Dystrophin deficiency disrupts muscle clock expression and mitochondrial quality control in mdx mice.

Impaired oxidative capacity and mitochondrial function contribute to the dystrophic pathology in muscles of patients with Duchenne muscular dystrophy (DMD) and in relevant mouse models of the disease. Emerging evidence suggests an association between disrupted core clock expression and mitochondrial quality control, but this has not been established in muscles lacking dystrophin. We examined the diurnal regulation of muscle core clock and mitochondrial quality control expression in dystrophin-deficient C57BL/10ScSn-Dmdmdx (mdx) mice, an established model of DMD. Male C57BL/10 (BL/10; n = 18) and mdx mice (n = 18) were examined every 4 h beginning at the dark cycle. Throughout the entire light-dark cycle, extensor digitorum longus (EDL) muscles from mdx mice had decreased core clock mRNA expression (Arntl, Cry1, Cry2, Nr1d2; P < 0.05) and disrupted mitochondrial quality control mRNA expression related to biogenesis (decreased; Ppargc1a, Esrra; P < 0.05), fission (increased; Dnm1l; P < 0.01), fusion (decreased; Opa1, Mfn1; P < 0.05), and autophagy/mitophagy (decreased: Bnip3; P < 0.05; increased: Becn1; P < 0.05). Cosinor analysis revealed a decrease in the rhythmicity parameters mesor and amplitude for Arntl, Cry1, Cry2, Per2, and Nr1d1 (P < 0.001) in mdx mice. Diurnal oscillations in Esrra, Sirt1, Map1lc3b, and Sqstm1 were absent in mdx mice, along with decreased mesor and amplitude of Ppargc1a mRNA expression (P < 0.01). The expression of proteins involved in mitochondrial biogenesis (decreased: PPARGC1A, P < 0.05) and autophagy/mitophagy (increased: MAP1LC3BII, SQSTM1, BNIP3; P < 0.05) were also dysregulated in tibialis anterior muscles of mdx mice. These findings suggest that dystrophin deficiency in mdx mice impairs the regulation of the core clock and mitochondrial quality control, with relevance to DMD and related disorders.

Mitochondrial hydrogen sulfide supplementation improves health in the C. elegans Duchenne muscular dystrophy model.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle degeneration and weakness due to mutations in the dystrophin gene. The symptoms of DMD share similarities with those of accelerated aging. Recently, hydrogen sulfide (H2S) supplementation has been suggested to modulate the effects of age-related decline in muscle function, and metabolic H2S deficiencies have been implicated in affecting muscle mass in conditions such as phenylketonuria. We therefore evaluated the use of sodium GYY4137 (NaGYY), a H2S-releasing molecule, as a possible approach for DMD treatment. Using the dys-1(eg33) Caenorhabditis elegans DMD model, we found that NaGYY treatment (100 µM) improved movement, strength, gait, and muscle mitochondrial structure, similar to the gold-standard therapeutic treatment, prednisone (370 µM). The health improvements of either treatment required the action of the kinase JNK-1, the transcription factor SKN-1, and the NAD-dependent deacetylase SIR-2.1. The transcription factor DAF-16 was required for the health benefits of NaGYY treatment, but not prednisone treatment. AP39 (100 pM), a mitochondria-targeted H2S compound, also improved movement and strength in the dys-1(eg33) model, further implying that these improvements are mitochondria-based. Additionally, we found a decline in total sulfide and H2S-producing enzymes in dystrophin/utrophin knockout mice. Overall, our results suggest that H2S deficit may contribute to DMD pathology, and rectifying/overcoming the deficit with H2S delivery compounds has potential as a therapeutic approach to DMD treatment.

Xanthine oxidase is hyper-active in Duchenne muscular dystrophy.

