P2Y2 purinergic receptors are highly expressed in cardiac and diaphragm muscles of mdx mice,and their expression is decreased by suramin

Introduction: In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin leads to increased calcium influx and muscle necrosis. Patients suffer progressive muscle loss, and cardiomyopathy is an important determinant of morbidity. P2 purinergic receptors participate in the increased calcium levels in dystrophic skeletal muscles. Methods: In this study, we evaluated whether P2 receptors are involved in cardiomyopathy in mdx mice at later stages of the disease. Results: Western blotting revealed that P2Y₂ receptor levels were upregulated (54%) in dystrophic heart compared with a normal heart. Suramin reduced the levels of P2Y₂ to almost normal values. Suramin also decreased heart necrosis (reduced CK‐MB) and the expression of the stretch‐activated calcium channel TRPC1. Conclusions: This study suggests that P2Y₂ may participate in cardiomyopathy in mdx mice. P2‐selective drugs with specific actions in the dystrophic heart may ameliorate cardiomyopathy in dystrophinopathies. Muscle Nerve 55 : 116–121, 2017     

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     

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     

Long‐term therapy with deflazacort decreases myocardial fibrosis in mdx mice

We evaluated the effects of long‐term administration of deflazacort (DFZ) on the progression of myocardial fibrosis in mdx mice. Mdx mice (6 months old) were treated with DFZ for 15 months. Myocardial fibrosis (MF) was evaluated by histomorphometric methods, and treated and untreated mdx mice of the same age (21 months) were compared. DFZ significantly decreased MF. We conclude that long‐term therapy with DFZ is effective in slowing down the progression of fibrosis in the dystrophin‐deficient heart. Muscle Nerve, 2009     

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     

Prevention of muscle fibrosis and myonecrosis in mdx mice by suramin, a TGF-β1 blocker

Fibrosis is a pathological feature observed in patients with Duchenne muscular dystrophy (DMD) and in mdx mice, the experimental model of DMD. We evaluated the effect of suramin, a transforming growth factor-beta 1 (TGF-β1) blocker, on fibrosis in mdx mice. mdx mice (6 months old) received suramin for 7 weeks. Suramin- and saline-treated (control) mdx mice performed exercise on a treadmill to worsen disease progression. Immunoblotting showed an increase of TGF-β1 in mdx diaphragm, limb, and cardiac muscles. Suramin decreased creatine kinase in mdx mice and attenuated fibrosis in all muscles studied, except for cardiac muscle. Suramin protected limb muscles against damage and reduced the exercise-induced loss of strength over time. These findings support a role for TGF-β1 in fibrinogenesis and myonecrosis during the later stages of disease in mdx mice. Suramin might be a useful therapeutic alternative for the treatment of dystrophinopathies.     

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     

Timeline of cardiac dystrophy in 3–18‐month‐old MDX mice

The dystrophin‐deficient (mdx) mouse remains the most commonly used model for Duchenne muscular dystrophy (DMD). Mdx mice show a predominantly covert cardiomyopathy, the hallmark of which is fibrosis. We compared mdx and normal mice at six ages (3, 6, 9, 12, 15, and 18 months) using in vivo assessment of cardiac function, selective collagen staining, and measures of TGF‐β mRNA, Evans blue dye infiltration, macrophage infiltration, and aortic wall thickness. Clear temporal progression was demonstrated, including early fragility of cardiomyocyte membranes, which has an unrelated impact on cardiac function but is associated with macrophage infiltration and fibrosis. Aortic wall thickness is less in older mdx mice. Mdx mice display impaired responses to inotropic challenge from a young age; this is indicative of altered adrenoreceptor function. We draw attention to the paradox of ongoing fibrosis in mdx hearts without a strong molecular signature (in the form of TGF‐β mRNA expression). Muscle Nerve, 2010     

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     

Cardiomyopathy in the dystrophin/utrophin-deficient mouse model of severe muscular dystrophy is characterized by dysregulation of matrix metalloproteinases

Cardiomyopathy is a significant component in Duchenne muscular dystrophy. Although mdx mice are deficient in dystrophin, they only develop mild indicators of cardiomyopathy before 1 year-of-age, making therapeutic investigations using this model lengthy. In contrast, mdx mice also lacking utrophin (utrn^−/−;mdx) show severely reduced cardiac contractile function and histological indicators of cardiomyopathy by 8–10 weeks-of-age. Here we demonstrate that utrn^−/−;mdx mice show a similar pattern of cardiac damage to that in dystrophic patients. Matrix metalloproteinases required for ventricular remodeling during the evolution of heart failure are upregulated in utrn^−/−;mdx mice concurrent with the onset of cardiac pathology by 10 weeks-of-age. Matrix metalloproteinase activity is further dysregulated due to reduced levels of endogenous tissue inhibitors and co-localizes with fibroblasts and collagen I-containing scars. utrn^−/−;mdx mice are therefore a very useful model for investigating potential cardiac therapies.     

