Dystrophin-deficient Mdx Mice Research Articles

Abstract 4118084: Targeting Monoamine Oxidase B in Dystrophic mdx Hearts Dampens Inflammation and Fibrosis

Background: Cardiomyopathy is a major cause of death for Duchenne muscular dystrophy (DMD) patients. Current treatments cannot prevent cardiac tissue remodeling. Oxidative stress, inflammation and fibrosis are early events in DMD, preceding heart dysfunction. Monoamine Oxidase B (MAOB), which forms H 2 O 2 by oxidizing amines, is overactivated in inflammatory conditions and overcomes cell antioxidant defenses, thus altering redox homeostasis and eliciting harmful effects. We showed that targeting MAOB with inhibitors (iMAOB) improves skeletal muscle function in dystrophic mice by lowering oxidative stress, and reduces inflammation in murine models of sepsis and arthritis by dampening NF-kB, a redox-sensitive transcription factor. Aim: We explore the therapeutic potential of iMAOB to alleviate cardiomyopathy using dystrophin-deficient mdx mice. We hypothesize that iMAOB treatment could dampen oxidative stress, inflammation and fibrosis in dystrophic mdx hearts by modulating the phenotype of cells that are crucial in cardiac remodeling. Methods: Three-month-old mdx mice, that already show signs of fibrosis but no cardiac dysfunction, were orally treated with iMAOB or vehicle for one month (n≥ 6). Heart ventricular mononucleated cells were obtained by enzymatic digestion. Myeloid, endothelial and fibroblast cells were isolated by cell sorting by FACS and analysed by RT-PCR. Oxidative stress, inflammation and fibrosis were measured in whole tissue by immunohistochemistry. Results: The expression of proinflammatory and profibrotic genes was markedly increased in myeloid [Interleukin (Il)-1b, Il-6, Tgf-b and Spp1, the gene coding for osteopontin], endothelial [Il-1b, Il-6, mmp2 and nos3] and cardiac fibroblast cells [Tgf-b, Spp1, Timp1 and Col1] isolated from mdx hearts, as compared to wild type mice. All these genes were significantly dampened by iMAOB treatment. In parallel, once again iMAOB treatment blunted the increase in oxidative stress, inflammation and fibrosis observed in mdx cardiac sections. Of notice, the differences were not linked to changes in the percentage of the various cell types, as they were unmodified amongst wild type, mdx and iMAOB-treated mdx hearts. Conclusions: We show that iMAOB can positively affect the phenotype of cells that are important in cardiac tissue remodeling. Our data suggest that iMAOB can be a viable therapeutic tool in DMD cardiomyopathy. As iMAOB are already in clinical use, such approach could be easily translated to patients.

Empagliflozin treatment rescues abnormally reduced Na+ currents in ventricular cardiomyocytes from dystrophin-deficient mdx mice

Abstract Background Cardiac arrhythmias represent a major cause of death in Duchenne muscular dystrophy (DMD), a devastating muscle disease caused by dystrophin deficiency. An important source for arrhythmia vulnerability in DMD patients is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, and the development of reentrant circuits. Using the dystrophin-deficient mdx mouse model for DMD, we and others previously reported that the absence of dystrophin results in a significant peak Na+ current (INa) loss both in ventricular cardiomyocytes of the working myocardium, and in Purkinje fibers, ventricular myocytes specialized for rapid electrical impulse conduction. This finding provided a mechanistic explanation for ventricular conduction defects and concomitant arrhythmias in the dystrophic heart. Purpose In the present study, we tested the hypothesis that empagliflozin (EMPA), an inhibitor of sodium/glucose cotransporter 2 in clinical use to treat type II diabetes and nondiabetic heart failure, rescues the loss of peak INa in dystrophin-deficient ventricular cardiomyocytes. Methods In a first set of experiments, we treated a cohort of mdx mice with 15 mg/kg body weight/day EMPA via the drinking water. After 4 weeks of treatment, ventricular cardiomyocytes were isolated from these mice, as well as from untreated wild-type and mdx control mice, via Langendorff heart perfusion and enzymatic digestion. Peak INa was measured using the whole cell patch clamp technique. In a second experimental approach, peak INa was compared after a 24 hour exposure of isolated mdx ventricular cardiomyocytes with 1 µM EMPA, 10 µM of the Na+-H+ exchanger 1 inhibitor cariporide, or 10 µM cariporide in combination with 1 µM EMPA. In a third set of experiments, we superfused freshly isolated mdx ventricular cardiomyocytes with 1 or 10 µM EMPA in order to examine the acute action of the drug on peak INa. Results Consistent with the literature, we found peak INa of mdx ventricular cardiomyocytes to be substantially decreased compared to wild-type. Peak INa of ventricular cardiomyocytes derived from mdx mice, which had received EMPA for 4 weeks, was restored to wild-type level. Moreover, incubation of isolated mdx ventricular cardiomyocytes with EMPA for 24 hours significantly increased their peak INa. This effect did not depend on Na+-H+ exchanger 1 inhibition by the drug. The voltage dependencies of INa activation and fast inactivation were neither modified by long-term treatment nor incubation with EMPA. Finally, acute application of EMPA did not influence peak INa. Conclusion Our findings indicate that EMPA treatment can correct abnormally reduced peak INa of dystrophin-deficient ventricular cardiomyocytes. Long-term EMPA administration may diminish arrhythmia vulnerability in DMD patients.

