Decreased mAKAP, ryanodine receptor, and SERCA2a gene expression in mdx hearts

Duchenne muscular dystrophy (DMD) is a common genetic disease resulting from mutations in the dystrophin gene. The lack of dystrophin function as a cytoskeletal protein leads to abnormal intracellular Ca(2+) homeostasis, the actual source and functional consequences of which remain obscure. The mdx mouse, a mouse model of DMD, revealed alterations in contractile properties that are likely due to defective Ca(2+) handling. However, the exact mechanisms of the Ca(2+) handling defect are unclear. We performed suppressive subtractive hybridization to isolate differentially expressed genes between 5-month-old mdx and control mice. We observed a decrease in muscle A-kinase anchoring protein (mAKAP) in the mdx hearts. We noticed not only down-regulation of mAKAP mRNA but also decreased mRNA level of the molecules involved in Ca(2+) handling and excitation-contraction (E-C) coupling in the sarcoplasmic reticulum (SR), the cardiac ryanodine receptor, and the sarcoplasmic reticulum Ca(2+) ATPase. Therefore, dystrophin deficiency may cause an impairment of SR Ca(2+) homeostasis and E-C coupling in mdx hearts, in part, by decreased gene expression of molecules involved in SR Ca(2+) handling.     

iNOS expression in dystrophinopathies can be reduced by somatic gene transfer of dystrophin or utrophin

BACKGROUND: Nitric oxide (NO) is an inorganic gas produced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sections from control, DMD, and BMD patients. MATERIALS AND METHODS: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in muscles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. RESULTS: In control patients, few fibers (<2%) demonstrated focal accumulation of iNOS in sarcolemma. In DMD patients, a strong iNOS immunoreactivity was observed in some necrotic muscle fibers as well as in some mononuclear cells, and regenerating muscle fibers had diffusely positive iNOS immunoreactivity. In DMD patients, NADPH-d reactivity was increased and mainly localized in regenerating muscle fibers. In mdx mice quadriceps, iNOS expression was mainly observed in regenerating muscle fibers, but not prior to 4 weeks postnatal, and was still present 8 weeks after birth. The expression of dystrophin and the overexpression of utrophin using adenovirus-mediated constructs reduced the number of iNOS-positive fibers in mdx quadriceps muscles. The correction of some pathology in mdx by dystrophin expression or utrophin overexpression was independent of the presence of nNOS. CONCLUSIONS: These results suggest that iNOS could play a role in the physiopathology of DMD and that the abnormal expression of iNOS could be corrected by gene therapy.     

Intracellular calcium handling in ventricular myocytes from mdx mice

Duchenne muscular dystrophy (DMD) is a lethal degenerative disease of skeletal muscle, characterized by the absence of the cytoskeletal protein dystrophin. Some DMD patients show a dilated cardiomyopathy leading to heart failure. This study explores the possibility that dystrophin is involved in the regulation of a stretch-activated channel (SAC), which in the absence of dystrophin has increased activity and allows greater Ca(2+) into cardiomyocytes. Because cardiac failure only appears late in the progression of DMD, we examined age-related effects in the mdx mouse, an animal model of DMD. Ca(2+) measurements using a fluorescent Ca(2+)-sensitive dye fluo-4 were performed on single ventricular myocytes from mdx and wild-type mice. Immunoblotting and immunohistochemistry were performed on whole hearts to determine expression levels of key proteins involved in excitation-contraction coupling. Old mdx mice had raised resting intracellular Ca(2+) concentration ([Ca(2+)](i)). Isolated ventricular myocytes from young and old mdx mice displayed abnormal Ca(2+) transients, increased protein expression of the ryanodine receptor, and decreased protein expression of serine-16-phosphorylated phospholamban. Caffeine-induced Ca(2+) transients showed that the Na(+)/Ca(2+) exchanger function was increased in old mdx mice. Two SAC inhibitors streptomycin and GsMTx-4 both reduced resting [Ca(2+)](i) in old mdx mice, suggesting that SACs may be involved in the Ca(2+)-handling abnormalities in these animals. This finding was supported by immunoblotting data, which demonstrated that old mdx mice had increased protein expression of canonical transient receptor potential channel 1, a likely candidate protein for SACs. SACs may play a role in the pathogenesis of the heart failure associated with DMD. Early in the disease process and before the onset of clinical symptoms increased, SAC activity may underlie the abnormal Ca(2+) handling in young mdx mice.     

Presence of dystrophine-like protein at the neuromuscular junction in Duchenne muscular dystrophy and in "mdx" mutant mice

We have studied by indirect immunofluorescence, using three different polyclonal antidystrophin antibodies raised against fusion proteins, the neuromuscular junctions (NMJs) in muscle biopsies from Duchenne muscular dystrophy (DMD) patients, from human controls and mutant "mdx" mice and normal mice. In controls the periphery of all muscle fibres was strongly labelled by the three dystrophin antibodies and there was a high concentration of labelling at the NMJs (where it was co-localized with acetylcholine receptor labelled by the alpha-bungarotoxin). In DMD and in "mdx" mice the NMJs were equally labelled whereas there was an absence of reaction at the periphery of all (DMD) or most ("mdx" mice) muscle fibers. These findings suggest that a dystrophin-like protein, which was identified by the antibodies we have used, is present at the NMJs in the Duchenne's myopathy and "mdx" mice.     

