Electrical impedance myography for the in vivo and ex vivo assessment of muscular dystrophy (mdx) mouse muscle

Introduction: Sensitive, non‐invasive techniques are needed that can provide biomarkers of disease status and the effects of therapy in muscular dystrophy. Methods: We evaluated electrical impedance myography (EIM) to serve in this role by studying 2‐month‐old and 18‐month‐old mdx and wild‐type (WT) animals (10 animals in each of 4 groups). Results: Marked differences were observed in EIM values between mdx and WT animals; the differences were more pronounced between the older age groups (e.g., reactance of 92.6 ±4.3 Ω for mdx animals vs. 130 ± 4.1 Ω for WT animals, P < 0.001). In addition, in vivo EIM parameters correlated significantly with the extent of connective tissue deposition in the mdx animals. Conclusions: EIM has the potential to serve as a valuable non‐invasive method for evaluating muscular dystrophy. It can be a useful biomarker to assist with therapeutic testing in both pre‐clinical and clinical studies. Muscle Nerve 49 : 829–835, 2014     

Electrical impedance myography in facioscapulohumeral muscular dystrophy: A 1‐year follow‐up study

Introduction: Electrical impedance myography (EIM) is a noninvasive technique for measuring muscle composition and a potential physiological biomarker for facioscapulohumeral muscular dystrophy (FSHD). Methods: Thirty‐two participants with genetically confirmed and clinically affected FSHD underwent EIM in 7 muscles bilaterally. Correlations between EIM and baseline clinical measures were used to select EIM variables of interest in FSHD, and EIM and clinical measures were followed for 1 year. Results: There were no significant changes in the EIM variables. Although 50‐kHZ reactance correlated the strongest with clinical measures at baseline, the 50–211‐kHZ phase ratio demonstrated lower within‐subject 12‐month variability, potentially offering sample size savings for FSHD clinical trial planning. Discussion: EIM did not identify significant disease progression over 12 months. It is currently unclear whether this is because of limitations of the technology or the slow rate of disease progression in this cohort of FSHD patients over this period of time. Muscle Nerve 58 : 213–218, 2018     

Functional in situ assessment of muscle contraction in wild‐type and mdx mice

Introduction: Reports of muscle testing are frequently limited to maximal force alone. The experiments reported here show that force generation and relaxation rates can be obtained from the same experiments and provide a more complete functional characterization. Methods: Partial in situ testing was performed on the tibialis anterior of young wild‐type (WT) mice, young mdx mice, and old mdx mice. Force, force generation rate, and relaxation rates were measured during a fatigue test, 2 frequency–force tests, and a passive tension test. Results: We measured increased force but decreased force generation rate in WT compared with mdx muscles, and increased force but decreased relaxation rate of old compared with young mdx muscles. Young mdx muscles were the most sensitive to increases in passive tension. Conclusions: These measurements offer an improved understanding of muscle capability and are readily acquired by further analysis of the same tests used to obtain force measurements. Muscle Nerve 53: 260–268, 2016     

Electrophysiological subtypes and prognosis of Guillain–Barré syndrome in Northeastern China

Introduction: The dystrophic features in hindlimb skeletal muscles of female mdx mice are unclear. Methods: We analyzed force‐generating capacity and force decline after lengthening contraction‐induced damage (fragility). Results: Young (6‐month‐old) female mdx mice displayed reduced force‐generating capacity (?18%) and higher fragility (23% force decline) compared with female age‐matched wild‐type mice. These 2 dystrophic features were less accentuated in young female than in young male mdx mice (?32% and 42% force drop). With advancing age, force‐generating capacity decreased and fragility increased in old (20 month) female mdx mice (?21% and 57% force decline), but they were unchanged in old male mdx mice. Moreover, estradiol treatment had no effect in old female mdx mice. Conclusions: Female gender–related factors mitigate dystrophic features in young but not old mdx mice. Further studies are warranted to identify the beneficial gender‐related factor in dystrophic muscle. Muscle Nerve, 2013     

Gender differences in contractile and passive properties of mdx extensor digitorum longus muscle

Introduction: Duchenne muscular dystrophy (DMD) is a severe, muscle‐wasting disease caused by mutations in the dystrophin gene. The mdx mouse is the first and perhaps the most commonly used animal model for study of DMD. Both male and female mdx mice are used. However, it is not completely clear whether gender influences contraction and the passive mechanical properties of mdx skeletal muscle. Methods: We compared isometric tetanic forces and passive forces of the extensor digitorum longus muscle between male and female mdx mice. Results: At age 6 months, female mdx mice showed better‐preserved specific tetanic force. Interestingly, at 20 months of age, female mdx muscle appeared stiffer. Conclusions: Our results suggest that gender may profoundly influence physiological measurement outcomes in mdx mice. Gender should be considered when using the mdx model. Muscle Nerve, 2012     