Reduced Na current in dystrophin-deficient ventricular cardiomyocytes is rescued by tenascin-C inhibition

Abstract Background Duchenne muscular dystrophy (DMD) is an X-linked hereditary disease triggered by the deficiency of the structural protein dystrophin, which leads to muscular degeneration mainly affecting young males. Major contributors to early death in DMD patients are cardiac arrhythmias and dystrophic cardiomyopathy. One cause for arrhythmias is impaired ventricular impulse conduction, which leads to ventricular asynchrony and reentrant mechanisms. We recently showed that the disruption of dystrophin results in a significant reduction of Na current in ventricular cardiomyocytes (vCMs) of the dystrophin-deficient mdx mouse model for human DMD. Na current reduction provides a mechanistic explanation for the impaired ventricular conduction and accompanying arrhythmias in the dystrophic heart. The extracellular matrix protein tenascin-C (TN-C) is a significant remodeling factor in the injured and diseased heart and is strongly upregulated in dystrophic cardiomyopathy. To this date, it is unknown how the upregulation of TN-C in DMD patients affects dystrophic cardiomyopathy. Purpose In this study, we examined the effect of TN-C inhibition on diminished Na currents in dystrophin-deficient vCMs. Methods We compared four different mouse genotypes with each other, namely wild-type, dystrophin-deficient mdx, TN-C-deficient and dystrophin- plus TN-C-deficient mice. Furthermore, a cohort of mdx mice was injected with TN-C siRNA twice a week for 9 weeks to investigate the effect of TN-C knockdown. Hearts from adult male mice were enzymatically digested using a Langendorff system to isolate single vCMs. Na currents were then measured with the whole cell patch clamp technique. Results Na current densities were increased in TN-C deficient vCMs compared to wild-type vCMs. Accordingly, 24-hour incubation of wild-type vCMs with human recombinant TN-C resulted in a significant decrease in Na current. Na currents of vCMs from mdx mice were reduced, but restored to the wild-type level in vCMs from TN-C-deficient mdx mice. Moreover, vCMs of TN-C siRNA-treated mdx mice had significantly increased Na currents compared to control mdx vCMs. Conclusion Upregulation of TN-C in dystrophin-deficient vCMs reduces Na currents, whereas inhibition of TN-C prevents this reduction. Therefore, inhibition of TN-C in DMD patients may be considered as a potential new therapeutic strategy to improve ventricular conduction and reduce arrhythmia vulnerability.

