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.     

Structure of neuromuscular junctions and differentiation of striated muscle fibers in mdx mice after bone-marrow stem cell therapy

Mdx mice are an experimental model of Duchenne muscular dystrophy caused by mutations in the dystrophin gene. Repeated cycles of muscle degeneration-regeneration are common for mdx mice. Disrupted neuromuscular junctions also characterize mdx mice. The structure of mdx mice neuromuscular junctions and the differentiation of striated muscle fibers were investigated 4, 8, 16, and 24 weeks after transplantation of C57BL/6 Lin(−) bone-marrow stem cells. We found that the death of striated muscle fibers decreased 4 weeks after the transplantation of bone-marrow stem cells. Accumulation of muscle fibers without centrally located nuclei began in 8 weeks and dystrophin synthesis increased in 16–24 weeks after the bone-marrow stem cells transplantation. On the longitudinal sections of quadriceps muscle of mdx mice 4 weeks after transplantation, we observed a reduced quantity of acetylcholine receptor clusters and an increase in their area in neuromuscular junctions. Sixteen weeks after the transplantation, the total area of neuromuscular junctions increased due to an enlarged number of acethylcholine receptors and their extended area. The single intramuscular transplantation of C57BL/6 Lin(−) bone-marrow stem cells induces the differentiation of mdx mice striated muscle fibers and improves the structure of neuromuscular junctions.     

Therapeutic gene transfer to dystrophic diaphragm by an adenoviral vector deleted of all viral genes

Duchenne muscular dystrophy is caused by defects in the dystrophin gene, and the mdx mouse is the most frequently employed genetic model of this disease. It is well known that different muscle groups do not respond in the same way to dystrophin deficiency. In particular, the mdx mouse diaphragm exhibits severe morphological and functional changes not found in other mdx muscles. Use of early generation adenoviral vectors to deliver genes to the diaphragm in immunocompetent mdx mice has been associated with substantial functional toxicity and a rapid loss of transgene expression. Here we determined the response to dystrophin gene replacement in the mdx diaphragm using a "gutted" adenoviral vector that contains the coding sequence of two full-length dystrophin genes and is deleted of most viral DNA sequences. At 1 wk postdelivery of the vector, 23.6 +/- 4% of total fibers in the injected diaphragm bundle expressed dystrophin at the sarcolemma, which remained stable over the study duration of 30 days without the need for continuous immunosuppression. Treated diaphragms showed a significantly improved resistance to the abnormal force deficits induced by high-stress muscle contractions, the latter being a functional hallmark of dystrophin-deficient muscle. This functional amelioration was achieved despite the presence of mildly increased inflammation (CD4+ and CD8+ lymphocytes) within the vector-treated diaphragms. To our knowledge, this is the first demonstration that a viral vector can achieve reversal of functional abnormalities in the dystrophic diaphragm via therapeutic dystrophin gene transfer without the need for sustained immunosuppressive therapy.     

Gene hACR-1 suppresses apoptosis of striated muscle fibres of m. quadriceps femoris after ballistic transfection of mdx mice

Human minidystrophin gene (pSG5dys plasmid) and hACR-1 gene (pRc-CMV-10.1 plasmid) were cotransfected by means of "gene-gun" to M. quadriceps femoris of mdx mice. Effects of transfection on dystrophin expression and survival of striated muscle fibres (SMF) were studied on the 21st day after shots. In the control mdx dystrophin-positive muscular fibers [D(+)] SMF and destroyed SMF made 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. In mice transfected with pSG5dys plasmid (20 mkg of DNA per mouse), the shares of D(+) SMF and dead SMF raised, respectively, up to 5.6 +/- 1.4 and 4.5 +/- 0.9%. Transfection of mice with pRc-CMV-10.1 (DNA dose is 20 mkg per mouse) reduced the levels of apoptosis in SMF and D(+) SMF level to 1.6 +/- 0.6 and 1.1 +/- 0.4%, respectively. Cotransfection by pSG5dys and pRc-CMV-10.1 plasmids (10 and 10 mkg of each plasmids DNA per mouse) reduced the share of D(+) SMF to 1.1 +/- 0.5% and SMF destruction to 0.9 +/- 0.3%. pSG5dys transfection considerably reduced the share of SMF having peripherally located nuclei, thus indicating a decrease in SMF differentiation level after transfection. Cotransfection of ACR-1 gene and a dystrophin minigene did not suppress further cytodifferentiation of mdx muscle fibers. A conclusion is made that ballistic transfection by hACR-1 gene reduces the level of apoptosis in mdx mice SMF without changing the level of SMF differentiation. The cotransfection of mdx mice muscle by hACR-1 and human minidystrophin gene reduces SMF destruction and supports SMF differentiation, too.     