Combined XIL‐6R and urocortin‐2 treatment restores MDX diaphragm muscle force

Introduction: Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration leading to immobility, respiratory failure, and premature death. As chronic inflammation and stress are implicated in DMD pathology, the efficacy of an anti‐inflammatory and anti‐stress intervention strategy in ameliorating diaphragm dysfunction was investigated. Methods: Diaphragm muscle contractile function was compared in wild‐type and dystrophin‐deficient mdx mice treated with saline, anti‐interleukin‐6 receptor antibodies (xIL‐6R), the corticotrophin‐releasing factor receptor 2 (CRFR2) agonist, urocortin 2, or both xIL‐6R and urocortin 2. Results: Combined treatment with xIL‐6R and urocortin 2 rescued impaired force in mdx diaphragms. Mechanical work production and muscle shortening was also improved by combined drug treatment. Discussion: Treatment which neutralizes peripheral IL‐6 signaling and stimulates CRFR2 recovers force‐generating capacity and the ability to perform mechanical work in mdx diaphragm muscle. These findings may be important in the search for therapeutic targets in DMD. Muscle Nerve 56 : E134–E140, 2017     

MRC sum-score in the ICU: good reliability does not necessarily reflect "true reliability"

Introduction: Muscle and cardiac metabolism are dependent on the oxidation of fats and glucose for adenosine triphosphate production, for which L ‐carnitine is an essential cofactor. Methods: We measured muscle carnitine concentrations in skeletal muscles, diaphragm, and ventricles of C57BL/10ScSn‐DMDmdx/J mice (n = 10) and compared them with wild‐type C57BL/6J (n = 3), C57BL/10 (n = 10), and C3H (n = 12) mice. Citrate synthase (CS) activity was measured in quadriceps/gluteals and ventricles of mdx and wild‐type mice. Results: We found significantly lower tissue carnitine in quadriceps/gluteus (P < 0.05) and ventricle (P < 0.05), but not diaphragm of mdx mice, when compared with controls. CS activity was increased in mdx quadriceps/gluteus (P < 0.03) and ventricle (P < 0.02). This suggests compensatory mitochondrial biogenesis. Conclusions: Decreased tissue carnitine has implications for reduced fatty acid and glucose oxidation in mdx quadriceps/gluteus and ventricle. The mdx mouse may be a useful model for studying the role of muscle carnitine deficiency in DMD bioenergetic insufficiency and providing a targeted and timed rationale for L ‐carnitine therapy. Muscle Nerve 46: 767–772, 2012     

An octaguanidine-morpholino oligo conjugate improves muscle function of mdx mice

Introduction: Skeletal muscles of mdx mice lack functional levels of dystrophin due to a mutation in Dmd exon 23. Morpholino antisense oligomers can induce expression of a truncated dystrophin by redirecting splicing to skip processing of exon 23. Methods: We tested whether systemic administration of Vivo‐Morpholino, an octaguanidine delivery moiety–Morpholino conjugate that targets exon 23 (VMO23), restored function to muscles of mdx mice. Results: Extensor digitorum longus (EDL) muscles of mdx mice were weaker, less powerful, and showed greater functional deficits after eccentric contractions than normal. VMO23 treatment normalized EDL force and power of mdx mice and eliminated their exaggerated sensitivity to eccentric contractions. Diaphragm muscle strips from mdx mice also produced lower‐than‐normal force and power, and these variables were restored to normal, or near‐normal, levels by VMO23 treatment. Conclusion: These results provide a functional basis for continuing development of VMO23 as a treatment for Duchenne muscular dystrophy. Muscle Nerve, 2011     

Muscle magnetic resonance imaging and histopathology in ACTA1‐related congenital nemaline myopathy

Introduction: Muscle biopsy is usually diagnostic in nemaline myopathy (NM), but some patients may show nonspecific findings, leading to pitfalls in diagnosis. Muscle MRI is a helpful complementary tool. Methods: We assessed the clinical, histopathological, MRI, and molecular findings in a 19‐year‐old patient with NM in whom 2 muscle biopsies with ultrastructural examination showed no nemaline bodies. We analyzed the degree and pattern of muscle MRI involvement of the entire body, including the tongue and pectoral muscles. Results: Muscle MRI abnormalities in sartorius, adductor magnus, and anterior compartment muscles of the leg suggested NM. A previously unreported fatty infiltration of the tongue was found. A third biopsy after the muscle MRI showed scant nemaline bodies. A novel heterozygous de novo ACTA1 c.611C>T/p.Thr204Ile mutation was detected. Conclusions: We highlight the contribution of muscle imaging in addressing the genetic diagnosis of ACTA1‐related NM. Muscle Nerve 50: 1011–1016, 2014     

