Cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD), however, in the mdx mouse model of DMD, the cardiac phenotype differs from that seen in DMD-associated cardiomyopathy. Although some have used pharmacologic stress to enhance the cardiac phenotype in the mdx model, many methods lead to high mortality, variable cardiac outcomes, and do not recapitulate the structural and functional cardiac changes seen in human disease. Here, we describe a simple and effective method to enhance the cardiac phenotype model in mdx mice using advanced 2D and 4D high-frequency ultrasound to monitor cardiac dysfunction progression in vivo. For our study, mdx and wild-type (WT) mice received daily low-dose (2 mg/kg/day) isoproterenol injections for 10 days. Histopathologic assessment showed that isoproterenol treatment increased myocyte injury, elevated serum cardiac troponin I levels, and enhanced fibrosis in mdx mice. Ultrasound revealed reduced ventricular function, decreased wall thickness, increased volumes, and diminished cardiac reserve in mdx mice compared to wild-type. Our findings highlight the utility of low-dose isoproterenol in mdx mice as a valuable model for exploring therapies targeting DMD-associated cardiac complications.

The progressive degeneration of the skeletal musculature in Duchenne muscular dystrophy is accompanied by reactive myofibrosis, fat substitution, and chronic inflammation. Fibrotic changes and reduced tissue elasticity correlate with the loss in motor function in this X-chromosomal disorder. Thus, although dystrophinopathies are due to primary abnormalities in the DMD gene causing the almost-complete absence of the cytoskeletal Dp427-M isoform of dystrophin in voluntary muscles, the excessive accumulation of extracellular matrix proteins presents a key histopathological hallmark of muscular dystrophy. Animal model research has been instrumental in the characterization of dystrophic muscles and has contributed to a better understanding of the complex pathogenesis of dystrophinopathies, the discovery of new disease biomarkers, and the testing of novel therapeutic strategies. In this article, we review how mass-spectrometry-based proteomics can be used to study changes in key components of the endomysium, perimysium, and epimysium, such as collagens, proteoglycans, matricellular proteins, and adhesion receptors. The mdx-4cv mouse diaphragm displays severe myofibrosis, making it an ideal model system for large-scale surveys of systematic alterations in the matrisome of dystrophic fibers. Novel biomarkers of myofibrosis can now be tested for their appropriateness in the preclinical and clinical setting as diagnostic, pharmacodynamic, prognostic, and/or therapeutic monitoring indicators.

NADPH oxidase (NOX2) is responsible for reactive oxygen species (ROS) production in neutrophils and has been recognized as a key mediator in inflammatory and cardiovascular pathologies. Nevertheless, there is a lack of specific NOX2 pharmacological inhibitors. In medicinal chemistry, heterocyclic compounds are essential scaffolds for drug design, and among them, indole is a very versatile pharmacophore. We tested the hypothesis that indole heteroaryl-acrylonitrile derivatives may serve as NOX2 inhibitors by evaluating the capacity of 19 of these molecules to inhibit NOX2-derived ROS production in human neutrophils (HL-60 cells). Of these compounds, C6 and C14 exhibited concentration-dependent inhibition of NOX2 (IC50~1 µM). These molecules also reduced NOX2-derived oxidative stress in cardiomyocytes and prevented cardiac damage induced by ischemia-reperfusion. Compound C6 significantly reduced the membrane translocation of p47phox, a cytosolic subunit that is required for NOX2 activation. Molecular docking analyses of the binding modes of these molecules with p47phox indicated that C6 and C14 interact with specific residues in the inner part of the groove of p47phox, the binding cavity for p22phox. This combination of methods showed that novel indole heteroaryl acrylonitriles represent interesting lead compounds for developing specific and potent NOX2 inhibitors.

Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet-to-be-fully deciphered molecular perturbations. Emerging evidence implicates RhoA/Rho-associated protein kinase (ROCK) signalling in DMD pathology, yet its direct role in DMD muscle function, and related mechanisms, are unknown.
Three-dimensionally engineered dystrophin-deficient mdx skeletal muscles and mdx mice were used to test the role of ROCK in DMD muscle function in vitro and in situ, respectively. The role of ARHGEF3, one of the RhoA guanine nucleotide exchange factors (GEFs), in RhoA/ROCK signalling and DMD pathology was examined by generating Arhgef3 knockout mdx mice. The role of RhoA/ROCK signalling in mediating the function of ARHGEF3 was determined by evaluating the effects of wild-type or GEF-inactive ARHGEF3 overexpression with ROCK inhibitor treatment. To gain more mechanistic insights, autophagy flux and the role of autophagy were assessed in various conditions with chloroquine.
Inhibition of ROCK with Y-27632 improved muscle force production in 3D-engineered mdx muscles (+25% from three independent experiments, P < 0.05) and in mice (+25%, P < 0.001). Unlike suggested by previous studies, this improvement was independent of muscle differentiation or quantity and instead related to increased muscle quality. We found that ARHGEF3 was elevated and responsible for RhoA/ROCK activation in mdx muscles, and that depleting ARHGEF3 in mdx mice restored muscle quality (up to +36%, P < 0.01) and morphology without affecting regeneration. Conversely, overexpressing ARHGEF3 further compromised mdx muscle quality (-13% vs. empty vector control, P < 0.01) in GEF activity- and ROCK-dependent manner. Notably, ARHGEF3/ROCK inhibition exerted the effects by rescuing autophagy which is commonly impaired in dystrophic muscles.
Our findings uncover a new pathological mechanism of muscle weakness in DMD involving the ARHGEF3-ROCK-autophagy pathway and the therapeutic potential of targeting ARHGEF3 in DMD.