Generation of superoxide by xanthine oxidase can be stimulated under ischemic and aberrant calcium homeostasis. Because patients and mice with Duchenne muscular dystrophy (DMD) suffer from ischemia and excessive calcium influx, we tested the hypothesis that xanthine oxidase activity is elevated and contributes to disease pathology. Xanthine oxidase activity was measured by urinary isoxanthopterin in DMD patients at rest and in response to exercise. Urinary isoxanthopterin/creatinine was elevated compared to age-matched controls and Becker muscular dystrophy (BMD) patients. Concentrations were also increased after a six minute walk test in ambulatory patients. We also measured urinary isoxanthopterin in wildtype mice and a number of dystrophic mouse models; the DMD mouse model (mdx), mdx mice overexpressing a variety of transgenic miniaturized and chimeric skeletal muscle-specific dystrophins and utrophin and the ß-sarcoglycan deficient (Scgb-/-) mouse which represents type 2E human limb-girdle muscular dystrophy. Mdx and Scgb-/-mice had greater urinary isoxanthopterin/creatinine than wildtype mice while mdx mice expressing dystrophin or utrophin linking the extracellular matrix to the actin cytoskeleton were not different than wildtype. We also measured higher levels of urinary ortho-tyrosine in humans and mice deficient for dystrophin to confirm elevated oxidative stress. Surprisingly, mdx had lower xanthine oxidase protein levels and higher mRNA in gastrocnemius muscle compared to wildtype mice, however, the enzymatic activity of skeletal muscle xanthine oxidase was elevated above wildtype and a transgenic rescued mdx mouse (DysΔMTB-mdx). Downhill treadmill running also caused significant increases in mdx urinary isoxanthopterin that was prevented with the xanthine oxidase inhibitor allopurinol. Similarly, in vitro eccentric contraction-induced force drop of mdx muscle was attenuated by the allopurinol metabolite, oxypurinol. Together, our data suggests hyper-activity of xanthine oxidase in DMD, identifies xanthine oxidase activity as a contributing factor in eccentric contraction-induced force drop of dystrophin-deficient skeletal muscle and highlights the potential of isoxanthopterin as a noninvasive biomarker in DMD.

Increased constitutive nitric oxide production by whole body periodic acceleration ameliorates alterations in cardiomyocytes associated with utrophin/dystrophin deficiency.

Duchenne Muscular Dystrophy (DMD) cardiomyopathy is a progressive lethal disease caused by the lack of the dystrophin protein in the heart. The most widely used animal model of DMD is the dystrophin-deficient mdx mouse; however, these mice exhibit a mild dystrophic phenotype with heart failure only late in life. In contrast, mice deficient for both dystrophin and utrophin (mdx/utrn-/-, or dKO) can be used to model severe DMD cardiomyopathy where pathophysiological indicators of heart failure are detectable by 8-10weeks of age. Nitric oxide (NO) is an important signaling molecule involved in vital functions of regulating rhythm, contractility, and microcirculation of the heart, and constitutive NO production affects the function of proteins involved in excitation-contraction coupling. In this study, we explored the efficacy of enhancing NO production as a therapeutic strategy for treating DMD cardiomyopathy using the dKO mouse model of DMD. Specifically, NO production was induced via whole body periodic acceleration (pGz), a novel non-pharmacologic intervention which enhances NO synthase (NOS) activity through sinusoidal motion of the body in a headward-footward direction, introducing pulsatile shear stress to the vascular endothelium and cardiomyocyte plasma membrane. Male dKO mice were randomized at 8weeks of age to receive daily pGz (480cpm, Gz±3.0m/s2, 1h/d) for 4weeks or no treatment, and a separate age-matched group of WT animals (pGz-treated and untreated) served as non-diseased controls. At the conclusion of the protocol, cardiomyocytes from untreated dKO animals had, respectively, 4.3-fold and 3.5-fold higher diastolic resting concentration of Ca2+ ([Ca2+]d) and Na+ ([Na+]d) compared to WT, while pGz treatment significantly reduced these levels. For dKO cardiomyocytes, pGz treatment also improved the depressed contractile function, decreased oxidative stress, blunted the elevation in calpain activity, and mitigated the abnormal increase in [Ca2+]d upon mechanical stress. These improvements culminated in a significant reduction in circulating cardiac troponin T (cTnT) and an extension of the median lifespan of dKO mice from 16 to 31weeks. Treatment with L-NAME (NOS inhibitor) significantly decreased overall lifespan and abolished the cardioprotective properties elicited by pGz. Our results provide evidence that enhancement of NO synthesis by pGz can ameliorate cellular dysfunction in dKO cardiomyocytes and may represent a novel therapeutic intervention in DMD cardiomyopathy patients.

Inhibition of Human Glioma Cell Proliferation Caused by Knockdown of Utrophin Using a Lentivirus-Mediated System.