Effect of voluntary physical activity initiated at age 7 months on skeletal hindlimb and cardiac muscle function in mdx mice of both genders

Introduction: The effects of voluntary activity initiated in adult mdx (C57BL/10ScSc‐DMD^mdx/J) mice on skeletal and cardiac muscle function have not been studied extensively. Methods: We studied the effects of 3 months of voluntary wheel running initiated at age 7 months on hindlimb muscle weakness, increased susceptibility to muscle contraction?induced injury, and left ventricular function in mdx mice. Results: We found that voluntary wheel running did not worsen the deficit in force‐generating capacity and the force drop after lengthening contractions in either mdx mouse gender. It increased the absolute maximal force of skeletal muscle in female mdx mice. Moreover, it did not affect left ventricular function, structural heart dimensions, cardiac gene expression of inflammation, fibrosis, or remodeling markers. Conclusion: These results indicate that voluntary activity initiated at age 7 months had no detrimental effects on skeletal or cardiac muscles in either mdx mouse gender. Muscle Nerve 52 : 788–794, 2015     

Use of evans blue dye to compare limb muscles in exercised young and old mdx mice

Evans blue dye (EBD) is used to mark damaged and permeable muscle fibers in mouse models of muscular dystrophy and as an endpoint in therapeutic trials. We counted EBD‐positive muscle fibers and extracted EBD from muscles sampled throughout the hindlimbs in young adult and old mdx mice to determine if the natural variability in morphology would allow measurement of a functional improvement in one limb compared to the contralateral limb. Following one bout of rotarod or treadmill exercise that greatly increased serum creatine kinase levels, the number of EBD⁺ muscle fibers in 12–19‐month‐old mdx mice increased 3‐fold, EBD in the muscles increased, and, importantly, contralateral pairs of muscles contained similar amounts of EBD. In contrast, the intra‐ and interlimb amounts of EBD in 2–7‐month‐old mdx mice were much too variable. A therapeutic effect can more readily be measured in old mdx mice. These results will be useful in the design of therapy protocols using the mdx mouse. Muscle Nerve, 2010     

The magnetic resonance imaging spectrum of facioscapulohumeral muscular dystrophy

Introduction: Facioscapulohumeral muscular dystrophy (FSHD) is associated with a repeat contraction in the D4Z4 gene locus on chromosome 4q35. We used a one‐step quantitative magnetic resonance imaging (MRI) method to evaluate muscle, edema, and fat in patients spanning the range of severity. Methods: Fifteen patients with FSHD were compared with 10 healthy subjects using non‐negative linear least‐squares fitting of 32‐echo relaxation data (T2). The results were compared with a biexponential approach for characterizing muscle/fat ratio and T2 relaxation measurements from fat‐suppressed inversion recovery. Results: Increased T2 signal consistent with edema was common in FSHD subjects, a pattern not present in healthy controls. A varied pattern of edema and fatty replacement in muscles was shown. Conclusions: As a discrete biomarker, edema may be useful for following the clinical course of FSHD. Future work toward optimizing measurement is discussed. Muscle Nerve, 2012     

Soluble activin receptor type IIB increases forward pulling tension in the mdx mouse

Introduction: In this study we investigated the action of RAP‐031, a soluble activin receptor type IIB (ActRIIB) comprised of a form of the ActRIIB extracellular domain linked to a murine Fc, and the NF‐κB inhibitor, ursodeoxycholic acid (UDCA), on the whole body strength of mdx mice. Methods: The whole body tension (WBT) method of assessing the forward pulling tension (FPT) exerted by dystrophic (mdx) mice was used. Results: RAP‐031 produced a 41% increase in body mass and a 42.5% increase in FPT without altering the FPT normalized for body mass (WBT). Coadministration of RAP‐031 with UDCA produced increases in FPT that were associated with an increase in WBT. Conclusions: Myostatin inhibition increases muscle mass without altering the fundamental weakness characteristic of dystrophic muscle. Cotreatment with an NF‐κB inhibitor potentiates the effects of myostatin inhibition in improving FPT in mdx mice. Muscle Nerve, 2011     

Diffusion-weighted magnetic resonance imaging is useful for assessing inflammatory myopathies

Introduction: Short tau inversion recovery (STIR) sequences in whole-body MRI are usually used for detecting muscle edema (ME) in inflammatory myopathies. We evaluated b-value 800 diffusion-weighted imaging (b800 DWI).Methods: Two radiologists independently and a consensus reader retrospectively reexamined 60 patients with inflammatory myopathies and 15 controls. For each participant, 78 muscles were analyzed with 3 sets of imaging acquisitions: T1-weighted (T1) turbo spin echo and STIR; T1 and DWI; and T1, STIR and DWI. Mean edema per patient was compared between sequences. Agreement was evaluated. Results: Diffusion-weighted imaging detected more ME compared with STIR (P < 0.001). Agreement between readers was better with both sequences (k = 0.94) than with b800 DWI (k = 0.89) or STIR (k = 0.84) alone. Discussion: Diffusion-weighted imaging is a valuable add-on for the study of inflammatory myopathies. Muscle Nerve 59:555–555, 2019     