Histone deacetylase 6 inhibition promotes microtubule acetylation and facilitates autophagosome-lysosome fusion in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy is a progressive muscle-wasting disease caused by mutations in the dystrophin gene. Despite progress in dystrophin-targeted gene therapies, it is still a fatal disease requiring novel therapeutics that can be used synergistically or alternatively to emerging gene therapy. Defective autophagy and disorganized microtubule networks contribute to dystrophic pathogenesis, yet the mechanisms by which microtubule alterations regulate autophagy remain elusive. The present study was designed to uncover possible mechanisms underpinning the role of microtubules in regulating autophagy in dystrophic mice. Mdx mice were also supplemented with Tubastatin A, a pharmacological inhibitor of histone deacetylase 6, and pathophysiology was assessed. Mdx mice with a genetic deletion of the Nox-2 scaffolding subunit p47phox were used to assess redox dependence on tubulin acetylation. Our data show decreased acetylation of α-tubulin with enhanced histone deacetylase 6 expression. Tubastatin A increases tubulin acetylation and Q-SNARE complex formation but does not alter microtubule organization or density, indicating improved autophagosome-lysosome fusion. Tubastatin A increases the acetylation of peroxiredoxin and protects it from hyper-oxidation, hence modulating intracellular redox status in mdx mice. Tubastatin A reduces muscle damage and enhances force production. Genetic down regulation of Nox2 activity in the mdx mice promotes autophagosome maturation but not autolysosome formation. Our data highlight that autophagy is differentially regulated by redox and acetylation in mdx mice. By improving autophagy through promoting tubulin acetylation, Tubastatin A decreases the dystrophic phenotype and improves muscle function, suggesting a great potential for clinical translation and treating dystrophic patients.

Reduction of Mitochondrial Calcium Overload via MKT077-Induced Inhibition of Glucose-Regulated Protein 75 Alleviates Skeletal Muscle Pathology in Dystrophin-Deficient mdx Mice.

Duchenne muscular dystrophy is secondarily accompanied by Ca2+ excess in muscle fibers. Part of the Ca2+ accumulates in the mitochondria, contributing to the development of mitochondrial dysfunction and degeneration of muscles. In this work, we assessed the effect of intraperitoneal administration of rhodacyanine MKT077 (5 mg/kg/day), which is able to suppress glucose-regulated protein 75 (GRP75)-mediated Ca2+ transfer from the sarcoplasmic reticulum (SR) to mitochondria, on the Ca2+ overload of skeletal muscle mitochondria in dystrophin-deficient mdx mice and the concomitant mitochondrial dysfunction contributing to muscle pathology. MKT077 prevented Ca2+ overload of quadriceps mitochondria in mdx mice, reduced the intensity of oxidative stress, and improved mitochondrial ultrastructure, but had no effect on impaired oxidative phosphorylation. MKT077 eliminated quadriceps calcification and reduced the intensity of muscle fiber degeneration, fibrosis level, and normalized grip strength in mdx mice. However, we noted a negative effect of MKT077 on wild-type mice, expressed as a decrease in the efficiency of mitochondrial oxidative phosphorylation, SR stress development, ultrastructural disturbances in the quadriceps, and a reduction in animal endurance in the wire-hanging test. This paper discusses the impact of MKT077 modulation of mitochondrial dysfunction on the development of skeletal muscle pathology in mdx mice.

Glucocorticoid Deflazacort Normalizes the Ultrastructure of Skeletal Muscles and the State of the Colon Microbiota in Dystrophin-Deficient Mice.

We studied the effect of enteral administration of the glucocorticoid deflazacort (DFC, 1.2 mg/kg per day, 28 days) on the state of skeletal muscles and tissue ultrastructure, as well as the composition of the colon microbiota in dystrophin-deficient mdx mice. DFC has been shown to reduce the intensity of degeneration/regeneration cycles in muscle fibers of mdx mice. This effect of DFC was accompanied by normalization of the size of sarcomeres of skeletal muscles of mdx mice, improvement of the ultrastructure of the subsarcolemmal population of mitochondria, and an increase in the number of organelles, as well as normalization of the number of contact interactions between the sarcoplasmic reticulum and mitochondria. In addition, DFC had a corrective effect on the colon microbiota of mdx mice, which manifested in an increase in the number of the Bifidobacterium genus microorganisms and a decrease in the level of E. coli with reduced enzymatic activity.

Mitochondrial Transplantation Therapy Ameliorates Muscular Dystrophy in mdx Mouse Model.