BACKGROUND: Glioma is the most devastating brain tumor worldwide. Previous studies showed that UTRN (utrophin) was related to cancers, but its role in glioma cells remains uncovered. MATERIALS AND METHODS: RNAi was used to knockdown UTRN in U251 cells using lentivirus system. The knockdown efficiency was validated by real-time quantitative PCR. Cell proliferation, cell cycle, and apoptosis progression were determined by MTT, colony formation analysis, and flow cytometry analysis. Furthermore, some apoptotic markers were examined by Western blot assay. RESULTS: Most cells were infected. Cell proliferation and colony formation ability were suppressed in U251 cells lacking UTRN. Moreover, there was an obvious increase in cell percentage in the G2/M phases and a significant apoptosis in U251 cells after UTRN silencing. Further investigation demonstrated that UTRN knockdown activated caspase and PARP pathways. CONCLUSIONS: Knockdown of UTRN expression by shRNA evidently inhibited cell proliferation and promoted cell apoptosis in glioma cells.

Chronic Dosing with Membrane Sealant Poloxamer 188 NF Improves Respiratory Dysfunction in Dystrophic Mdx and Mdx/Utrophin-/- Mice.

Poloxamer 188 NF (national formulary (NF) grade of P-188) improves cardiac muscle function in the mdx mouse and golden retriever muscular dystrophy models. However in vivo effects on skeletal muscle have not been reported. We postulated that P-188 NF might protect diaphragm muscle membranes from contraction-induced injury in mdx and mdx/utrophin-/- (dko) muscular dystrophy models. In the first study 7-month old mdx mice were treated for 22 weeks with subcutaneous (s.c.) injections of saline or P-188 NF at 3 mg/Kg. In the second, dkos were treated with saline or P-188 NF (1 mg/Kg) for 8 weeks beginning at age 3 weeks. Prednisone was the positive control in both studies. Respiratory function was monitored using unrestrained whole body plethysmography. P-188 NF treatment affected several respiratory parameters including tidal volume/BW and minute volume/BW in mdx mice. In the more severe dko model, P-188 NF (1 mg/Kg) significantly slowed the decline in multiple respiratory parameters compared with saline-treated dko mice. Prednisone's effects were similar to those seen with P-188 NF. Diaphragms from P-188 NF or prednisone treated mdx and dko mice showed signs of muscle fiber protection including less centralized nuclei, less variation in fiber size, greater fiber density, and exhibited a decreased amount of collagen deposition. P-188 NF at 3 mg/Kg s.c. also improved parameters of systolic and diastolic function in mdx mouse hearts. These results suggest that P-188 NF may be useful in treating respiratory and cardiac dysfunction, the leading causes of death in Duchenne muscular dystrophy patients.

Metabolic dysfunction and altered mitochondrial dynamics in the utrophin-dystrophin deficient mouse model of duchenne muscular dystrophy.

The utrophin-dystrophin deficient (DKO) mouse model has been widely used to understand the progression of Duchenne muscular dystrophy (DMD). However, it is unclear as to what extent muscle pathology affects metabolism. Therefore, the present study was focused on understanding energy expenditure in the whole animal and in isolated extensor digitorum longus (EDL) muscle and to determine changes in metabolic enzymes. Our results show that the 8 week-old DKO mice consume higher oxygen relative to activity levels. Interestingly the EDL muscle from DKO mouse consumes higher oxygen per unit integral force, generates less force and performs better in the presence of pyruvate thus mimicking a slow twitch muscle. We also found that the expression of hexokinase 1 and pyruvate kinase M2 was upregulated several fold suggesting increased glycolytic flux. Additionally, there is a dramatic increase in dynamin-related protein 1 (Drp 1) and mitofusin 2 protein levels suggesting increased mitochondrial fission and fusion, a feature associated with increased energy demand and altered mitochondrial dynamics. Collectively our studies point out that the dystrophic disease has caused significant changes in muscle metabolism. To meet the increased energetic demand, upregulation of metabolic enzymes and regulators of mitochondrial fusion and fission is observed in the dystrophic muscle. A better understanding of the metabolic demands and the accompanied alterations in the dystrophic muscle can help us design improved intervention therapies along with existing drug treatments for the DMD patients.

