Development of Approaches to Improve Cell Survival in Myoblast Transfer Therapy

Myoblast transplantation has been extensively studied as a gene complementation approach for genetic diseases such as Duchenne Muscular Dystrophy. This approach has been found capable of delivering dystrophin, the product missing in Duchenne Muscular Dystrophy muscle, and leading to an increase of strength in the dystrophic muscle. This approach, however, has been hindered by numerous limitations, including immunological problems, and low spread and poor survival of the injected myoblasts. We have investigated whether antiinflammatory treatment and use of different populations of skeletal muscle–derived cells may circumvent the poor survival of the injected myoblasts after implantation. We have observed that different populations of muscle-derived cells can be isolated from skeletal muscle based on their desmin immunoreactivity and differentiation capacity. Moreover, these cells acted differently when injected into muscle: 95% of the injected cells in some populations died within 48 h, while others richer in desmin-positive cells survived entirely. Since pure myoblasts obtained from isolated myofibers and myoblast cell lines also displayed a poor survival rate of the injected cells, we have concluded that the differential survival of the populations of muscle-derived cells is not only attributable to their content in desmin-positive cells. We have observed that the origin of the myogenic cells may influence their survival in the injected muscle. Finally, we have observed that myoblasts genetically engineered to express an inhibitor of the inflammatory cytokine, IL-1, can improve the survival rate of the injected myoblasts. Our results suggest that selection of specific muscle-derived cell populations or the control of inflammation can be used as an approach to improve cell survival after both myoblast transplantation and the myoblast-mediated ex vivo gene transfer approach.     

Dystrophic mdx mouse myoblasts exhibit elevated ATP/UTP-evoked metabotropic purinergic responses and alterations in calcium signalling

Pathophysiology of Duchenne Muscular Dystrophy (DMD) is still elusive. Although progressive wasting of muscle fibres is a cause of muscle deterioration, there is a growing body of evidence that the triggering effects of DMD mutation are present at the earlier stage of muscle development and affect myogenic cells. Among these abnormalities, elevated activity of P2X7 receptors and increased store-operated calcium entry myoblasts have been identified in mdx mouse. Here, the metabotropic extracellular ATP/UTP-evoked response has been investigated. Sensitivity to antagonist, effect of gene silencing and cellular localization studies linked these elevated purinergic responses to the increased expression of P2Y2 but not P2Y4 receptors. These alterations have physiological implications as shown by reduced motility of mdx myoblasts upon treatment with P2Y2 agonist. However, the ultimate increase in intracellular calcium in dystrophic cells reflected complex alterations of calcium homeostasis identified in the RNA seq data and with significant modulation confirmed at the protein level, including a decrease of Gq11 subunit α, plasma membrane calcium ATP-ase, inositol-2,4,5-trisphosphate-receptor proteins and elevation of phospholipase Cβ, sarco-endoplamatic reticulum calcium ATP-ase and sodium‑calcium exchanger. In conclusion, whereas specificity of dystrophic myoblast excitation by extracellular nucleotides is determined by particular receptor overexpression, the intensity of such altered response depends on relative activities of downstream calcium regulators that are also affected by Dmd mutations. Furthermore, these phenotypic effects of DMD emerge as early as in undifferentiated muscle. Therefore, the pathogenesis of DMD and the relevance of current therapeutic approaches may need re-evaluation.     

Programmed cell death in dystrophic (mdx) muscle is inhibited by IGF-II

The pathology of Duchenne Muscular Dystrophy (DMD) is characterised by unstable muscle fibres and by increased cell turnover due to the absence of functional dystrophin protein. We have used skeletal muscle, primary muscle stem cell cultures (Smith and Schofield, 1994; Smith et al., paper submitted) and clonal cell lines of the mouse DMD model (mdx) and its congenic control (C57BI) to demonstrate that programmed cell death (PCD) and apoptotic morphology is increased in dystrophic (mdx) muscle and in cultured muscle cells. We also show that the peptide growth factor (IGF-II), which is thought to play a role in mammalian myogenesis, reduces PCD in mammalian skeletal muscle myoblasts both in vivo and in vitro. This is the first time that apoptosis or PCD have been demonstrated in normal mammalian skeletal muscle. We discuss the potential of this system in determining the role of PCD in mammalian myogenesis and skeletal muscle maturation, its significance in dystrophic muscle, and suggest a novel therapeutic route whereby the pathology of DMD may be alleviated using the survival properties of IGF-II.     