Duchenne muscular dystrophy is caused by loss of the dystrophin protein. This pathology is accompanied by mitochondrial dysfunction contributing to muscle fiber instability. It is known that mitochondria-targeted in vivo therapy mitigates pathology and improves the quality of life of model animals. In the present work, we applied mitochondrial transplantation therapy (MTT) to correct the pathology in dystrophin-deficient mdx mice. Intramuscular injections of allogeneic mitochondria obtained from healthy animals into the hind limbs of mdx mice alleviated skeletal muscle injury, reduced calcium deposits in muscles and serum creatine kinase levels, and improved the grip strength of the hind limbs and motor activity of recipient mdx mice. We noted normalization of the mitochondrial ultrastructure and sarcoplasmic reticulum/mitochondria interactions in mdx muscles. At the same time, we revealed a decrease in the efficiency of oxidative phosphorylation in the skeletal muscle mitochondria of recipient mdx mice accompanied by a reduction in lipid peroxidation products (MDA products) and reduced calcium overloading. We found no effect of MTT on the expression of mitochondrial signature genes (Drp1, Mfn2, Ppargc1a, Pink1, Parkin) and on the level of mtDNA. Our results show that systemic MTT mitigates the development of destructive processes in the quadriceps muscle of mdx mice.

Transcriptional dysregulation of autophagy in the muscle of a mouse model of Duchenne muscular dystrophy

It has been reported that autophagic activity is disturbed in the skeletal muscles of dystrophin-deficient mdx mice and patients with Duchenne muscular dystrophy (DMD). Transcriptional regulations of autophagy by FoxO transcription factors (FoxOs) and transcription factor EB (TFEB) play critical roles in adaptation to cellular stress conditions. Here, we investigated whether autophagic activity is dysregulated at the transcription level in dystrophin-deficient muscles. Expression levels of autophagy-related genes were globally decreased in tibialis anterior and soleus muscles of mdx mice compared with those of wild-type mice. DNA microarray data from the NCBI database also showed that genes related to autophagy were globally downregulated in muscles from patients with DMD. These downregulated genes are known as targets of FoxOs and TFEB. Immunostaining showed that nuclear localization of FoxO1 and FoxO3a was decreased in mdx mice. Western blot analyses demonstrated increases in phosphorylation levels of FoxO1 and FoxO3a in mdx mice. Nuclear localization of TFEB was also reduced in mdx mice, which was associated with elevated phosphorylation levels of TFEB. Collectively, the results suggest that autophagy is disturbed in dystrophin-deficient muscles via transcriptional downregulation due to phosphorylation-mediated suppression of FoxOs and TFEB.

Empagliflozin treatment rescues abnormally reduced Na+ currents in ventricular cardiomyocytes from dystrophin-deficient mdx mice.

Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a severe muscle illness caused by mutations in the gene encoding for the intracellular protein dystrophin. A major source for arrhythmia vulnerability in patients with DMD is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, decreased cardiac output, and the development of reentrant circuits. Using the dystrophin-deficient mdx mouse model for human DMD, we previously reported that the lack of dystrophin causes a significant loss of peak Na+ current (INa) in ventricular cardiomyocytes. This finding provided a mechanistic explanation for ventricular conduction defects and concomitant arrhythmias in the dystrophic heart. In the present study, we explored the hypothesis that empagliflozin (EMPA), an inhibitor of sodium/glucose cotransporter 2 in clinical use to treat type II diabetes and nondiabetic heart failure, rescues peak INa loss in dystrophin-deficient ventricular cardiomyocytes. We found that INa of cardiomyocytes derived from mdx mice, which had received clinically relevant doses of EMPA for 4 wk, was restored to wild-type level. Moreover, incubation of isolated mdx ventricular cardiomyocytes with 1 µM EMPA for 24 h significantly increased their peak INa. This effect was independent of Na+-H+ exchanger 1 inhibition by the drug. Our findings imply that EMPA treatment can rescue abnormally reduced peak INa of dystrophin-deficient ventricular cardiomyocytes. Long-term EMPA administration may diminish arrhythmia vulnerability in patients with DMD.NEW & NOTEWORTHY Dystrophin deficiency in cardiomyocytes leads to abnormally reduced Na+ currents. These can be rescued by long-term empagliflozin treatment.

Uridine Administration Promotes Normalization of Heart Mitochondrial Function in Dystrophin-Deficient Mice and Decreases Tissue Fibrosis.