SERCA1 overexpression minimizes skeletal muscle damage in dystrophic mouse models.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting secondary to repeated muscle damage and inadequate repair. Elevations in intracellular free Ca²âº have been implicated in disease progression, and sarcoplasmic/endoplasmic reticulum Ca²âº-ATPase 1 (SERCA1) overexpression has been shown to ameliorate the dystrophic phenotype in mdx mice. The purpose of this study was to assess the effects of SERCA1 overexpression in the more severe mdx/Utr(-/-) mouse model of DMD. Mice overexpressing SERCA1 were crossed with mdx/Utr ± mice to generate mdx/Utr(-/-)/+SERCA1 mice and compared with wild-type (WT), WT/+SERCA1, mdx/+SERCA1, and genotype controls. Mice were assessed at ∼12 wk of age for changes in Ca²âº handling, muscle mass, quadriceps torque, markers of muscle damage, and response to repeated eccentric contractions. SERCA1-overexpressing mice had a two- to threefold increase in maximal sarcoplasmic reticulum Ca²âº-ATPase activity compared with WT which was associated with normalization in body mass for both mdx/+SERCA1 and mdx/Utr(-/-)/+SERCA1. Torque deficit in the quadriceps after eccentric injury was 2.7-fold greater in mdx/Utr(-/-) vs. WT mice, but only 1.5-fold greater in mdx/Utr(-/-)/+SERCA1 vs. WT mice, an attenuation of 44%. Markers of muscle damage (% centrally nucleated fibers, necrotic area, and serum creatine kinase levels) were higher in both mdx and mdx/Utr(-/-) vs. WT, and all were attenuated by overexpression of SERCA1. These data indicate that SERCA1 overexpression ameliorates functional impairments and cellular markers of damage in a more severe mouse model of DMD. These findings support targeting intracellular Ca²âº control as a therapeutic approach for DMD.

The role of proteases in excitation-contraction coupling failure in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is one of the most frequent types of muscular dystrophy. Alterations in intracellular calcium (Ca(2+)) handling are thought to contribute to the disease severity in DMD, possibly due to the activation of Ca(2+)-activated proteases. The purpose of this study was twofold: 1) to determine whether prolonged excitation-contraction (E-C) coupling disruption following repeated contractions is greater in animals lacking both dystrophin and utrophin (mdx/Utr(-/-)) compared with mice lacking only dystrophin (mdx); and 2) to assess whether protease inhibition can prevent E-C coupling failure following repeated tetani in these dystrophic mouse models. Excitation-contraction coupling was assessed using Fura-2 ratio, as an index of intracellular free Ca(2+) concentration, in response to electrical stimulation of single muscle fibers from the flexor digitorum brevis muscle. Resting Fura-2 ratio was higher in dystrophic compared with control (Con) fibers, but peak Fura-2 ratios during stimulation were similar in dystrophic and Con fibers. One hour after a series of repeated tetani, peak Fura-2 ratios were reduced by 30 ± 5.6%, 23 ± 2%, and 36 ± 3.1% in mdx, mdx/Utr(+/-), and mdx/Utr(-/-), respectively, with the greatest reduction in mdx/Utr(-/-) fibers (P < 0.05). Protease inhibition attenuated this decrease in peak Fura-2 ratio. These data indicate that E-C coupling impairment after repeated contractions is greatest in fibers lacking both dystrophin and utrophin and that prevention of protease activation can mitigate the prolonged E-C coupling impairment. These data further suggest that acute protease inhibition may be useful in reducing muscle weakness in DMD.

Systemic delivery of human bone marrow embryonic-like stem cells improves motor function of severely affected dystrophin/utrophin-deficient mice.

