a database of 45 MRI scans from 8 normal and 10 GRMD dogs in a longitudinal natural history study. We first segmented six proximal pelvic limb muscles using a semiautomated full muscle segmentation method. We then performed preprocessing, including intensity inhomogeneity correction, spatial registration of different image sequences, intensity calibration of T2-weighted and T2-weighted fat-suppressed images, and calculation of MRI biomarker maps. Finally, for each of the segmented muscles, we automatically measured MRI biomarkers of muscle volume, intensity statistics over MRI biomarker maps, and statistical image texture features. Results The muscle volume and the mean intensities in T2 value, fat, and water maps showed group differences between normal and GRMD dogs. For the statistical texture biomarkers, both the histogram and run-length matrix features showed obvious group differences between normal and GRMD dogs. The full muscle segmentation showed significantly less error and variability in the proposed biomarkers when compared to the standard, limited muscle range segmentation. Conclusion The experimental results demonstrated that this quantification tool could reliably quantify MRI biomarkers in GRMD dogs, suggesting that it would also be useful for quantifying disease progression and measuring therapeutic effect in DMD patients.

Background: Despite the advancement in drug and surgical interventions, myocardial damage and associated cardiac dysfunction lead to heart failure that remains common cause of death following myocardial infarction (MI). Spiny mice (Acomys cahirinus, SM) have been shown to possess regenerating capacity following deep tissue injury without scarring ( Nature 2013 ). This led us to investigate if this regenerative property would also be preserved in the heart. Methods and Results: Adult CD1 and SM were subject to left anterior descending coronary artery ligation or sham surgeries. Proliferative cells were identified by nuclear incorporation of 5-bromodeoxyuridine (BrdU, daily, i.p.) and injection was started from 3d post MI continued to 2wks post MI. Cardiac function was assessed using echocardiography and MRI. SM exhibited 3-fold smaller infarct size (SM-MI 18.6±3.4% vs CD1-MI 76.2±3.4%, p+ cells in left ventricle after MI while CD1 mice had 4-fold increase (CD1-sham 11±3.5 vs CD1-MI 44±9.1 and SM-sham 16±9.8 vs SM-MI 101.1±30.9, p Conclusions: SM are protected from ischemia induced cardiac damage and dysfunction. This involves increased proliferating cardiac cells and reduction in infarct size. Thus SM could be an ideal animal model for identification of molecular and genetic circuits involved in preservation/regeneration of cardiac function with translational implication to human MI.

There is a pressing need for biomarkers and outcomes that can be used across disease stages in Duchenne muscular dystrophy (DMD), to facilitate the inclusion of a wider range of participants in clinical trials and to improve our understanding of the natural history of DMD. Quantitative magnetic resonance imaging (qMRI) and spectroscopy (MRS) biomarkers show considerable promise in both the legs and forearms of individuals with DMD, but have not yet been examined in functionally important proximal upper extremity muscles such as the biceps brachii and deltoid. The primary objective of this study was to examine the feasibility of implementing qMRI and MRS biomarkers in the proximal upper extremity musculature, and the secondary objective was to examine the relationship between MR measures of arm muscle pathology and upper extremity functional endpoints. Biomarkers included MRS and MRI measures of fat fraction and transverse relaxation time (T 2). The MR exam was well tolerated in both ambulatory and non-ambulatory boys. qMR biomarkers differentiated affected and unaffected participants and correlated strongly with upper extremity function (r = 0.91 for biceps brachii T 2 versus performance of upper limb score). These qMR outcome measures could be highly beneficial to the neuromuscular disease community, allowing measurement of the quality of functionally important muscles across disease stages to understand the natural history of DMD and particularly to broaden the opportunity for clinical trial participation.