Analysis of fibronectin expression during human muscle differentiation

Fibronectin expression during human muscle differentiation was investigated by determining its distribution in foetal, normal adult and dystrophic muscle and in foetal, normal adult and dystrophic muscle cultures during myogenesis. Muscle sections and muscle cultures were studied by indirect immunofluorescence staining using polyclonal and monoclonal anti-human antibodies. Mass and clonal muscle cultures were prepared from foetal, adult and dystrophic muscle tissue. Immunofluorescence staining detected fibronectin on the epimysium, perimysium and endomysium of transverse sections of normal adult muscle, while sarcoplasm was devoid of this glycoprotein. In foetal muscle, some fibers showed a prominent ring of fibronectin. In mass and clonal cultures, myoblasts were found to synthesize and accumulate fibronectin while myotubes did not. No difference in fibronectin distribution was observed between Duchenne Muscular Dystrophy (DMD) and control myotubes. An enzyme-linked immunoassay (ELISA), performed on homogenated muscle, sonicated fibroblasts and muscle cells, showed a high fibronectin level in fibroblasts when compared with the other samples tested.     

MMP1 gene expression enhances myoblast migration and engraftment following implanting into mdx/SCID mice

Myoblast transplantation (MT) is a method to introduce healthy genes into abnormal skeletal muscle. It has been considered as a therapeutic modality in the last few decades for diseases such as Duchenne Muscular Dystrophy (DMD). However, challenges including cell death and poor graft engraftment have limited its application. The current experiment utilizes MMP1 gene transfer to improve the efficacy of myoblast transplantation into the diseased dystrophic skeletal muscle of mdx mice. Our results indicated that MMP1 expression can promote myogenic differentiation and fusion capacities, increase migration of MMP1 expressing myoblasts in vitro, as well as improve engraftment of dystrophin positive myofibers in vivo. Taken together, our observation suggests that the addition of MMP1 can overcome limitations in MT and improve its clinical efficacy.     

Genetic correction of splice site mutation in purified and enriched myoblasts isolated from mdx5cv mice

Background  

Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disorder that results in the production of a dysfunctional form of the protein, dystrophin. The mdx^5cv mouse is a model of DMD in which a point mutation in exon 10 of the dystrophin gene creates an artificial splice site. As a result, a 53 base pair deletion of exon 10 occurs with a coincident creation of a frameshift and a premature stop codon. Using primary myoblasts from mdx^5cv mice, single-stranded DNA oligonucleotides were designed to correct this DNA mutation.     

Defects in Mitochondrial ATP Synthesis in Dystrophin-Deficient Mdx Skeletal Muscles May Be Caused by Complex I Insufficiency

Duchenne Muscular Dystrophy is a chronic, progressive and ultimately fatal skeletal muscle wasting disease characterised by sarcolemmal fragility and intracellular Ca²⁺ dysregulation secondary to the absence of dystrophin. Mounting literature also suggests that the dysfunction of key energy systems within the muscle may contribute to pathological muscle wasting by reducing ATP availability to Ca²⁺ regulation and fibre regeneration. No study to date has biochemically quantified and contrasted mitochondrial ATP production capacity by dystrophic mitochondria isolated from their pathophysiological environment such to determine whether mitochondria are indeed capable of meeting this heightened cellular ATP demand, or examined the effects of an increasing extramitochondrial Ca²⁺ environment. Using isolated mitochondria from the diaphragm and tibialis anterior of 12 week-old dystrophin-deficient mdx and healthy control mice (C57BL10/ScSn) we have demonstrated severely depressed Complex I-mediated mitochondrial ATP production rate in mdx mitochondria that occurs irrespective of the macronutrient-derivative substrate combination fed into the Kreb’s cycle, and, which is partially, but significantly, ameliorated by inhibition of Complex I with rotenone and stimulation of Complex II-mediated ATP-production with succinate. There was no difference in the MAPR response of mdx mitochondria to increasing extramitochondrial Ca²⁺ load in comparison to controls, and 400 nM extramitochondrial Ca²⁺ was generally shown to be inhibitory to MAPR in both groups. Our data suggests that DMD pathology is exacerbated by a Complex I deficiency, which may contribute in part to the severe reductions in ATP production previously observed in dystrophic skeletal muscle.     