Alterations in the permeability of dystrophic fibers during neuromuscular junction development

In the mdx mice, lack of dystrophin leads to increases in calcium influx and myonecrosis, followed by muscle regeneration. Synapse elimination is faster in mdx than in controls, suggesting that increases in calcium influx during development could be involved. In the present study, we evaluated whether dystrophic fibers display changes in permeability to Evans Blue Dye (EBD) during development of the neuromuscular junction. EBD is a sensitive label for the early detection of increased myofiber permeability and sarcolemmal damage. After intraperitoneal injection of EBD, sternomastoid (STN) and tibialis anterior (T. anterior) muscles were analyzed with fluorescence microscopy. At 01, 07 and 14 days of age, STN and TA mdx myofibers were not stained with EBD. At 21 days of age, positive labeling of TA and STN mdx myofibers was seen, suggesting permeability modification and myonecrosis. Adult muscles showed a decrease (T. anterior) or no changes (STN) in the amount of EBD-positive fibers. These results suggest that there is no sarcolemmal damage detected by EBD during development of dystrophic neuromuscular junctions and other factors may contribute to the earlier synapse elimination seen in dystrophic muscle.     

The short MCK1350 promoter/enhancer allows for sufficient dystrophin expression in skeletal muscles of mdx mice

First-generation adenovirus vectors (AdV) have been used successfully to transfer a human dystrophin minigene to skeletal muscle of mdx mice. In most studies, strong viral promoters such as the cytomegalovirus promoter/enhancer (CMV) were used to drive dystrophin expression. More recently, a short version of the muscle creatine kinase promoter (MCK1350) has been shown to provide muscle-specific reporter gene expression after AdV-mediated gene delivery. Therefore, we generated a recombinant AdV where dystrophin expression is controlled by MCK1350 (AdVMCKdys). AdVMCKdys was injected by the intramuscular route into anterior tibialis muscle of mdx mice shortly after birth. Dystrophin expression was assessed at 20, 30, and 60 days after AdV-injection. At 20 days, muscles of AdVMCKdys-injected mdx mice showed a high number of dystrophin-positive fibers (mean: 365). At 60 days, the number of dystrophin-positive fibers was not only maintained, but increased significantly (mean: 600). In conclusion, MCK1350 allows for sustained dystrophin expression after AdV-mediated gene transfer to skeletal muscle of newborn mdx mice. In contrast to previous studies, where strong viral promoters were used, dystrophin expression driven by MCK1350 peaks at later time points. This may have implications for the future use of muscle-specific promoters for gene therapy of Duchenne muscular dystrophy.     

Fetal muscle-derived cells can repair dystrophic muscles in mdx mice

We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.     

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.     

Myogenic potential of muscle side and main population cells after intravenous injection into sub-lethally irradiated mdx mice.

Muscle side population (SP) cells have demonstrated hematopoietic and myogenic activities in vivo upon intravenous (IV) injection into lethally irradiated mdx mice. In contrast, muscle main population (MP) cells were unable to rescue the bone marrow of lethally irradiated mice and, consequently, their in vivo myogenic potential could not be assessed using this method. In the current study, muscle SP or MP cells derived from syngeneic wild-type male mice were delivered to sub-lethally irradiated mdx female mice by single or serial IV injections. Recipient mice were euthanized 12 weeks after transplantation at which time the quadriceps and diaphragm muscles were analyzed for the presence of donor-derived cells. Mice injected with 10(4) muscle SP cells or with 10(6) MP cells appeared to have similar numbers of dystrophin-positive myofibers containing fused donor nuclei. Analysis of the remaining tissue via real-time quantitative PCR indicated that mice injected with muscle SP cells had a higher percentage of donor-derived Y-DNA in the quadriceps than mice injected with MP cells, suggesting that muscle SP cells may be enriched for progenitors able to engraft dystrophic skeletal muscles from the circulation. Although the overall engraftment did not reach therapeutically significant levels, these results indicate that further optimization of cell delivery techniques may lead to improved efficacy of cell-mediated therapy using muscle SP cells.     

