Biomedical subjects
Publications and source records attributed to Miranda D Grounds.
There is currently no effective treatment for the devastating muscle-wasting disease Duchenne muscular dystrophy (DMD). Cossu and colleagues report in a recent Nature paper that transplantation of mesoangioblast stem cells may hold promise for treating DMD. Further studies are required to fully evaluate the clinical potential of these blood-vessel-associated stem cells.
Necrosis of skeletal muscle fibres in the lethal childhood myopathy Duchenne Muscular Dystrophy results from deficiency of the cell membrane associated protein, dystrophin. We test the hypothesis in dystrophin-deficient mice, that the initial sarcolemmal breakdown resulting from dystrophin deficiency is exacerbated by inflammatory cells, specifically neutrophils, and that cytokines, specifically Tumour Necrosis Factor alpha (TNFalpha), contribute to myofibre necrosis. Antibody depletion of host neutrophils resulted in a delayed and significantly reduced amount of skeletal muscle breakdown in young dystrophic mdx mice. A more striking and prolonged protective effect was seen after pharmacological blockade of TNFalpha bioactivity using Etanercept. The extent of exercise induced myofibre necrosis in adult mdx mice after voluntarily wheel exercise was also reduced after Etanercept administration. These data show a clear role for neutrophils and TNFalpha in necrosis of dystrophic mdx muscle in vivo. Etanercept is a highly specific anti-inflammatory drug, widely used clinically, and potential application to muscular dystrophies is suggested by this reduced breakdown of mdx skeletal muscle.
Duchenne muscular dystrophy is a lethal muscle wasting disorder, resulting from mutations in the gene encoding for the skeletal muscle protein dystrophin. The absence of functional dystrophin leaves the muscle membrane vulnerable to damage during contraction. Damage initially occurs as 'tears' in the membrane, this damage can be exacerbated by the inflammatory response leading to myofibre necrosis rather than repair. Mast cells resident within skeletal muscle represent an immediate source of pro-inflammatory cytokines. We hypothesise that blockade of mast cell degranulation would reduce the extent of myofibre necrosis in the mdx mouse. Daily cromolyn injections were performed on young and exercised adult mdx mice and histological analysis confirmed that mast cell degranulation contributes to myofibre necrosis. This research identified high biological variation between individual mdx mice in the severity of the dystrophic pathology, and supported a relationship between extent of muscle damage in adult mdx mice and their individual enthusiasm for voluntary wheel running.
The hypothesis that intracellular Ca(2+) is elevated in dystrophic (mdx) skeletal muscle due to increased Ca(2+) influx is controversial. As the sub-sarcolemmal Ca(2+) ([Ca(2+)](mem)) should be even higher than the global cytosolic Ca(2+) in the presence of increased Ca(2+) influx, we investigated [Ca(2+)](mem) levels in collagenase-isolated adult flexor digitorum brevis (FDB) myofibres and myotubes of mdx and normal mice with the near-membrane Ca(2+) indicator FFP-18. Confocal imaging showed strong localization of FFP-18 to the sarcolemma only. No significant difference in [Ca(2+)](mem) was found in FDB myofibres of normal (77.3+/-3.8 nM, n=68) and mdx (79.3+/-5.6 nM, n=21, p=0.89) mice using FFP-18. Increasing external Ca(2+) to 18 mM did not significantly affect [Ca(2+)](mem) in either the normal or mdx myofibres. In the myotubes, the FFP-18 was non-selectively incorporated, distributing throughout the cytoplasm, and FFP-18-derived [Ca(2+)] values were similar to values obtained with Fura-2. Nevertheless, in the mdx myotubes, the [Ca(2+)] measured with FFP-18 increased linearly to a level approximately 2.75 times that of controls as the time of culture was prolonged. In older mdx myotubes (>or=8 days in culture), 18 mM extracellular Ca(2+) increased the steady state cytosolic [Ca(2+)] to approximately 22 times greater level than controls. This study suggests that the sub-sarcolemmal Ca(2+) homeostasis is well maintained in isolated adult mdx myofibers and also further supports the hypothesis that cytosolic Ca(2+) handling is compromised in mdx myotubes.