Sarcoplasmic reticulum Ca2+ permeation explored from the lumen side in mdx muscle fibers under voltage control

Under resting conditions, external Ca(2+) is known to enter skeletal muscle cells, whereas Ca(2+) stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the sarcolemmal Ca(2+) entry and in the SR Ca(2+) leak is still a matter of debate, but several lines of evidence suggest that these Ca(2+) fluxes are up-regulated in Duchenne muscular dystrophy. We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the mouse model of Duchenne muscular dystrophy. Using the cytosolic Ca(2+) dye Fura2, we first demonstrated that the rate of Ca(2+) increase in response to cyclopiazonic acid (CPA)-induced inhibition of SR Ca(2+)-ATPases at resting potential was significantly higher in mdx fibers, which suggests an elevated SR Ca(2+) leak. However, removal of external Ca(2+) reduced the rate of CPA-induced Ca(2+) increase in mdx and increased it in control fibers, which indicates an up-regulation of sarcolemmal Ca(2+) influx in mdx fibers. Fibers were then loaded with the low-affinity Ca(2+) dye Fluo5N-AM to measure intraluminal SR Ca(2+) changes. Trains of action potentials, chloro-m-cresol, and depolarization pulses evoked transient Fluo5N fluorescence decreases, and recovery of voltage-induced Fluo5N fluorescence changes were inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR Ca(2+) changes. Voltage dependence and magnitude of depolarization-induced SR Ca(2+) depletion were found to be unchanged in mdx fibers, but the rate of the recovery phase that followed depletion was found to be faster, indicating a higher SR Ca(2+) reuptake activity in mdx fibers. Overall, CPA-induced SR Ca(2+) leak at -80 mV was found to be significantly higher in mdx fibers and was potentiated by removal of external Ca(2+) in control fibers. The elevated passive SR Ca(2+) leak may contribute to alteration of Ca(2+) homeostasis in mdx muscle.     

The role of reactive oxygen species in the hearts of dystrophin-deficient mdx mice

Duchenne muscular dystrophy (DMD) is caused by deficiency of the cytoskeletal protein dystrophin. Oxidative stress is thought to contribute to the skeletal muscle damage in DMD; however, little is known about the role of oxidative damage in the pathogenesis of the heart failure that occurs in DMD patients. The dystrophin-deficient (mdx) mouse is an animal model of DMD that also lacks dystrophin. The current study investigates the role of the antioxidant N-acetylcysteine (NAC) on mdx cardiomyocyte function, Ca(2+) handling, and the cardiac inflammatory response. Treated mice received 1% NAC in their drinking water for 6 wk. NAC had no effect on wild-type (WT) mice. Immunohistochemistry experiments revealed that mdx mice had increased dihydroethidine (DHE) staining, an indicator of superoxide production; NAC-treatment reduced DHE staining in mdx hearts. NAC treatment attenuated abnormalities in mdx cardiomyocyte Ca(2+) handling. Mdx cardiomyocytes had decreased fractional shortening and decreased Ca(2+) sensitivity; NAC treatment returned mdx fractional shortening to WT values but did not affect the Ca(2+) sensitivity. Immunohistochemistry experiments revealed that mdx hearts had increased levels of collagen type III and the macrophage-specific protein, CD68; NAC-treatment returned collagen type III and CD68 expression close to WT values. Finally, mdx hearts had increased NADPH oxidase activity, suggesting it could be a possible source of increased reactive oxygen species in mdx mice. This study is the first to demonstrate that oxidative damage may be involved in the pathogenesis of the heart failure that occurs in mdx mice. Therapies designed to reduce oxidative damage might be beneficial to DMD patients with heart failure.     

Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2.

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.     

Nav1.4 deregulation in dystrophic skeletal muscle leads to Na+ overload and enhanced cell death

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca(2+) concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na(+) concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Na(v)1.4, leading to an increased Na(+) concentration under the sarcolemma. Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4. Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.     

Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice

In Duchenne muscular dystrophy (DMD), dystrophin mutation leads to progressive lethal skeletal muscle degeneration. For unknown reasons, dystrophin deficiency does not recapitulate DMD in mice (mdx), which have mild skeletal muscle defects and potent regenerative capacity. We postulated that human DMD progression is a consequence of loss of functional muscle stem cells (MuSC), and the mild mouse mdx phenotype results from greater MuSC reserve fueled by longer telomeres. We report that mdx mice lacking the RNA component of telomerase (mdx/mTR) have shortened telomeres in muscle cells and severe muscular dystrophy that progressively worsens with age. Muscle wasting severity parallels a decline in MuSC regenerative capacity and is ameliorated histologically by transplantation of wild-type MuSC. These data show that DMD progression results, in part, from a cell-autonomous failure of?MuSC to maintain the damage-repair cycle initiated by dystrophin deficiency. The essential role of MuSC function has therapeutic implications for DMD.     