Muscular dystrophies are inherited neuromuscular diseases, resulting in progressive disability and often affecting life expectancy. The most severe, common types are Duchenne muscular dystrophy (DMD) and Limb-girdle sarcoglycanopathy, which cause advancing muscle weakness and wasting. These diseases share a common pathomechanism where, due to the loss of the anchoring dystrophin (DMD, dystrophinopathy) or due to mutations in sarcoglycan-encoding genes (LGMDR3 to LGMDR6), the α-sarcoglycan ecto-ATPase activity is lost. This disturbs important purinergic signaling: An acute muscle injury causes the release of large quantities of ATP, which acts as a damage-associated molecular pattern (DAMP). DAMPs trigger inflammation that clears dead tissues and initiates regeneration that eventually restores normal muscle function. However, in DMD and LGMD, the loss of ecto-ATPase activity, that normally curtails this extracellular ATP (eATP)-evoked stimulation, causes exceedingly high eATP levels. Thus, in dystrophic muscles, the acute inflammation becomes chronic and damaging. The very high eATP over-activates P2X7 purinoceptors, not only maintaining the inflammation but also tuning the potentially compensatory P2X7 up-regulation in dystrophic muscle cells into a cell-damaging mechanism exacerbating the pathology. Thus, the P2X7 receptor in dystrophic muscles is a specific therapeutic target. Accordingly, the P2X7 blockade alleviated dystrophic damage in mouse models of dystrophinopathy and sarcoglycanopathy. Therefore, the existing P2X7 blockers should be considered for the treatment of these highly debilitating diseases. This review aims to present the current understanding of the eATP-P2X7 purinoceptor axis in the pathogenesis and treatment of muscular dystrophies.

The pathology in Duchenne muscular dystrophy (DMD) is characterized by degenerating muscle fibers, inflammation, fibro-fatty infiltrate, and edema, and these pathological processes replace normal healthy muscle tissue. The mdx mouse model is one of the most commonly used preclinical models to study DMD. Mounting evidence has emerged illustrating that muscle disease progression varies considerably in mdx mice, with inter-animal differences as well as intra-muscular differences in pathology in individual mdx mice. This variation is important to consider when conducting assessments of drug efficacy and in longitudinal studies. Magnetic resonance imaging (MRI) is a non-invasive method that can be used qualitatively or quantitatively to measure muscle disease progression in the clinic and in preclinical models. Although MR imaging is highly sensitive, image acquisition and analysis can be time intensive. The purpose of this study was to develop a semi-automated muscle segmentation and quantitation pipeline that can quickly and accurately estimate muscle disease severity in mice. Herein, we show that the newly developed segmentation tool accurately divides muscle. We show that measures of skew and interdecile range based on segmentation sufficiently estimate muscle disease severity in healthy wildtype and diseased mdx mice. Moreover, the semi-automated pipeline reduced analysis time by nearly 10-fold. Use of this rapid, non-invasive, semi-automated MR imaging and analysis pipeline has the potential to transform preclinical studies, allowing for pre-screening of dystrophic mice prior to study enrollment to ensure more uniform muscle disease pathology across treatment groups, improving study outcomes.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease that results in muscle wasting, wheelchair dependence, and eventual death due to cardiac and respiratory complications. In addition to muscle fragility, dystrophin deficiency also results in multiple secondary dysfunctions, which may lead to the accumulation of unfolded proteins causing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The purpose of this investigation was to understand how ER stress and the UPR are modified in muscle from D2-mdx mice, an emerging DMD model, and from humans with DMD. We hypothesized that markers of ER stress and the UPR are upregulated in D2-mdx and human dystrophic muscles compared to their healthy counterparts. Immunoblotting in diaphragms from 11-month-old D2-mdx and DBA mice indicated increased ER stress and UPR in dystrophic diaphragms compared to healthy, including increased relative abundance of ER stress chaperone CHOP, canonical ER stress transducers ATF6 and pIRE1α S724, and transcription factors that regulate the UPR such as ATF4, XBP1s, and peIF2α S51. The publicly available Affymetrix dataset (GSE38417) was used to analyze the expression of ER stress and UPR-related transcripts and processes. Fifty-eight upregulated genes related to ER stress and the UPR in human dystrophic muscles suggest pathway activation. Further, based on analyses using iRegulon, putative transcription factors that regulate this upregulation profile were identified, including ATF6, XBP1, ATF4, CREB3L2, and EIF2AK3. This study adds to and extends the emerging knowledge of ER stress and the UPR in dystrophin deficiency and identifies transcriptional regulators that may be responsible for these changes and be of therapeutic interest.