Background Cardiomyopathy is a leading cause of mortality among Duchenne muscular dystrophy patients and lacks effective therapies. Phosphodiesterase type 5 is implicated in dystrophic pathology, and the phosphodiesterase type 5 inhibitor tadalafil has recently been studied in a clinical trial for Duchenne muscular dystrophy. Methods and Results Tadalafil was evaluated for the prevention of cardiomyopathy in the mdx mouse and golden retriever muscular dystrophy dog models of Duchenne muscular dystrophy. Tadalafil blunted the adrenergic response in mdx hearts during a 30‐minute dobutamine challenge, which coincided with cardioprotective signaling, reduced induction of μ‐calpain levels, and decreased sarcomeric protein proteolysis. Dogs with golden retriever muscular dystrophy began daily tadalafil treatment prior to detectable cardiomyopathy and demonstrated preserved cardiac function, as assessed by echocardiography and magnetic resonance imaging at ages 18, 21, and 25 months. Tadalafil treatment improved golden retriever muscular dystrophy histopathological features, decreased levels of the cation channel TRPC 6, increased total threonine phosphorylation status of TRPC 6, decreased m‐calpain levels and indicators of calpain target proteolysis, and elevated levels of utrophin. In addition, we showed that Duchenne muscular dystrophy patient myocardium exhibited increased TRPC 6, m‐calpain, and calpain cleavage products compared with control human myocardium. Conclusions Prophylactic use of tadalafil delays the onset of dystrophic cardiomyopathy, which is likely attributed to modulation of TRPC 6 levels and permeability and inhibition of protease content and activity. Consequently, phosphodiesterase type 5 inhibition is a candidate therapy for slowing the development of cardiomyopathy in Duchenne muscular dystrophy patients.

Purpose Skeletal muscles of children with Duchenne muscular dystrophy (DMD) have enhanced susceptibility to damage and progressive lipid infiltration, which contribute to an increase in magnetic resonance proton transverse relaxation time (T2). Therefore, examining T2 changes in individual muscles may be useful for monitoring disease progression in DMD. In this study we utilized mean T2, percent elevated pixels, and T2 heterogeneity to assess changes in composition of dystrophic muscles. In addition, we used fat saturation (fatsat) to distinguish T2 changes due to edema and inflammation from fat infiltration in muscles.

The purpose of this study was to evaluate skeletal muscle composition of lower extremity muscles in a manifesting female carrier of Duchenne muscular dystrophy (MFC DMD ) using magnetic resonance imaging (MRI) and spectroscopy (MRS). MRI/MRS was performed on the lower extremities and heart of a MFC DMD (47years, 51kg) on four occasions within 21months and in a control subject. Heterogeneity and asymmetry among muscles in the MFC DMD was observed in lipid fraction and mean transverse relaxation time (T 2 ) of lower extremity muscles with some muscles presenting as unaffected (e.g., rectus femoris) and others showing substantial deterioration and lipid infiltration (e.g., vasti muscles). There was an association of abnormal MRI findings and strength and motor function. Over the 21months a small decrease in CSA max and increase in lipid fraction and T 2 was observed in the MFC DMD in some muscles. In summary, this MFC DMD revealed significant imaging evidence of pathologic heterogeneity among muscles. Furthermore, this study shows the feasibility of combining various quantitative MRI and MRS approaches to monitor skeletal muscle involvement.

The aim of this study was to use the rat moderate spinal cord contusion model to investigate the effects of incomplete spinal cord injury (SCI) on the muscle regeneration process, comparing regeneration of slow-twitch plantarflexor soleus muscle and fast-twitch dorsiflexor tibialis anterior (TA) muscle. Additionally, we wanted to examine the effect of a week of locomotor training following incomplete SCI on the muscle regeneration process in these muscles and also determine if a week of similar locomotor training is sufficient to initiate muscle regeneration in control, non-injured rats. Thirty-two, adult, female, Sprague-Dawley rats were chosen for the study. Moderate, midthoracic contusion SCIs were produced using a NYU (New York University) impactor in all rats except controls. Animals were randomly assigned to treadmill training or untrained groups. Rats in the treadmill training group were manually treadmill trained starting at 1 week after SCI, for 10 bouts (2 sessions of 20 min of actual stepping) over 5 days and control rats in the training group received similar training. Our results indicate that a muscle regenerative response was initiated only in the slow-twitch soleus muscle in the initial 2 weeks following SCI, the addition of 1 week of locomotor treadmill training led to a significant increase in soleus regenerative process. No significant regenerative process was observed in the fast-twitch TA. Increased muscle regeneration in soleus is suggested by our findings of increased expression of (1) insulin-like growth factor-1, involved in the activation of satellite cells; (2) Pax7, a marker of satellite cell activation; (3) myogenin, a muscle regulatory protein; and (4) embryonic myosin, an indicator of new muscle fiber formation. Locomotor training in control, non-injured animals did not induce similar changes towards the regenerative process.