Dramatic clinical success in the treatment of chronic inflammatory diseases has resulted from the use of anti-cytokine therapies including specific blocking antibodies, soluble receptors and traps to silence the actions of inflammatory cytokines such as tumour necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1). Two agents used clinically to block the functional activity of TNFalpha protein are Remicade (an antibody) and Enbrel (a soluble TNF receptor). These tools are now being extended to many other clinical disorders. We have a specific interest in the treatment of muscle diseases. In order to study the effects of novel anti-cytokine drugs on mouse models of human disease, such drugs must be investigated to determine whether they are indeed effective in blocking the inflammatory response in mouse. This has been carried out by means of a simple in vivo bioassay. Histological examination of transverse sections from whole muscle autografts in C57BL/10ScSn mice sampled at 5 days after transplantation provides an excellent assay model and clearly shows that Remicade and Enbrel block the acute inflammatory cell response in vivo. This graft model has also been used to show that a single intraperitoneal injection of Remicade (10 microg/g) is long-lived and effective when administered at 1 week and even 4 weeks prior to the assay. Enbrel is highly effective when injected twice at -3 days and -1 day (2 x 100 microg) before muscle grafting but shows no inhibition of the inflammatory response after a single injection (100 microg) 1 week prior to grafting. This striking ablation of inflammation by pharmacological blockage of TNFalpha is in marked contrast to the lack of any effect in TNFalpha null mice. This simple reproducible in vivo assay model in mice can be used to evaluate the efficacy of many novel anti-cytokine interventions designed to block inflammation.
Transgenic mice that overexpress insulin-like growth factor-1 (IGF-I) specifically in skeletal muscle have generated much information about the role of this factor for muscle growth and remodelling and provide insight for therapeutic applications of IGF-I for different pathological states and ageing. However, difficulties arise when attempting to critically compare the significance of data obtained in vivo by using different genetically engineered mouse lines and various experimental models. Complications arise due to complexity of the IGF-I system, since multiple transcripts of the IGF-I gene encode different isoforms generated by alternate promoter usage, differential splicing and post-translational modification, and how IGF-I gene expression relates to its diverse autocrine, paracrine and endocrine modes of action in vivo has still to be elucidated. In addition, there are problems related to specification of the exact IGF-I isoform used, expression patterns of the promoters, and availability of the transgene product under different experimental conditions. This review discusses the factors that must be considered when reconciling data from cumulative studies on IGF-I in striated muscle growth and differentiation using genetically modified mice. Critical evaluation of the literature focuses specifically on: (1) the importance of detailed information about the IGF-I isoforms and their mode of action (local, systemic or both); (2) expression pattern and strength of the promoters used to drive transgenic IGF-I in skeletal muscle cells (mono and multi-nucleated); (3) local compared with systemic action of the transgene product and possible indirect effects of transgenic IGF-I due to upregulation of other genes within skeletal muscle; (4) re-interpretation of these results in light of the most recent approaches to the dissection of IGF-I function. Full understanding of these complex in vivo issues is essential, not only for skeletal muscle but for many other tissues, in order to effectively extend observations derived from transgenic studies into potential clinical situations.
Loss of the nerve supply to skeletal muscle results in a relentless loss of muscle mass (atrophy) over time. The ability of insulin-like growth factor-1 to reduce atrophy resulting from denervation was examined after transection of the sciatic nerve in transgenic MLC/mIGF-1 mice that over-express mIGF-1 specifically in differentiated myofibres. The cross sectional area (CSA) of all types of myofibres and specifically type IIB myofibres was measured in tibialis anterior muscles from transgenic and wild-type mice at 28 days after denervation. There was a marked myofibre atrophy ( approximately 60%) in the muscles of wild-type mice over this time with increased numbers of myofibres with small CSA. In the muscles of MLC/mIGF-1 mice, over-expression of mIGF-1 reduced the rate of denervation induced myofibre atrophy by approximately 30% and preserved myofibres with larger CSA, compared to wild-type muscles. It is proposed that the protective effect of mIGF-1 on denervated myofibres might be due to reduced protein breakdown.
Stem cells have been proposed as a wonder solution for tissue repair in many situations and have attracted much attention in the media for both their therapeutic potential and ethical implications. In addition to the excitement generated by embryonic stem cells, research has now identified a number of stem cells within adult tissues which pose much more realistic targets for therapeutic interventions. Myoblast transfer therapy (MTT) has long been viewed as a potential therapy for the debilitating muscle-wasting disorder Duchenne Muscular Dystrophy. This technique relies on the transplantation of committed muscle precursor cells directly into the muscle fibres but has had little success in clinical trials. The recent discovery of a population of cells within adult muscle with stem cell-like characteristics has interesting implications for the future of such putative cell transplantation therapies. This review focuses on the characterization and application of these potential muscle-derived stem cells (MDSC) to MTT.