Increased expression of deltaCaMKII isoforms in skeletal muscle regeneration: Implications in dystrophic muscle disease

The expression of delta isoforms of calcium-calmodulin/dependent protein kinase II (CaMKII) has been reported in mammalian skeletal muscle; however, their functions in this tissue are largely unknown. This study was conducted to determine if deltaCaMKII expression was altered during regeneration of skeletal muscle fibers in two distinct models. In the first model, necrosis and regeneration were induced in quadriceps of normal mice by intramuscular administration of 50% glycerol. Immunostaining and confocal microscopy revealed that deltaCaMKII expression was clearly enhanced in fibers showing centralized nuclei. The second model was the mdx mouse, which undergoes enhanced muscle necrosis and regeneration due to a mutation in the dystrophin gene. sern blot analysis of hind leg extracts from 4 to 6 week old mdx mice revealed that deltaCaMKII content was decreased when compared to age-matched control mice. This loss in delta kinase content was seen in myofibrillar and membrane fractions and was in contrast to unchanged deltaCaMKII levels in cardiac and brain extracts from dystrophic mice. Confocal microscopy of mdx quadriceps and tibialis muscle showed that deltaCaMKII expression was uniformly decreased in most fibers from dystrophic mice; however, enhanced kinase expression was observed in regenerating muscle fibers. These data support a fundamental role for deltaCaMKII in the regeneration process of muscle fibers in normal and mdx skeletal muscle and may have important implications in the reparative process following muscle death.     

Participation of bone-marrow stem cells in the differentiation of mdx mice striated muscle

Two sets of experiments were carried out. The first one involved chimeric mice, obtained by intravenously injections of bone marrow derived cells taken from transgenic C57BL/6 mice, expressing GFP, to 5 Gy X-ray irradiated mdx or C57BL/6 mice. In 2 months M. quadriceps femoris of chimeric mice were destroyed by surgical clamp. Following the next 4-5 weeks, the same muscles were studied for the presence of GFP-positive striated muscle fibres. In the case of chimeric C57BL/6 mice GFP-positive striated muscle fibres were observed in 0.3 +/- 0.5 and in 0.2 +/- 0.3 % of destroyed muscle, and in lateral (control) muscle, consequently. In the case of chimeric mdx mice, positive results were observed in 1.7 +/- 0.4 and in 0.5 +/- 0.3 % of destroyed and control muscles, respectively. In the second set of experiments, the GFP-positive bone marrow cells were used for multiple intramuscular injections to M. quadriceps femoris of C57BL/6 or mdx mice in a dose of 2 x 10(5)-5 x 10(5) cells per mouse. Before injection, GFP-positive bone marrow cells were fractionated in a 63 % Percoll solution and then were exhausted from differentiated cells by magnetic manner using CD4, CD8, CD38, CD45R, CD119, Ly-6G, and F4/80 antibodies. After 2-3 weeks, as many as 0.15 +/- 0.40 and 0.1 +/- 0.2 % of GFP-positive muscle fibres were found in injected and control muscles of C57BL/6 mice, respectively. In the case of mdx mice, the frequency of GFP-positive striated muscle fibres was 2.0 +/- 0.8 and 1.2 +/- 0.6 % for injected and control muscles, respectively. A conclusion is made that bone marrow stem cells can take part in differentiation of mdx mouse muscles after their delivery by needle injections.     