Studies have shown promise in using various approaches of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy to evaluate skeletal muscle involvement in Duchenne muscular dystrophy. However, these studies have mainly been performed using a cross-sectional design, and the correlation of these MRI changes with disease progression and disease severity has not been fully elucidated. Overall, skeletal muscle MRI is a powerful and sensitive technique in the evaluation of muscle disease, and its use as a biomarker for disease progression or therapeutic response in clinical trials deserves further study.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked recessive disease affecting 1 in 3,500 newborn boys for which there is no effective treatment or cure. One novel strategy that has therapeutic potential for DMD is inhibition of myostatin, a negative regulator of skeletal muscle mass that may also promote fibrosis. Therefore, our goal in this study was to evaluate systemic myostatin inhibition in the golden retriever model of DMD (GRMD). GRMD canines underwent liver-directed gene transfer of a self-complementary adeno-associated virus type 8 vector designed to express a secreted dominant-negative myostatin peptide (n = 4) and were compared with age-matched, untreated GRMD controls (n = 3). Dogs were followed with serial magnetic resonance imaging (MRI) for 13 months to assess cross-sectional area and volume of skeletal muscle, then euthanized so that tissue could be harvested for morphological and histological analysis. We found that systemic myostatin inhibition resulted in increased muscle mass in GRMD dogs as assessed by MRI and confirmed at tissue harvest. We also found that hypertrophy of type IIA fibers was largely responsible for the increased muscle mass and that reductions in serum creatine kinase and muscle fibrosis were associated with long-term myostatin inhibition in GRMD. This is the first report describing the effects of long-term, systemic myostatin inhibition in a large-animal model of DMD, and we believe that the simple and effective nature of our liver-directed gene-transfer strategy makes it an ideal candidate for evaluation as a novel therapeutic approach for DMD patients.

Insulin-like growth factor I (IGF-I) is a potent myogenic factor that plays a critical role in muscle regeneration and muscle hypertrophy. The purpose of this study was to evaluate the effect of IGF-I overexpression on the recovery of muscle size and function during reloading/reambulation after a period of cast immobilization in predominantly fast twitch muscles. In addition, we investigated concomitant molecular responses in IGF-I receptor and binding proteins (BPs). Recombinant adeno-associated virus vector for IGF-I (rAAV-IGF-IA) was injected into the anterior compartment of one of the hindlimbs of young (3 wk) C57BL6 female mice. At 20 wk of age, both hindlimbs were cast immobilized in a shortened position for 2 wk to unload the tibialis anterior (TA) and extensor longus digitorum (EDL) muscles. The TA and EDL muscles were removed bilaterally after 2 wk of cast immobilization and after 1 and 3 wk of free cage reambulation. Increases in IGF-I mRNA and protein levels with IGF-I overexpression were associated with significant increases in muscle wet weight, fiber size, and tetanic force, although overexpression did not protect against cast immobilization-induced muscle atrophy. After 1 wk of reambulation, evidence of enhanced muscle regeneration was noted in IGF-I-overexpressing muscles with an increased prevalence of central nuclei, embryonic myosin, and Pax7 positive fibers. We also observed larger relative gains in muscle size (wet weight and fiber area), but not force, during the 3-wk reambulation period in hindlimb muscles overexpressing IGF-I compared with contralateral control legs. Changes in IGFBP-5 mRNA expression during cast immobilization and reambulation paralleled those of IGF-I, whereas IGFBP-3 expression changed inversely to IGFBP-5.