Defective growth in vitro of Duchenne muscular dystrophy myoblasts: The molecular and biochemical basis

As the molecular basis of Duchenne Muscular Dystrophy (DMD) was being discovered, increasing focus was placed on the mechanisms of progressive failure of myoregeneration. In this study, we propose a pathogenesis model for DMD, where an autocrine growth factor release of TGF‐β1—from necrotic myofibers—could contribute to the increasing loss of muscle regeneration. In fact, we report evidence that DMD myoblasts reduce their proliferation rate, in time and later cultures; in connection with this, we observed TGF‐β1 increase in conditioned media of DMD myoblasts, able to control the myoblast growth by reducing fusion and differentiation of DMD satellite cells. J. Cell. Biochem. 76:118–132, 1999. © 1999 Wiley‐Liss, Inc.     

Motor Physical Therapy Affects Muscle Collagen Type I and Decreases Gait Speed in Dystrophin-Deficient Dogs

Golden Retriever Muscular Dystrophy (GRMD) is a dystrophin-deficient canine model genetically homologous to Duchenne Muscular Dystrophy (DMD) in humans. Muscular fibrosis secondary to cycles of degeneration/regeneration of dystrophic muscle tissue and muscular weakness leads to biomechanical adaptation that impairs the quality of gait. Physical therapy (PT) is one of the supportive therapies available for DMD, however, motor PT approaches have controversial recommendations and there is no consensus regarding the type and intensity of physical therapy. In this study we investigated the effect of physical therapy on gait biomechanics and muscular collagen deposition types I and III in dystrophin-deficient dogs. Two dystrophic dogs (treated dogs-TD) underwent a PT protocol of active walking exercise, 3×/week, 40 minutes/day, 12 weeks. Two dystrophic control dogs (CD) maintained their routine of activities of daily living. At t0 (pre) and t1 (post-physical therapy), collagen type I and III were assessed by immunohistochemistry and gait biomechanics were analyzed. Angular displacement of shoulder, elbow, carpal, hip, stifle and tarsal joint and vertical (Fy), mediolateral (Fz) and craniocaudal (Fx) ground reaction forces (GRF) were assessed. Wilcoxon test was used to verify the difference of biomechanical variables between t0 and t1, considering p<.05. Type I collagen of endomysium suffered the influence of PT, as well as gait speed that had decreased from t0 to t1 (p<.000). The PT protocol employed accelerates morphological alterations on dystrophic muscle and promotes a slower velocity of gait. Control dogs which maintained their routine of activities of daily living seem to have found a better balance between movement and preservation of motor function.     

Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells

Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy- and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the pre-dominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC₅₀), even if limitations remain from endosomal escape.     

Defective growth in vitro of Duchenne Muscular Dystrophy myoblasts: the molecular and biochemical basis

As the molecular basis of Duchenne Muscular Dystrophy (DMD) was being discovered, increasing focus was placed on the mechanisms of progressive failure of myoregeneration. In this study, we propose a pathogenesis model for DMD, where an autocrine growth factor release of TGF-beta1-from necrotic myofibers-could contribute to the increasing loss of muscle regeneration. In fact, we report evidence that DMD myoblasts reduce their proliferation rate, in time and later cultures; in connection with this, we observed TGF-beta1 increase in conditioned media of DMD myoblasts, able to control the myoblast growth by reducing fusion and differentiation of DMD satellite cells.     

Activation of JNK1 contributes to dystrophic muscle pathogenesis

Duchenne Muscular Dystrophy (DMD) originates from deleterious mutations in the dystrophin gene, with a complete loss of the protein product. Subsequently, the disease is manifested in severe striated muscle wasting and death in early adulthood. Dystrophin provides a structural base for the assembly of an integral membrane protein complex. As such, dystrophin deficiency leads to an altered mechanical integrity of the myofiber and a predisposition to contraction-induced damage. However, the development of myofiber degeneration prior to an observed mechanical defect has been documented in various dystrophic models. Although activation of a detrimental signal transduction pathway has been suggested as a probable cause, a specific cellular cascade has yet to be defined. Here, it is shown that murine models of DMD displayed a muscle-specific activation of JNK1. Independent activation of JNK1 resulted in defects in myotube viability and integrity in vitro, similar to a dystrophic phenotype. In addition, direct muscle injection of an adenoviral construct containing the JNK1 inhibitory protein, JIP1, dramatically attenuated the progression of dystrophic myofiber destruction. Taken together, these results suggest that a JNK1-mediated signal cascade is a conserved feature of dystrophic muscle and contributes to the progression of the disease pathogenesis.     