Protein turnover is elevated in muscle of mdx mice in vivo.

mdx mice lack the protein dystrophin, the absence of which causes Duchenne muscular dystrophy in humans. To examine how mdx mice maintain muscle mass despite dystrophin deficiency, we measured protein turnover rates in muscles of mdx and wild-type (C57BL/10) mice in vivo. At all ages studied, rates of muscle protein synthesis and degradation were higher in mdx than in C57BL/10 mice.     

Acute pathophysiological effects of muscle-expressed Dp71 transgene on normal and dystrophic mouse muscle.

products of the dystrophin gene range from the 427-kDa full-length dystrophin to the 70.8-kDa Dp71. Dp427 is expressed in skeletal muscle, where it links the actin cytoskeleton with the extracellular matrix via a complex of dystrophin-associated proteins (DAPs). Dystrophin deficiency disrupts the DAP complex and causes muscular dystrophy in humans and the mdx mouse. Dp71, the major nonmuscle product, consists of the COOH-terminal part of dystrophin, including the binding site for the DAP complex but lacks binding sites for microfilaments. Dp71 transgene (Dp71tg) expressed in mdx muscle restores the DAP complex but does not prevent muscle degeneration. In wild-type (WT) mouse muscle, Dp71tg causes a mild muscular dystrophy. In this study, we tested, using isolated extensor digitorum longus muscles, whether Dp71tg exerts acute influences on force generation and sarcolemmal stress resistance. In WT muscles, there was no effect on isometric twitch and tetanic force generation, but with a cytomegalovirus promotor-driven transgene, contraction with stretch led to sarcolemmal ruptures and irreversible loss of tension. In MDX muscle, Dp71tg reduced twitch and tetanic tension but did not aggravate sarcolemmal fragility. The adverse effects of Dp71 in muscle are probably due to its competition with dystrophin and utrophin (in MDX muscle) for binding to the DAP complex.     

Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract.

Duchenne muscular dystrophy is characterized by the absence of dystrophin from muscle cells. Dystrophic muscle cells are susceptible to oxidative stress. We tested the hypothesis that 3 wk of endurance exercise starting at age 21 days in young male mdx mice would blunt oxidative stress and improve dystrophic skeletal muscle function, and these effects would be enhanced by the antioxidant green tea extract (GTE). In mice fed normal diet, average daily running distance increased 300% from week 1 to week 3, and total distance over 3 wk was improved by 128% in mice fed GTE. Running, independent of diet, increased serum antioxidant capacity, extensor digitorum longus tetanic stress, and total contractile protein content, heart citrate synthase, and heart and quadriceps beta-hydroxyacyl-CoA dehydrogenase activities. GTE, independent of running, decreased serum creatine kinase and heart and gastrocnemius lipid peroxidation and increased gastrocnemius citrate synthase activity. These data suggest that both endurance exercise and GTE may be beneficial as therapeutic strategies to improve muscle function in mdx mice.     

Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca²⁺ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Pathological pattern of Mdx mice diaphragm correlates with gradual expression of the short utrophin isoform Up71

Utrophin gene is transcribed in a large mRNA of 13 kb that codes for a protein of 395 kDa. It shows amino acid identity with dystrophin of up to 73% and is widely expressed in muscle and non-muscle tissues. Up71 is a short utrophin product of the utrophin gene with the same cysteine-rich and C-terminal domains as full-length utrophin (Up395). Using RT-PCR, Western blots analysis, we demonstrated that Up71 is overexpressed in the mdx diaphragm, the most pathological muscle in dystrophin-deficient mdx mice, compared to wild-type C57BL/10 or other mdx skeletal muscles. Subsequently, we demonstrated that this isoform displayed an increased expression level up to 12 months, whereas full-length utrophin (Up395) decreased. In addition, beta-dystroglycan, the transmembrane glycoprotein that anchors the cytoplasmic C-terminal domain of utrophin, showed similar increase expression in mdx diaphragm, as opposed to other components of the dystrophin-associated protein complex (DAPC) such as alpha-dystrobrevin1 and alpha-sarcoglycan. We demonstrated that Up71 and beta-dystroglycan were progressively accumulated along the extrasynaptic region of regenerating clusters in mdx diaphragm. Our data provide novel functional insights into the pathological role of the Up71 isoform in dystrophinopathies.