BACKGROUND AIMS: Embryonic-like stem cells (ELSCs) express embryonic stem cell-specific marker genes, such as SSEA-4, Oct-4 and Nanog, and can be induced to differentiate into cells of all 3 germ layers. Our preliminary data showed that ELSCs isolated from human bone marrow express multipotent antigen markers and differentiate into multinucleated myotube-like cells more efficiently than do mesenchymal stromal cells (MSCs) isolated from the same source. We investigated the therapeutic effect of ELSCs in dystrophin/utrophin double knock-out (dko) mice, one of the Duchenne muscular dystrophy animal models, by systemically transplanting them through tail-vein injection. METHODS: ELSCs and MSCs were both isolated from human bone marrow. Two months after equal amounts of ELSCs or MSCs were injected through tail-vein injection, we evaluated skeletal muscle motor function and serum creatine kinase activity and measured dystrophin expression by means of immunostaining, Western blotting and semi-quantitative reverse transcriptase-polymerase chain reaction. RESULTS: ELSCs positive for Oct-4 and Nanog-3 expressed higher levels of SSEA-4, FZD-9 and CD105 and were induced to differentiate into myotube-like cells more efficiently than did MSCs in vitro. Transplantation of ELSCs through the tail vein improved motor function and decreased serum creatine kinase activity at 2 months after cell transplantation. In addition, dystrophin protein and messenger RNA were upregulated and the skeletal muscle histology was improved in these dko mice transplanted with ELSCs. CONCLUSIONS: ELSCs could be more efficiently induced to differentiate into myotubes than were MSCs in vitro, and systematically transplanting ELSCs improved muscle motor function and muscle histology in dko mice.

Prednisolone attenuates improvement of cardiac and skeletal contractile function and histopathology by lisinopril and spironolactone in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an inherited disease that causes striated muscle weakness. Recently, we showed therapeutic effects of the combination of lisinopril (L), an angiotensin converting enzyme (ACE) inhibitor, and spironolactone (S), an aldosterone antagonist, in mice lacking dystrophin and haploinsufficient for utrophin (utrn(+/-);mdx, het mice); both cardiac and skeletal muscle function and histology were improved when these mice were treated early with LS. It was unknown to what extent LS treatment is effective in the most commonly used DMD murine model, the mdx mouse. In addition, current standard-of-care treatment for DMD is limited to corticosteroids. Therefore, potentially useful alternative or additive drugs need to be both compared directly to corticosteroids and tested in presence of corticosteroids. We evaluated the effectiveness of this LS combination in the mdx mouse model both compared with corticosteroid treatment (prednisolone, P) or in combination (LSP). We tested the additional combinatorial treatment containing the angiotensin II receptor blocker losartan (T), which is widely used to halt and treat the developing cardiac dysfunction in DMD patients as an alternative to an ACE inhibitor. Peak myocardial strain rate, assessed by magnetic resonance imaging, showed a negative impact of P, whereas in both diaphragm and extensor digitorum longus (EDL) muscle contractile function was not significantly impaired by P. Histologically, P generally increased cardiac damage, estimated by percentage area infiltrated by IgG as well as by collagen staining. In general, groups that only differed in the presence or absence of P (i.e. mdx vs. P, LS vs. LSP, and TS vs. TSP) demonstrated a significant detrimental impact of P on many assessed parameters, with the most profound impact on cardiac pathology.

Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy.

New Findings What is the central question of this study? The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD). What is the main finding and its importance? Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD. Abstract In Duchenne muscular dystrophy (DMD), muscle damage and impaired regeneration lead to progressive muscle wasting, weakness and premature death. The Notch signalling pathway represents a central regulator of gene expression and is critical for cellular proliferation, differentiation and apoptotic signalling during all stages of embryonic muscle development. Notch activation improves muscle regeneration in aged mice, but its potential to restore regeneration and function in muscular dystrophy is unknown. We performed a comprehensive examination of several genes involved in Notch signalling in muscles from dystrophin-deficient mdx and dko (utrophin- and dystrophin-null) mice and DMD patients. A reduction of Notch1 and Hes1 mRNA in tibialis anterior muscles of dko mice and quadriceps muscles of DMD patients and a reduction of Hes1 mRNA in the diaphragm of the mdx mice were observed, with other targets being inconsistent across species. Activation and inhibition of Notch signalling, followed by measures of muscle regeneration and function, were performed in the mouse models of DMD. Notch activation had no effect on functional regeneration in C57BL/10, mdx or dko mice. Notch inhibition significantly depressed the frequency-force relationship in regenerating muscles of C57BL/10 and mdx mice after injury, indicating reduced force at each stimulation frequency, but enhanced the frequency-force relationship in muscles from dko mice. We conclude that while Notch inhibition produces slight functional defects in dystrophic muscle, Notch activation does not significantly improve muscle regeneration in murine models of muscular dystrophy. Furthermore, the inconsistent expression of Notch targets between murine models and DMD patients suggests caution when making interspecies comparisons.

Alterations of dystrophin-associated glycoproteins in the heart lacking dystrophin or dystrophin and utrophin.