BACKGROUND: Myoblast transfer therapy (MTT) is a strategy designed to compensate for the defective gene in myopathies such as Duchenne muscular dystrophy (DMD). Experimental MTT in the mdx mouse (an animal model of DMD) has used donor myoblasts derived from mice of various ages; however, to date, there has been no direct quantitative comparison between the efficacy of MTT using myoblasts isolated from adult and neonate donor muscle. METHODS: Donor normal male myoblasts were injected into Tibialis Anterior muscles of dystrophic female host mice and the survival and proliferation of male myoblasts quantitated using Y-chromosome specific real-time quantitative polymerase chain reaction. The survival of late preplate (PP6) myoblasts derived from neonatal (3-5 days old) or adult (6-8 weeks old) donor mice after MTT were compared. The influence of the number of tissue culture passages, on survival post-MTT, was also evaluated for both types of myoblasts. RESULTS: Surprisingly, superior transplantation efficiency was observed for adult-derived compared with neonatal myoblasts (both early and late passage). Extended expansion (>17 passages) in tissue culture resulted in inferior survival and proliferation of both adult and neonatal myoblasts; however, proliferation of early passage myoblasts (both adult and neonate) was evident between 3 weeks and 3 months. CONCLUSIONS: Myoblasts derived from neonatal mice were inferior for transplantation, and early passage donor myoblasts from adult mice are recommended for MTT in this model.
BACKGROUND: Myoblast transfer therapy (MTT) is a cell-based gene therapy representing a potential treatment for Duchenne muscular dystrophy. The rapid disappearance of donor myoblasts from transplanted muscles after MTT is one of the most controversial and significant obstacles facing research in this area. Dystrophin-deficient muscles show constitutively high levels of inflammation, thus necessitating an examination of whether inflammatory cells, specifically natural killer (NK) cells, neutrophils, and macrophages, within dystrophic muscle are responsible for poor graft survival. METHODS: Female mdx mice were treated with RB6-8C5 monoclonal antibody, PK136 monoclonal antibody, or clodronate liposomes to systemically deplete neutrophils, NK cells, and macrophages, respectively. After each depletion regimen, the mice and age-matched controls received 5.0 x 10 male myoblasts injected longitudinally into each tibialis anterior muscle. Donor myoblast survival was assessed by Y-chromosome specific quantitative real-time polymerase chain reaction analysis. RESULTS.: The systemic depletion of host neutrophils and NK cells resulted in a transient improvement in donor myoblast survival at 72 hr and 7 days post-MTT, respectively. Systemic depletion of macrophages had no significant beneficial effect on myoblast survival. Overall, the number of detectable male donor myoblasts was similar at time 0 and 1 hr post-MTT; however, there was significant loss by 24 hr (approximately 50%-70%) followed by a continual decline in donor cell numbers. CONCLUSIONS: Neutrophils and macrophages do not seem to play a major role in the rapid death of donor myoblasts after transplantation into dystrophic muscle. NK cells similarly seem to have no significant effect, contrary to earlier findings reported by our group.
Necrosis of dystrophic myofibers in Duchenne muscular dystrophy and mdx mice results from defects in the subsarcolemmal protein dystrophin that cause membrane fragility and tears in the sarcolemma, and these lead to the destruction of the myofibers. The present study specifically tests whether overexpression of mIGF-1 in mdx/mIGF-1 transgenic mice reduces myofiber breakdown during the acute onset phase of dystrophy (at 21 days). The extent of muscle damage and Evans blue dye (EBD) staining of myofibers was quantitated histologically for mdx/mIGF-1 and their mdx littermates from 15 to 30 days of age. Overexpression of mIGF-1 strikingly reduced the extent of myofiber damage (histology and EBD staining) by up to 97% in tibialis anterior and quadriceps muscles at 21-22 days after birth. In the mdx diaphragm, the onset of muscle breakdown was earlier (by 15 days after birth) but no significant protective effect of IGF-1 was apparent within the first month of age in mdx/IGF-1 mice. These novel observations show that increased mIGF-1 within mdx myofibers specifically reduces the breakdown of dystrophic muscle during the acute onset of muscle degeneration. This mechanism of action can account for the long-term reduced severity of the dystropathology in mdx mice that overexpress mIGF-1 and provides promising opportunities for therapeutic strategies.
Necrosis of skeletal muscle fibers in the lethal childhood myopathy Duchenne muscular dystrophy (DMD) results from defects in the cell membrane-associated protein, dystrophin. This study tests the novel hypothesis that the initial sarcolemmal breakdown resulting from dystrophin deficiency is exacerbated by inflammatory cells and that cytokines, specifically tumor necrosis factor-alpha (TNFalpha), contribute to muscle necrosis. To block in vivo TNFalpha bioactivity, young dystrophic mdx mice (a model for DMD) were injected weekly from 7 days of age with the anti-TNFalpha antibody Remicade before the onset of muscle necrosis and dystropathology that normally occurs at 21 days postnatally. The extent of inflammation, muscle necrosis, and myotube formation was measured by histological analysis from 18 to 28 days and muscle damage was also visualized by penetration of Evans blue dye into myofibers. Data from Remicade-treated and control mdx mice were compared with mdx/TNFalpha-/- mice that lack TNFalpha. Pharmacological blockade of TNFalpha activity with Remicade clearly delayed and greatly reduced the breakdown of dystrophic muscle, in marked contrast to the situation in mdx and mdx/TNFalpha-/- mice. Remicade had no adverse effect on new muscle formation. Remicade is a highly specific anti-inflammatory intervention, and clinical application to muscular dystrophies is suggested by this marked protective effect against skeletal muscle breakdown.