Menstrual blood-derived cells confer human dystrophin expression in the murine model of Duchenne muscular dystrophy via cell fusion and myogenic transdifferentiation

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood–derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood–derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood–derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood–derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.     

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.     

Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1

In skeletal muscle excitation-contraction (E-C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.     

Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice

Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx) mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD) patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv) mice express a near-full length dystrophin protein at ～5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.     

Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.     

In situ study of the sarcoplasmic reticulum function in control and mdx mouse diaphragm muscle.

Sarcoplasmic reticulum (SR) calcium handling in diaphragm was compared between mdx mice (7-8 weeks old) and age-matched controls. The total SR Ca2+ load was released from the SR by rapidly cooling muscle bundles from 22 to -1 degree C. The plateau amplitude of the rapid cooling contracture (RCC) was considered as an index of the SR Ca2+ content. The steady-state RCC amplitude was significantly lower by 50% in mdx bundles mainly because of a decreased capacity of the dystrophic diaphragm to generate maximal tension. There was no significant difference between either RCC time to peak or the time to half-relaxation of the transient, spike-like, contractile response induced by muscle rewarming. The recovery process of RCC was studied by using a paired RCC protocol. In both groups, at the shortest interval (10 s) between two RCCs, the amplitude of the second RCC was decreased by 25% compared with the first RCC. Increasing the time interval led to progressive monoexponential recovery of the second RCC with similar time constants in control and mdx diaphragm. These results indicate that the dystrophic process does not significantly alter SR Ca2+ uptake nor Ca2+ redistribution within the muscular cell.     

Mechanical function of dystrophin in muscle cells

We have directly measured the contribution of dystrophin to the cortical stiffness of living muscle cells and have demonstrated that lack of dystrophin causes a substantial reduction in stiffness. The inferred molecular structure of dystrophin, its preferential localization underlying the cell surface, and the apparent fragility of muscle cells which lack this protein suggest that dystrophin stabilizes the sarcolemma and protects the myofiber from disruption during contraction. Lacking dystrophin, the muscle cells of persons with Duchenne muscular dystrophy (DMD) are abnormally vulnerable. These facts suggest that muscle cells with dystrophin should be stiffer than similar cells which lack this protein. We have tested this hypothesis by measuring the local stiffness of the membrane skeleton of myotubes cultured from mdx mice and normal controls. Like humans with DMD mdx mice lack dystrophin due to an x-linked mutation and provide a good model for the human disease. Deformability was measured as the resistance to indentation of a small area of the cell surface (to a depth of 1 micron) by a glass probe 1 micron in radius. The stiffness of the membrane skeleton was evaluated as the increment of force (mdyne) per micron of indentation. Normal myotubes with an average stiffness value of 1.23 +/- 0.04 (SE) mdyne/micron were about fourfold stiffer than myotubes cultured from mdx mice (0.34 +/- 0.014 mdyne/micron). We verified by immunofluorescence that both normal and mdx myotubes, which were at a similar developmental stage, expressed sarcomeric myosin, and that dystrophin was detected, diffusely distributed, only in normal, not in mdx myotubes. These results confirm that dystrophin and its associated proteins can reinforce the myotube membrane skeleton by increasing its stiffness and that dystrophin function and, therefore, the efficiency of therapeutic restoration of dystrophin can be assayed through its mechanical effects on muscle cells.     

The passive mechanical properties of the extensor digitorum longus muscle are compromised in 2- to 20-mo-old mdx mice

Muscle rigidity and myotendinous junction (MTJ) deficiency contribute to immobilization in Duchenne muscular dystrophy (DMD), a lethal disease caused by the absence of dystrophin. However, little is known about the muscle passive properties and MTJ strength in a diseased muscle. Here, we hypothesize that dystrophin-deficient muscle pathology renders skeletal muscle stiffer and MTJ weaker. To test our hypothesis, we examined the passive properties of an intact noncontracting muscle-tendon unit in mdx mice, a mouse model for DMD. The extensor digitorum longus (EDL) muscle-tendon preparations of 2-, 6-, 14-, and 20-mo-old mdx and normal control mice were strained stepwisely from 110% to 160% of the muscle optimal length. The stress-strain response and failure position were analyzed. In support of our hypothesis, the mdx EDL preparation consistently developed higher stress before muscle failure. Postfailure stresses decreased dramatically in mdx but not normal preparations. Further, mdx showed a significantly faster stress relaxation rate. Consistent with stress-strain assay results, we observed significantly higher fibrosis in mdx muscle. In 2- and 6-mo-old mdx and 20-mo-old BL10 mice failure occurred within the muscle (2- to 14-mo-old BL10 preparations did not fail). Interestingly, in ≥14-mo-old mdx mice the failure site shifted toward the MTJ. Electron microscopy revealed substantial MTJ degeneration in aged but not young mdx mice. In summary, our results suggest that the passive properties of the EDL muscle and the strength of MTJ are compromised in mdx in an age-dependent manner. These findings offer new insights in studying DMD pathogenesis and developing novel therapies.