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     

Cognitive impairment appears progressive in the mdx mouse

Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disease caused by mutations in the DMD gene, which encodes the large cytoskeletal protein dystrophin. Approximately one-third of DMD patient's exhibit cognitive problems yet it is unknown if cognitive impairments worsen with age. The mdx mouse model is deficient in dystrophin demonstrates cognitive abnormalities, but no studies have investigated this longitudinally. We assessed the consequences of dystrophin deficiency on brain morphology and cognition in male mdx mice. We utilised non-invasive methods to monitor CNS pathology with an aim to identify changes longitudinally (between 4 and 18 months old) which could be used as outcome measures. MRI identified a total brain volume (TBV) increase in control mice with ageing (p < 0.05); but the mdx mice TBV increased significantly more (p < 0.01). Voxel-based morphometry (VBM) identified decreases in grey matter volume, particularly in the hippocampus of the mdx brain, most noticeable from 12 months onwards, as were enlarged lateral ventricles in mdx mice. The caudate putamen of older mdx mice showed increases in T₂- relaxometry which may be considered as evidence of increased water content. Hippocampal spatial learning and memory was decreased in mdx mice, particularly long-term memory, which progressively worsened with age. The novel object recognition (NOR) task highlighted elevated anxiety-related behaviour in older mdx mice. Our studies suggest that dystrophin deficiency causes a progressive cognitive impairment in mice (compared to ageing control mice), becoming evident at late disease stages, and may explain why progressive CNS symptoms are not obvious in DMD patients.     

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 weakness and atrophy are associated with decreased regenerative capacity and changes in mTOR signaling in skeletal muscles of venerable (18–24‐month‐old) dystrophic mdx mice

The muscles of mdx mice progressively deteriorate with age. We wanted to know whether this is associated with a decrease in regenerative capacity and/or changes in the mammalian target of rapamycin complex (mTOR) signaling pathway. Muscles of mdx mice aged 5 weeks, 5, 12, and 18–24 months were studied. Maximal force and muscle weight of the older mice were decreased as compared to younger adult mice. Activation of the mTOR signaling pathway, i.e., phosphorylation of Akt (also known as protein kinase B) and ribosomal protein S6 was also reduced in the older mice. Moreover, 14 days after cardiotoxin injury the degree of recovery of maximal force and muscle weight were less in the older mice. In contrast to younger mice, there was also activation of the mTOR pathway during regeneration in the older mice. Progressive muscle weakness and atrophy in mdx mouse muscle is associated with a decline in regenerative potential and changes in activation of the mTOR signaling pathway. Muscle Nerve 000: 000–000, 2010     

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     

P2 purinoceptor blocker suramin antagonises NMDA receptors and protects against excitatory behaviour caused by NMDA receptor agonist (RS)-(tetrazol-5-yl)-glycine in rats

It has been reported that suramin, an anthelminthic, trypanocidal agent and an inhibitor of P₂ receptors, may antagonise N-methyl-D-aspartate (NMDA) subtype of the excitatory amino acid receptors. Both NMDA receptors and P_2X subclass of P₂ receptors are ligand-gated Ca²⁺-selective channels and, since the increased influx of Ca²⁺ into neurons has been linked to neurotoxicity, simultaneous inhibition of P_2X and NMDA receptors in vivo by suramin could represent an effective neuroprotective treatment. We have found that suramin inhibited the binding of [³H]CGP 39653 to NMDA receptor binding sites in vitro and reduced the frequency of NMDA channel openings in patch-clamp studies. Suramin (1 mM) had no effect on [³H]kainate binding in vitro. In vivo, intracerebroventricular (ICV) injections of suramin (70 nmol/brain) antagonised convulsive effects of the NMDA agonist (RS)-(tetrazol-5-yl)-glycine (TZG, LY 285265). Suramin, however, did not prevent neurotoxic lesions in the hippocampus caused by ICV administration of TZG. Increasing the dose of suramin resulted in death from severe respiratory depression. J. Neurosci. Res. 49:627–638, 1997. © 1997 Wiley-Liss, Inc.     

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     

Evidence of mdx mouse skeletal muscle fragility in vivo by eccentric running exercise

Duchenne muscular dystrophy is an X-linked devastating disease due to the lack of expression of a functional dystrophin. Unfortunately, the dystrophin-deficient mdx mouse model does not present clinical signs of dystrophy before the age of 18 months, and the role of dystrophin in fiber integrity is not fully understood. The fragility of the skeletal muscle fibers was investigated in transgenic mice expressing β-galactosidase under the control of a muscle specific promoter. Adult mdx/β-galactosidase (dystrophin-negative) and normal/β-galactosidase (dystrophin-positive) mice were submitted to one short session of eccentric, downhill running exercise. The leakage of muscle enzymes creatine kinase and β-galactosidase was investigated before, 1 h after, and 3 days after the running session. A significant and transient rise in the level of these enzymes was noted in the serum of mdx mice following the exercise session. Thus, the lack of dystrophin in the mdx model led to local microdamages to the exercised muscle allowing leakage of proteins from the fibers. The peak leakage was transient, suggesting that muscle fiber lesions were rapidly repaired following this short, noninvasive eccentric running session. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:567–576, 1998.