Detection of truncated dystrophin lacking the C-terminal domain in a Chinese pedigree by next-generation sequencing

Dystrophin (DMD) gene is the largest gene containing 79 exons involving various mutation types and regions, and targeted next-generation sequencing (NGS) was employed in detecting DMD gene mutation in the present study. A literature-annotated disease nonsense mutation (c.10141C>T, NM_004006.1) in exon 70 that has been reported as Duchenne Muscular Dystrophy (DMD)-causing mutation was found in our two patients, the proband and his cousin. In the present study two main methods were used, the next-generation sequencing and the classic Sanger sequencing. The exon capture followed by HiSeq2000 sequencing was specifically used in this study. Combined applications of the next-generation sequencing platform and bioinformatics are proved to be effective methods for DMD diagnosis.     

Quantification of birefringence readily measures the level of muscle damage in zebrafish

Muscular dystrophies are a group of genetic disorders that progressively weaken and degenerate muscle. Many zebrafish models for human muscular dystrophies have been generated and analysed, including dystrophin-deficient zebrafish mutants dmd that model Duchenne Muscular Dystrophy. Under polarised light the zebrafish muscle can be detected as a bright area in an otherwise dark background. This light effect, called birefringence, results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres. Muscle damage, as seen in zebrafish models for muscular dystrophies, can readily be detected by a reduction in the birefringence. Therefore, birefringence is a very sensitive indicator of overall muscle integrity within larval zebrafish. Unbiased documentation of the birefringence followed by densitometric measurement enables the quantification of the birefringence of zebrafish larvae. Thereby, the overall level of muscle integrity can be detected, allowing the identification and categorisation of zebrafish muscle mutants. In addition, we propose that the establish protocol can be used to analyse treatments aimed at ameliorating dystrophic zebrafish models.     

Halofuginone improves muscle-cell survival in muscular dystrophies

Halofuginone has been shown to prevent fibrosis via the transforming growth factor-β/Smad3 pathway in muscular dystrophies. We hypothesized that halofuginone would reduce apoptosis—the presumed cause of satellite-cell depletion during muscle degradation—in the mdx mouse model of Duchenne muscular dystrophy. Six-week-old mdx mouse diaphragm exhibited fourfold higher numbers of apoptotic nuclei compared with wild-type mice as determined by a TUNEL assay. Apoptotic nuclei were found in macrophages and in Pax7-expressing cells; some were located in centrally-nucleated regenerating myofibers. Halofuginone treatment of mdx mice reduced the apoptotic nuclei number in the diaphragm, together with reduction in Bax and induction in Bcl2 levels in myofibers isolated from these mice. A similar effect was observed when halofuginone was added to cultured myofibers. No apparent effect of halofuginone was observed in wild-type mice. Inhibition of apoptosis or staurosporine-induced apoptosis by halofuginone in mdx primary myoblasts and C2 myogenic cell line, respectively, was reflected by less pyknotic/apoptotic cells and reduced Bax expression. This reduction was reversed by a phosphinositide-3-kinase and mitogen-activated protein kinase/extracellular signal-regulated protein kinase inhibitors, suggesting involvement of these pathways in mediating halofuginone's effects on apoptosis. Halofuginone increased apoptosis in α smooth muscle actin- and prolyl 4-hydroxylase β-expressing cells in mdx diaphragm and in myofibroblasts, the major source of extracellular matrix. The data suggest an additional mechanism by which halofuginone improves muscle pathology and function in muscular dystrophies.     

Oxidative stress and calcium homeostasis in dystrophic skin fibroblasts

Muscular dystrophy is a genetic disease that affects primarily skeletal muscle. The dystrophin absence has been related to the degeneration of muscle fibres. Indirect evidences suggest that oxidative stress may play a role in the pathogenesis of the disease, but the significance and precise extent of this contribution is poorly understood. In this paper we show that Becker Muscular Dystrophy (BMD) and Duchenne Muscular Dystrophy (DMD) skin fibroblasts are more susceptible to H2O2 treatment than are fibroblasts from unaffected persons. In particular, we found that, in growing DMD skin fibroblasts, the oxidative treatment resulted in significantly reduced growing capacity. We also investigated the concentrations of intracellular calcium during H2O2 treatment. The intracellular free calcium concentration increased by 22%, 35%, and 40% in unaffected, BMD, and DMD fibroblasts, respectively. However, the increase of the intracellular free calcium concentration is not related, as previously hypothesized, to a reduction of acylphosphatase concentrations, which seem to be unaffected by the H2O2 treatment, but rather to reduced enzyme activity.     