Heart disease is a leading cause of death in patients with Duchenne muscular dystrophy (DMD). Patients with DMD lack the protein dystrophin, which is widely expressed in striated muscle. In skeletal muscle, the loss of dystrophin results in dramatically decreased expression of the dystrophin associated glycoprotein complex (DGC). Interestingly, in the heart the DGC is normally expressed without dystrophin; this has been attributed to presence of the dystrophin homologue utrophin. We demonstrate here that neither utrophin nor dystrophin are required for the expression of the cardiac DGC. However, alpha-dystroglycan (α-DG), a major component of the DGC, is differentially glycosylated in dystrophin-(mdx) and dystrophin-/utrophin-(dko) deficient mouse hearts. In both models the altered α-DG retains laminin binding activity, but has an altered localization at the sarcolemma. In hearts lacking both dystrophin and utrophin, the alterations in α-DG glycosylation are even more dramatic with changes in gel migration equivalent to 24 ± 3 kDa. These data show that the absence of dystrophin and utrophin alters the processing of α-DG; however it is not clear if these alterations are a consequence of the loss of a direct interaction with dystrophin/utrophin or results from an indirect response to the presence of severe pathology. Recently there have been great advances in our understanding of the glycosylation of α-DG regarding its role as a laminin receptor. Here we present data that alterations in glycosylation occur in the hearts of animal models of DMD, but these changes do not affect laminin binding. The physiological consequences of these alterations remain unknown, but may have significant implications for the development of therapies for DMD.

Nestin expression in end-stage disease in dystrophin-deficient heart: implications for regeneration from endogenous cardiac stem cells.

Nestin(+) cardiac stem cells differentiate into striated cells following myocardial infarct. Transplantation of exogenous stem cells into myocardium of a murine model for Duchenne muscular dystrophy (DMD) increased proliferation of endogenous nestin(+) stem cells and resulted in the appearance of nestin(+) striated cells. This correlated with, and may be responsible for, prevention of dilated cardiomyopathy. We examined nestin(+) stem cells in the myocardium of dystrophin/utrophin-deficient (mdx/utrn(-/-)) mice, a model for DMD. We found that 92% of nestin(+) interstitial cells expressed Flk-1, a marker present on cardiac progenitor cells that differentiate into the cardiac lineage, and that a subset expressed Sca-1, present on adult cardiac cells that become cardiomyocytes. Nestin(+) interstitial cells maintained expression of Flk-1 but lost Sca-1 expression with age and were present in lower numbers in dystrophin-deficient heart than in wild-type heart. Unexpectedly, large clusters of nestin(+) striated cells ranging in size from 20 to 250 cells and extending up to 500 µm were present in mdx/utrn(-/-) heart near the end stage of disease. These cells were also present in dystrophin-deficient mdx/utrn(+/-) and mdx heart but not wild-type heart. Nestin(+) striated cells expressed cardiac troponin I, desmin, and Connexin 43 and correlated with proinflammatory CD68(+) macrophages. Elongated nestin(+) interstitial cells with striations were observed that did not express Flk-1 or the late cardiac marker cardiac troponin I but strongly expressed the early cardiac marker desmin. Nestin was also detected in endothelial and smooth muscle cells. These data indicate that new cardiomyocytes form in dystrophic heart, and nestin(+) interstitial cells may generate them in addition to other cells of the cardiac lineage.

RhoA mediates defective stem cell function and heterotopic ossification in dystrophic muscle of mice.

Heterotopic ossification (HO) and fatty infiltration (FI) often occur in diseased skeletal muscle and have been previously described in various animal models of Duchenne muscular dystrophy (DMD); however, the pathological mechanisms remain largely unknown. Dystrophin-deficient mdx mice and dystrophin/utrophin double-knockout (dKO) mice are mouse models of DMD; however, mdx mice display a strong muscle regeneration capacity, while dKO mice exhibit a much more severe phenotype, which is similar to patients with DMD. Our results revealed that more extensive HO, but not FI, occurred in the skeletal muscle of dKO mice versus mdx mice, and RhoA activation specifically occurred at the sites of HO. Moreover, the gene expression of RhoA, BMPs, and several inflammatory factors were significantly up-regulated in muscle stem cells isolated from dKO mice; while inactivation of RhoA in the cells with RhoA/ROCK inhibitor Y-27632 led to reduced osteogenic potential and improved myogenic potential. Finally, inactivation of RhoA signaling in the dKO mice with Y-27632 improved muscle regeneration and reduced the expression of BMPs, inflammation, HO, and intramyocellular lipid accumulation in both skeletal and cardiac muscle. Our results revealed that RhoA represents a major molecular switch in the regulation of HO and muscle regeneration in dystrophic skeletal muscle of mice.