The work shows the effect of the metabolic modulator uridine on the functioning and ultrastructure of heart mitochondria in dystrophin-deficient mdx mice. Intraperitoneal administration of uridine (30 mg/kg/day for 28 days) improved K+ transport and increased its content in the heart mitochondria of mdx mice to the level of wild-type animals. This was accompanied by a significant decrease in the level of malondialdehyde and an increase in the number of mitochondria in the heart of mdx mice. At the same time, uridine did not affect the hyperfunctionality of mitochondria in mdx mice, which manifested in an increase in the calcium retention capacity. Nevertheless, we noted that uridine causes a significant decrease in the level of fibrosis in the heart of mdx mice, which attested to a positive effect of therapy.

Loss of compensation afforded by accessory muscles of breathing leads to respiratory system compromise in the mdx mouse model of Duchenne muscular dystrophy.

Despite profound diaphragm weakness, peak inspiratory pressure-generating capacity is preserved in young mdx mice revealing adequate compensation by extra-diaphragmatic muscles of breathing in early dystrophic disease. We hypothesised that loss of compensation gives rise to respiratory system compromise in advanced dystrophic disease. Studies were performed in male wild-type (n=196) and dystrophin-deficient mdx mice (n=188) at 1, 4, 8, 12 and 16months of age. In anaesthetised mice, inspiratory pressure and obligatory and accessory respiratory EMG activities were recorded during baseline and sustained tracheal occlusion for up to 30-40s to evoke peak system activation to task failure. Obligatory inspiratory EMG activities were lower in mdx mice across the ventilatory range to peak activity, emerging in early dystrophic disease. Early compensation protecting peak inspiratory pressure-generating capacity in mdx mice, which appears to relate to transforming growth factor-β1-dependent fibrotic remodelling of the diaphragm and preserved accessory muscle function, was lost at 12 and 16months of age. Denervation and surgical lesion of muscles of breathing in 4-month-old mice revealed a greater dependency on diaphragm for peak inspiratory performance in wild-type mice, whereas mdx mice were heavily dependent upon accessory muscles (including abdominal muscles) for peak performance. Accessory EMG activities were generally preserved or enhanced in young mdx mice, but peak EMG activities were lower than wild-type by 12months of age. In general, ventilation was reasonably well protected in mdx mice until 16months of age. Despite the early emergence of impairments in the principal obligatory muscles of breathing, peak inspiratory performance is compensated in early dystrophic disease due to diaphragm remodelling and facilitated contribution by accessory muscles of breathing. Loss of compensation afforded by accessory muscles underpins the emergence of respiratory system morbidity in advanced dystrophic disease. KEY POINTS: Despite diaphragm weakness, peak inspiratory performance is preserved in young dystrophin-deficient mdx mice revealing adequate compensation by extra-diaphragmatic muscles. Peak obligatory muscle (diaphragm, external intercostal, and parasternal intercostal) EMG activities are lower in mdx mice, emerging early in dystrophic disease, before the temporal decline in peak performance. Peak EMG activities of some accessory muscles are lower, whereas others are preserved. There is greater recruitment of the trapezius muscle in mdx mice during peak system activation. In phrenicotomised mice with confirmed diaphragm paralysis, there is a greater contribution made by extra-diaphragmatic muscles to peak inspiratory pressure in mdx compared with wild-type mice. Surgical lesion of accessory (including abdominal) muscles adversely affects peak pressure generation in mdx mice. Diaphragm remodelling leading to stiffening provides a mechanical advantage to peak pressure generation via the facilitated action of extra-diaphragmatic muscles in early dystrophic disease. Peak accessory EMG activities are lower in 12-month-old mdx compared to wild-type mice. Peak inspiratory pressure declines in mdx mice with advanced disease. We conclude that compensation afforded by accessory muscles of breathing declines in advanced dystrophic disease precipitating the emergence of respiratory system dysfunction.