Expression of intercellular adhesion molecule‐1 by myofibers in mdx mice

Introduction: We investigated the extent to which intercellular adhesion molecule‐1 (ICAM‐1), a critical protein of the inflammatory response, is expressed in skeletal muscles of mdx mice (a murine model of Duchenne muscular dystrophy). Methods: Muscles were collected from control and mdx mice at 2‐24 weeks of age and analyzed for ICAM‐1 expression by means of Western blot and immunofluorescence. Results: Western blot revealed higher expression of ICAM‐1 in mdx compared with control muscles through 24 weeks of age. In contrast to control muscles, ICAM‐1 was expressed on the membrane of damaged, regenerating, and normal myofibers of mdx mice. CD11b+ myeloid cells also expressed ICAM‐1 in mdx muscles, and CD11b+ cells were closely associated with the membrane of myofibers expressing ICAM‐1. Conclusions: These findings support a paradigm in which ICAM‐1 and its localization to myofibers in muscles of mdx mice contributes to the dystrophic pathology. Muscle Nerve 52: 795–802, 2015     

Muscle–fat MRI: 1.5 tesla and 3.0 tesla versus histology

Introduction: We evaluated muscle/fat fraction (MFF) accuracy and reliability measured with an MR imaging technique at 1.5 Tesla (T) and 3.0T scanner strengths, using biopsy as reference. Methods: MRI was performed on muscle samples from pig and rabbit species (n = 8) at 1.5T and 3.0T. A chemical shift based 2‐point Dixon method was used, collecting in‐phase and out‐of‐phase data for fat/water of muscle samples. Values were compared with MFFs calculated from histology. Results: No significant difference was found between 1.5T and 3.0T (P values = 0.41–0.96), or between histology and imaging (P = 0.83) for any muscle tested. Conclusions: Results suggest that a 2‐point Dixon fat/water separation MRI technique may provide reliable quantification of MFFs at varying field strengths across different animal species, and consistency was established with biopsy. The results set a foundation for larger scale investigation of quantifying muscle fat in neuromuscular disorders. Muscle Nerve 50:170–176, 2014     

Metformin increases peroxisome proliferator–activated receptor γ Co‐activator‐1α and utrophin a expression in dystrophic skeletal muscle

Introduction: Metformin (MET) stimulates skeletal muscle AMP‐activated protein kinase (AMPK), a key phenotype remodeling protein with emerging therapeutic relevance for Duchenne muscular dystrophy (DMD). Our aim was to identify the mechanism of impact of MET on dystrophic muscle. Methods: We investigated the effects of MET in cultured C₂C₁₂ muscle cells and mdx mouse skeletal muscle. Expression of potent phenotypic modifiers was assessed, including peroxisome proliferator–activated receptor (PPAR)γ coactivator‐1α (PGC‐1α), PPARδ, and receptor‐interacting protein 140 (RIP140), as well as that of the dystrophin‐homolog, utrophin A. Results: In C₂C₁₂ cells, MET augmented expression of PGC‐1α, PPARδ, and utrophin A, whereas RIP140 content was reciprocally downregulated. MET treatment of mdx mice increased PGC‐1α and utrophin A and normalized RIP140 levels. Conclusions: In this study we identify the impact of MET on skeletal muscle and underscore the timeliness and importance of investigating MET and other AMPK activators as relevant therapeutics for DMD. Muscle Nerve 52 : 139–142, 2015     

Suramin affects metalloproteinase‐9 activity and increases beta‐dystroglycan levels in the diaphragm of the dystrophin‐deficient mdx MOUSE

Introduction: In Duchenne muscular dystrophy and in the mdx mouse, muscle fiber degeneration and subsequent fibrosis lead to cardiorespiratory failure. Previously, we demonstrated that the anti‐fibrotic agent suramin was effective in decreasing fibrosis in mdx muscles. In this study, we were interested to see whether suramin could affect metalloproteinases (MMP) and improve the functional activity of the mdx diaphragm muscle. Methods: Zymography was performed to evaluate MMP‐2 and MMP‐9 activity. Western blotting was used to analyze the levels of beta‐dystroglycan. Muscle function was assessed in hemidiaphragm in vitro preparations. Results: We found that suramin affects metalloproteinase‐9 activity and increases beta‐dystroglycan. Furthermore, suramin also protects against diaphragm muscle fatigue over time. Conclusions: These results show the potential benefits of suramin in maintaining the structure of the dystrophin‐glycoprotein complex. Muscle Nerve, 46:810–813, 2012