Low dystrophin levels increase survival and improve muscle pathology and function in dystrophin/utrophin double-knockout mice.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by the lack of functional dystrophin. There is no cure, but several clinical trials aimed to restore the synthesis of functional dystrophin are underway. The dystrophin levels needed for improvement of muscle pathology, function, and overall vitality are not known. Here, we describe the mdx/utrn(-/-)/Xist(Δhs) mouse model, which expresses a range of low dystrophin levels, depending on the degree of skewing of X inactivation in a utrophin-negative background. Mdx/utrn(-/-) mice develop severe muscle weakness, kyphosis, respiratory and heart failure, and premature death closely resembling DMD pathology. We show that at dystrophin levels < 4%, survival and motor function in these animals are greatly improved. In mice expressing >4% dystrophin, histopathology is ameliorated, as well. These findings suggest that the dystrophin levels needed to benefit vitality and functioning of patients with DMD might be lower than those needed for full protection against muscle damage.

Nitric oxide signalling pathway in Duchenne muscular dystrophy mice: up-regulation of L-arginine transporters.

DMD (Duchenne muscular dystrophy) is an incurable rapidly worsening neuromuscular degenerative disease caused by the absence of dystrophin. In skeletal muscle a lack of dystrophin disrupts the recruitment of neuronal NOS (nitric oxide synthase) to the sarcolemma thus affecting NO (nitric oxide) production. Utrophin is a dystrophin homologue, the expression of which is greatly up-regulated in the sarcolemma of dystrophin-negative fibres from mdx mice, a mouse model of DMD. Although cardiomyopathy is an important cause of death, little is known about the NO signalling pathway in the cardiac muscle of DMD patients. Thus we used cardiomyocytes and hearts from two month-old mdx and mdx:utrophin-/- (double knockout) mice (mdx:utr) to study key steps in NO signalling: L-arginine transporters, NOS and sGC (soluble guanylyl cyclase). nNOS did not co-localize with dystrophin or utrophin to the cardiomyocyte membrane. Despite this nNOS activity was markedly decreased in both mdx and mdx:utr mice, whereas nNOS expression was only decreased in mdx:utr mouse hearts, suggesting that utrophin up-regulation in cardiomyocytes maintains nNOS levels, but not function. sGC protein levels and activity remained at control levels. Unexpectedly, L-arginine transporter expression and function were significantly increased, suggesting a novel biochemical compensatory mechanism of the NO pathway and a potential entry site for therapeutics.

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 1year-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 8-10weeks-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 10weeks-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.

Sustaining cardiac claudin-5 levels prevents functional hallmarks of cardiomyopathy in a muscular dystrophy mouse model.

Identification of new molecular targets in heart failure could ultimately have a substantial positive impact on both the health and financial aspects of treating the large heart failure population. We originally identified reduced levels of the cell junction protein claudin-5 specifically in heart in the dystrophin/utrophin-deficient (Dmd(mdx);Utrn(-/-)) mouse model of muscular dystrophy and cardiomyopathy, which demonstrates physiological hallmarks of heart failure. We then showed that at least 60% of cardiac explant samples from patients with heart failure resulting from diverse etiologies also have reduced claudin-5 levels. These claudin-5 reductions were independent of changes in other cell junction proteins previously linked to heart failure. The goal of this study was to determine whether sustaining claudin-5 levels is sufficient to prevent the onset of histological and functional indicators of heart failure. Here, we show the proof-of-concept rescue experiment in the Dmd(mdx);Utrn(-/-) model, in which claudin-5 reductions were originally identified. Expression of claudin-5 4 weeks after a single administration of recombinant adeno-associated virus (rAAV) containing a claudin-5 expression cassette prevented the onset of physiological hallmarks of cardiomyopathy and improved histological signs of cardiac damage. This experiment demonstrates that claudin-5 may represent a novel treatment target for prevention of heart failure.