Sodium hydrosulfide moderately alleviates the hallmark symptoms of Duchenne muscular dystrophy in mdx mice

Duchenne muscular dystrophy (DMD) is an incurable disease caused by mutations in the X-linked DMD gene that encodes a structural muscle protein, dystrophin. This, in turn, leads to progressive degeneration of the skeletal muscles and the heart. Hydrogen sulfide (H2S), the pleiotropic agent with antioxidant, anti-inflammatory, and pro-angiogenic activities, could be considered a promising therapeutic factor for DMD.In this work, we studied the effect of daily intraperitoneal administration of the H2S donor, sodium hydrosulfide (NaHS, 100 μmol/kg/day for 5 weeks) on skeletal muscle (gastrocnemius, diaphragm and tibialis anterior) pathology in dystrophin-deficient mdx mice, characterized by decreased expression of H2S-generating enzymes.NaHS reduced the level of muscle damage markers in plasma (creatine kinase, lactate dehydrogenase and osteopontin). It lowered oxidative stress by affecting the GSH/GSSG ratio, up-regulating the level of cytoprotective heme oxygenase-1 (HO-1) and down-regulating the NF-κB pathway. In the gastrocnemius muscle, it also increased angiogenic vascular endothelial growth factor (Vegf) and its receptor (Kdr) expression, accompanied by the elevated number of α-SMA/CD31/lectin-positive blood vessels. The expression of fibrotic regulators, like Tgfβ, Col1a1 and Fn1 was decreased by NaHS in the tibialis anterior, while the level of autophagy markers (AMPKα signalling and Atg genes), was mostly affected in the gastrocnemius. Histological and molecular analysis showed no effect of H2S donor on regeneration and the muscle fiber type composition.Overall, the H2S donor modified the gene expression and protein level of molecules associated with the pathophysiology of DMD, contributing to the regulation of oxidative stress, inflammation, autophagy, and angiogenesis.

RANKL Inhibition Reduces Cardiac Hypertrophy in mdx Mice and Possibly in Children with Duchenne Muscular Dystrophy.

Cardiomyopathy has become one of the leading causes of death in patients with Duchenne muscular dystrophy (DMD). We recently reported that the inhibition of the interaction between the receptor activator of nuclear factor κB ligand (RANKL) and receptor activator of nuclear factor κB (RANK) significantly improves muscle and bone functions in dystrophin-deficient mdx mice. RANKL and RANK are also expressed in cardiac muscle. Here, we investigate whether anti-RANKL treatment prevents cardiac hypertrophy and dysfunction in dystrophic mdx mice. Anti-RANKL treatment significantly reduced LV hypertrophy and heart mass, and maintained cardiac function in mdx mice. Anti-RANKL treatment also inhibited NFκB and PI3K, two mediators implicated in cardiac hypertrophy. Furthermore, anti-RANKL treatment increased SERCA activity and the expression of RyR, FKBP12, and SERCA2a, leading possibly to an improved Ca2+ homeostasis in dystrophic hearts. Interestingly, preliminary post hoc analyses suggest that denosumab, a human anti-RANKL, reduced left ventricular hypertrophy in two patients with DMD. Taken together, our results indicate that anti-RANKL treatment prevents the worsening of cardiac hypertrophy in mdx mice and could potentially maintain cardiac function in teenage or adult patients with DMD.

The unconditioned fear response in dystrophin-deficient mice is associated with adrenal and vascular function

Loss of function mutations in the gene encoding dystrophin elicits a hypersensitive fear response in mice and humans. In the dystrophin-deficient mdx mouse, this behaviour is partially protected by oestrogen, but the mechanistic basis for this protection is unknown. Here, we show that female mdx mice remain normotensive during restraint stress compared to a hypotensive and hypertensive response in male mdx and male/female wildtype mice, respectively. Partial dystrophin expression in female mdx mice (heterozygous) also elicited a hypertensive response. Ovariectomized (OVX) female mdx mice were used to explain the normotensive response to stress. OVX lowered skeletal muscle mass and lowered the adrenal mass and zona glomerulosa area (aldosterone synthesis) in female mdx mice. During a restraint stress, OVX dampened aldosterone synthesis and lowered the corticosterone:11-dehydrocorticosterone. All OVX-induced changes were restored with replacement of oestradiol, except that oestradiol lowered the zona fasciculata area of the adrenal gland, dampened corticosterone synthesis but increased cortisol synthesis. These data suggest that oestrogen partially attenuates the unconditioned fear response in mdx mice via adrenal and vascular function. It also suggests that partial dystrophin restoration in a dystrophin-deficient vertebrate is an effective approach to develop an appropriate hypertensive response to stress.