Introduction: It has been proposed that an increased susceptivity to oxidative stress caused by the absence of the protein dystrophin from the inner surface of the sarcolemma is a trigger of skeletal muscle necrosis in the destructive dystrophin deficient muscular dystrophies. Here we use the mdx mouse model of human Duchenne Muscular Dystrophy to test the hypothesis that adding the antioxidant NAC at 2% to drinking water for six weeks will treat the inflammatory phase of the dystrophic process and reduce pathological muscle fiber branching and splitting resulting in a reduction of mass in mdx fast-twitch EDL muscles. Methods: Animal weight and water intake was recorded during the six weeks when 2% NAC was added to the drinking water. Post NAC treatment animals were euthanised and the EDL muscles dissected out and placed in an organ bath where the muscle was attached to a force transducer to measure contractile properties and susceptibility to force loss from eccentric contractions. After the contractile measurements had been made the EDL muscle was blotted and weighed. In order to assess the degree of pathological fiber branching mdx EDL muscles were treated with collagenase to release single fibers. For counting and morphological analysis single EDL mdx skeletal muscle fibers were viewed under high magnification on an inverted microscope. Results: During the six-week treatment phase NAC reduced body weight gain in three- to nine-week-old mdx and littermate control mice without effecting fluid intake. NAC treatment also significantly reduced the mdx EDL muscle mass and abnormal fiber branching and splitting. Discussion: We propose chronic NAC treatment reduces the inflammatory response and degenerative cycles in the mdx dystrophic EDL muscles resulting in a reduction in the number of complexed branched fibers reported to be responsible for the dystrophic EDL muscle hypertrophy.

Adeno-associated virus (AAV)-mediated systemic micro-dystrophin (μDys) therapy is currently in clinical trials. The hope is to permanently improve the life quality of Duchenne muscular dystrophy (DMD) patients. Numerous preclinical studies have been conducted to support these trials. However, none examined whether a single therapy at a young age can lead to lifelong disease amelioration. To address this critical question, we injected 1 × 1013 vg particles/mouse of an AAV serotype-9 μDys vector to 3-month-old mdx mice through the tail vein. Therapeutic outcomes were evaluated at the age of 11 months (adulthood, 8 months postinjection) and 21 months (terminal age, 18 months postinjection). Immunostaining and Western blot showed saturated supraphysiological levels of μDys expression in skeletal muscle and heart till the end of the study. Treatment significantly improved grip force and treadmill running, and significantly reduced the serum creatine kinase level at both time points. Since cardiac death is a major threat in late-stage patients, we evaluated cardiac electrophysiology and hemodynamics by ECG and the closed-chest cardiac catheter assay, respectively. Significant improvements were observed in these assays. Importantly, many ECG and hemodynamic parameters (heart rate, PR interval, QRS duration, QTc interval, end-diastolic/systolic volume, dP/dt max and min, max pressure, and ejection fraction) were completely normalized at 21 months of age. Our results have provided direct evidence that a single systemic AAV μDys therapy has the potential to provide lifelong benefits in the murine DMD model.

Bone marrow mesenchymal stem cells (BMSCs) are commonly used in regenerative medicine. However, it is not clear whether transplantation of BMSCs can improve cardiac function of the X-Linked Muscular Dystrophy Mice (mdx) and how to detect it. We aimed to investigate the role of speckle tracking echocardiography (STE) in detecting cardiac function of the BMSCs-transplanted mdx in comparison with the untreated mdx.
The experimental mice were divided into the BMSCs-transplanted mdx, untreated mdx, and control mice groups (n = 6 per group). The BMSCs were transplanted via tail vein injections into a subset of mdx at 20 weeks of age. After four weeks, the cardiac functional parameters of all the mice in the 3 groups were analyzed by echocardiography. Then, all the mice were sacrificed, and the cardiac tissues were harvested and analyzed by immunofluorescence. The serum biochemical parameters were also analyzed to determine the beneficial effects of BMSCs transplantation.
Traditional echocardiography parameters did not show statistically significant differences after BMSCs transplantation for the three groups of mice. In comparison with the control group, mdx showed significantly lower left ventricular (LV) STE parameters in both the long-axis and short-axis LV images (P < 0.05). However, BMSCs-transplanted mdx showed improvements in several STE parameters including significant increases in a few STE parameters (P < 0.05). Immunofluorescence staining of the myocardium tissues showed statistically significant differences between the mdx and the control mice (P < 0.05), and the mdx transplanted with BMSCs demonstrated significantly improvement compared with the untreated mdx (P < 0.05).
This study demonstrated that the early reduction in the LV systolic and diastolic function in the mdx were accurately detected by STE. Furthermore, our study demonstrated that the transplantation of BMSCs significantly improved myocardial function in the mdx.