Discrimination and consistency of five myosin ATPase stains in human normal and duchenne dystrophic muscle

Summary Limitations in the ability of the human visual system to assess accurately the relative staining densities of individual fibers in muscle tissue stained for myosin ATPase can complicate the objective evaluation of fiber type populations. In this study a novel approach is employed which utilizes human visual capabilities to provide accurate fiber classification. Using this approach, the ability of five ATPase staining techniques to discriminate fiber type categories in single samples of human normal and Duchenne dystrophic skeletal muscle is evaluated, as is the consistency of the fiber type classifications between stains. While no major discrepancies in fiber typing were observed in the sample of normal muscle, significant differences in classification, along with a decrease in the ability to discriminate fiber types were noted in the sample of Duchenne muscular dystrophy. For the most part, these discrepancies were resolved by a re-interpretation of the staining characteristics of fibers in one stain.This work was supported in part by NIH grant NS 15584, and by a grant from the Muscular Dystrophy Association     

Localization of donor nuclei in skeletal muscle grafts byin situ hybridization to a cDNA probe

Summary The development of therapies, based upon implantation of normal muscle cell precursors, for the treatment of skeletal muscle diseases such as Duchenne Muscular Dystrophy is in its infancy. Detailed analysis of the genetic and phenotypic contribution made by donor myoblasts to the regenerated muscle is critical. Using non-radioactivein situ hybridization of aY chromosome-specific DNA probe to sections of muscle, we have localized the position of male donor nuclei within female host muscles after myoblast implantation. These results were compared with the distribution of immunocytochemically-localized dystrophin and the expression of donor-specific glucose phosphate isomerase by isoelectric-focussing. We found consistent male-specific nuclear hybridization and a close spatial relationship between the distribution of male donor nuclei and dystrophin-positive muscle fibres within female, dystrophin-negative host muscles. This approach will be useful in the further analysis of myoblast implantation experiments.     

Effective Exon Skipping and Dystrophin Restoration by 2′-O-Methoxyethyl Antisense Oligonucleotide in Dystrophin-Deficient Mice

Antisense oligonucleotide (AO)–mediated exon-skipping therapy is one of the most promising therapeutic strategies for Duchenne Muscular Dystrophy (DMD) and several AO chemistries have been rigorously investigated. In this report, we focused on the effect of 2′-O-methoxyethyl oligonucleotides (MOE) on exon skipping in cultured mdx myoblasts and mice. Efficient dose-dependent skipping of targeted exon 23 was achieved in myoblasts with MOE AOs of different lengths and backbone chemistries. Furthermore, we established that 25-mer MOE phosphorothioate (PS) AOs provided the greatest exon-skipping efficacy. When compared with 2′O methyl phosphorothioate (2′OmePS) AOs, 25-mer MOE (PS) AOs also showed higher exon-skipping activity in vitro and in mdx mice after intramuscular injections. Characterization of uptake in vitro corroborated with exon-skipping results, suggesting that increased uptake of 25-mer MOE PS AOs might partly contribute to the difference in exon-skipping activity observed in vitro and in mdx mice. Our findings demonstrate the substantial potential for MOE PS AOs as an alternative option for the treatment of DMD.     

Epigallocatechin gallate counteracts oxidative stress in docosahexaenoxic acid-treated myocytes

Skeletal muscle is a key organ of mammalian energy metabolism, and its mitochondria are multifunction organelles that are targets of dietary bioactive compounds. The goal of this work was to examine the regulation of mitochondrial dynamics, functionality and cell energy parameters using docosahexaenoic acid (DHA), epigallocatechin gallate (EGCG) and a combination of both in L6 myocytes. Compounds (at 25 μM) were incubated for 4 h. Cells cultured with DHA displayed less oxygen consumption with higher ADP/ATP ratio levels concomitant with downregulation of Cox and Ant1 gene expression. The disruption of energetic homeostasis by DHA, increases intracellular reactive oxygen species (ROS) levels and decreases mitochondrial membrane potential. The defence mechanism to counteract the excess of ROS production was by the upregulation of Ucp2, Ucp3 and MnSod gene expression. Moreover myocytes cultured with DHA had a higher mitochondrial mass with a higher proportion of large and elongated mitochondria, whereas the fission genes Drp1 and Fiss1 and the fusion gene Mfn2 were downregulated. In myocytes co-incubated with DHA and EGCG, ROS levels and the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio were similar to untreated myocytes and the decrease of oxygen consumption, higher mitochondrial mass and the overexpression of Ucp2 and Ucp3 genes were similar to the DHA-treated cells with also a higher amount of mitochondrial deoxyribonucleic acid (DNA), and reduced Drp1 and Fiss1 gene expression levels. In conclusion the addition of EGCG to DHA returned the cells to the control conditions in terms of mitochondrial morphology, energy and redox status, which were unbalanced in the DHA-treated myocytes.