Effect of the large-conductance calcium-dependent k<sup>+</sup> channel activator NS1619 on the function of mitochondria in the heart of dystrophin-deficient mice

Dystrophin-deficient muscular dystrophy (Duchenne dystrophy) is characterized by impaired ion homeostasis, in which mitochondria play an important role. In the present work, using a model of dystrophin-deficient mdx mice, we revealed a decrease in the efficiency of potassium ion transport and the total content of this ion in heart mitochondria. We evaluated the effect of chronic administration of the benzimidazole derivative NS1619, which is an activator of the large-conductance Ca2+-dependent K+ channel (mitoBKCa) on the structure and function of organelles and the state of the heart muscle. It was shown that NS1619 improves K+ transport and increases the content of the ion in the heart mitochondria of mdx mice, but this is not associated with changes in the level of the mitoBKCa protein and the expression of the encoding gene. The effect of NS1619 was accompanied by a decrease in the intensity of oxidative stress, assessed by the level of lipid peroxidation products (MDA products) and normalization of the mitochondrial ultrastructure in the heart of mdx mice. In addition, we found positive changes in the tissue, expressed in a decrease in the level of fibrosis in the heart of dystrophin-deficient animals treated with NS1619. It was noted that NS1619 had no significant effect on the structure and function of heart mitochondria in wild-type animals. The paper discusses the mechanisms of influence of NS1619 on the function of mouse heart mitochondria in Duchenne muscular dystrophy and the prospects for applying this approach to correct pathology.

BKCa Activator NS1619 Improves the Structure and Function of Skeletal Muscle Mitochondria in Duchenne Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive hereditary disease caused by the absence of the dystrophin protein. This is secondarily accompanied by a dysregulation of ion homeostasis, in which mitochondria play an important role. In the present work, we show that mitochondrial dysfunction in the skeletal muscles of dystrophin-deficient mdx mice is accompanied by a reduction in K+ transport and a decrease in its content in the matrix. This is associated with a decrease in the expression of the mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) in the muscles of mdx mice, which play an important role in cytoprotection. We observed that the BKCa activator NS1619 caused a normalization of mitoBKCa expression and potassium homeostasis in the muscle mitochondria of these animals, which was accompanied by an increase in the calcium retention capacity, mitigation of oxidative stress, and improvement in mitochondrial ultrastructure. This effect of NS1619 contributed to the reduction of degeneration/regeneration cycles and fibrosis in the skeletal muscles of mdx mice as well as a normalization of sarcomere size, but had no effect on the leakage of muscle enzymes and muscle strength loss. In the case of wild-type mice, we noted the negative effect of NS1619 manifested in the inhibition of the functional activity of mitochondria and disruption of their structure, which, however, did not significantly affect the state of the skeletal muscles of the animals. This article discusses the role of mitoBKCa in the development of DMD and the prospects of the approach associated with the correction of its function in treatments of this secondary channelopathy.

The Effect of Uridine on the State of Skeletal Muscles and the Functioning of Mitochondria in Duchenne Dystrophy.

Duchenne muscular dystrophy is caused by the loss of functional dystrophin that secondarily causes systemic metabolic impairment in skeletal muscles and cardiomyocytes. The nutraceutical approach is considered as a possible complementary therapy for this pathology. In this work, we have studied the effect of pyrimidine nucleoside uridine (30 mg/kg/day for 28 days, i.p.), which plays an important role in cellular metabolism, on the development of DMD in the skeletal muscles of dystrophin deficient mdx mice, as well as its effect on the mitochondrial dysfunction that accompanies this pathology. We found that chronic uridine administration reduced fibrosis in the skeletal muscles of mdx mice, but it had no effect on the intensity of degeneration/regeneration cycles and inflammation, pseudohypetrophy, and muscle strength of the animals. Analysis of TEM micrographs showed that uridine also had no effect on the impaired mitochondrial ultrastructure of mdx mouse skeletal muscle. The administration of uridine was found to lead to an increase in the expression of the Drp1 and Parkin genes, which may indicate an increase in the intensity of organelle fission and the normalization of mitophagy. Uridine had little effect on OXPHOS dysfunction in mdx mouse mitochondria, and moreover, it was suppressed in the mitochondria of wild type animals. At the same time, uridine restored the transport of potassium ions and reduced the production of reactive oxygen species; however, this had no effect on the impaired calcium retention capacity of mdx mouse mitochondria. The obtained results demonstrate that the used dose of uridine only partially prevents mitochondrial dysfunction in skeletal muscles during Duchenne dystrophy, though it mitigates the development of destructive processes in skeletal muscles.

Activation of SIRT1 promotes membrane resealing via cortactin

Muscular dystrophies are inherited myopathic disorders characterized by progressive muscle weakness. Recently, several gene therapies have been developed; however, the treatment options are still limited. Resveratrol, an activator of SIRT1, ameliorates muscular function in muscular dystrophy patients and dystrophin-deficient mdx mice, although its mechanism is still not fully elucidated. Here, we investigated the effects of resveratrol on membrane resealing. We found that resveratrol promoted membrane repair in C2C12 cells via the activation of SIRT1. To elucidate the mechanism by which resveratrol promotes membrane resealing, we focused on the reorganization of the cytoskeleton, which occurs in the early phase of membrane repair. Treatment with resveratrol promoted actin accumulation at the injured site. We also examined the role of cortactin in membrane resealing. Cortactin accumulated at the injury site, and cortactin knockdown suppressed membrane resealing and reorganization of the cytoskeleton. Additionally, SIRT1 deacetylated cortactin and promoted the interaction between cortactin and F-actin, thus possibly enhancing the accumulation of cortactin at the injury site. Finally, we performed a membrane repair assay using single fiber myotubes from control and resveratrol-fed mice, where the oral treatment with resveratrol promoted membrane repair ex vivo. These findings suggest that resveratrol promotes membrane repair via the SIRT1/cortactin axis.

Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties

Therapies that restore dystrophin expression are presumed to correct Duchenne muscular dystrophy (DMD), with antisense-mediated exon skipping being the leading approach. Here we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. Dystrophin-deficient mdx mice were treated with a PPMO (Pip9b2-PMO) designed to induce Dmd exon 23 skipping at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed 2 weeks later. Dose-dependent exon skipping and dystrophin protein restoration were observed, with dystrophin uniformly distributed at the sarcolemma of corrected myofibers at all doses. Serum microRNA biomarkers (i.e., miR-1a-3p, miR-133a-3p, miR-206-3p, miR-483-3p) and creatinine kinase levels were restored toward wild-type levels after treatment in a dose-dependent manner. All biomarkers were strongly anti-correlated with both exon skipping level and dystrophin expression. Dystrophin rescue was also strongly positively correlated with muscle stiffness (i.e., Young’s modulus) as determined by atomic force microscopy (AFM) nanoindentation assay. These data demonstrate that PPMO-mediated exon skipping generates myofibers with uniform dystrophin expression and that both serum microRNA biomarkers and muscle AFM have potential utility as pharmacodynamic biomarkers of dystrophin restoration therapy in DMD.

Inactivation of Sirt6 ameliorates muscular dystrophy in mdx mice by releasing suppression of utrophin expression

The NAD+-dependent SIRT1-7 family of protein deacetylases plays a vital role in various molecular pathways related to stress response, DNA repair, aging and metabolism. Increased activity of individual sirtuins often exerts beneficial effects in pathophysiological conditions whereas reduced activity is usually associated with disease conditions. Here, we demonstrate that SIRT6 deacetylates H3K56ac in myofibers to suppress expression of utrophin, a dystrophin-related protein stabilizing the sarcolemma in absence of dystrophin. Inactivation of Sirt6 in dystrophin-deficient mdx mice reduced damage of myofibers, ameliorated dystrophic muscle pathology, and improved muscle function, leading to attenuated activation of muscle stem cells (MuSCs). ChIP-seq and locus-specific recruitment of SIRT6 using a CRISPR-dCas9/gRNA approach revealed that SIRT6 is critical for removal of H3K56ac at the Downstream utrophin Enhancer (DUE), which is indispensable for utrophin expression. We conclude that epigenetic manipulation of utrophin expression is a promising approach for the treatment of Duchenne Muscular Dystrophy (DMD).