Early myogenic events in regenerating whole muscle grafts were compared between transgenic MLC/mIGF-1 mice with skeletal muscle-specific overexpression of the Exon-1 Ea isoform of insulin-like growth factor-1 (mIGF-1) and control FVB mice, from day 3 to day 21 after transplantation. Immunocytochemistry with antibodies against desmin showed that skeletal muscle-specific overexpression of IGF-1 did not affect the pattern of myoblast activation or proliferation or the onset and number of myotubes formed in regenerating whole muscle grafts. Hypertrophied myotubes were observed in MLC/mIGF grafts at day 7 after transplantation, although such hypertrophy was transient, and the transgenic and control grafts had a similar appearance at later time points (days 10, 14, and 21). Immunostaining with antibodies to platelet endothelial cell adhesion molecule-1, which identifies endothelial cells, demonstrated no difference in the formation of new vascular network in grafts of transgenic and control mice. Skeletal muscle-specific overexpression of mIGF-1 does not appear to stimulate the early events associated with myogenesis during regeneration of whole muscle grafts.
The transplantation of muscle precursor cells (myoblasts) is a potential therapy for Duchenne muscular dystrophy. A commonly used method to detect cell survival is quantitation of the Y chromosome following transplantation of male donor cells into female hosts. This article presents a direct comparison between real-time quantitative PCR (Q-PCR) and the DNA hybridization (slot-blot) technique for quantitation of Y chromosome DNA. Q-PCR has a significantly greater linear quantitation range and is up to 40-fold more sensitive at low concentrations of male DNA, detecting as little as 1 ng of male DNA in each female tibialis anterior (TA) muscle. At high male DNA concentrations, accurate quantitation by Q-PCR is 2.5 times higher than the maximum possible with slot-blot. In conclusion, Q-PCR has a higher dynamic range and is more efficient than slot-blot analysis for the detection of donor cell engraftment in a transsexual transplantation model.
Skeletal myogenesis is a precise procedure marked by specific changes in muscle cell morphology and cytoarchitecture. Cessation of proliferation by skeletal muscle precursor cells (myoblasts) coincides with the induction of fusion to form multinucleated myotubes and the initiation of differentiation, the process through which sarcomeres are formed. Concurrently, there is a distinct upregulation in expression of muscle-specific isoforms and an extreme downregulation of non-muscle-specific cytoskeletal isoforms. The sarcomere is the contractile unit of the cell and is comprised of a number of different proteins aggregated and aligned in very ordered arrays along the myotube. It is this rigorously controlled alignment that gives striated muscle its characteristic "striped" appearance. Previous studies, conducted predominantly in cardiac muscle, propose models for the development of the sarcomere that attribute little of the differentiative process to the myoblast morphology and cytoskeletal arrangement. In this study, perturbation of myoblast morphology and cytoskeletal arrangement by transfection with nonmuscle actin genes in the mouse skeletal muscle cell line C2 resulted in myotubes of both varied morphology and sarcomeric structure. The results presented herein not only provide novel insights into the formation of the sarcomere in skeletal muscle, but also suggest a role for myoblast morphology and cytoskeletal structure in the subsequent differentiation of the myotube.
There is a massive and rapid death of donor myoblasts (<20% surviving) within hours after intramuscular injection in myoblast transfer therapy (MTT), due to host immune cells, especially natural killer (NK) cells. To investigate the role of host immune cells in the dramatic death of donor myoblasts, MTT experiments were performed in irradiated host mice. Cultured normal C57BL/10ScSn male donor myoblasts were injected into muscles of female C57BL/10ScSn-Dmdmdx host mice after one of three treatments: whole body irradiation (WBI) to eliminate all circulating leukocytes, WBI and bone marrow reconstitution (BMR), or local irradiation (or protection) of one limb. Similar experiments were performed in host mice after antibody depletion of NK cells. Numbers of male donor myoblasts were quantified using a Y-chromosome-specific (male) probe following total DNA extraction of injected muscles. WBI prior to MTT resulted in dramatically enhanced survival (approximately 80%) of donor myoblasts at 1 hour after MTT, supporting a central role for host inflammatory cells in the initial death of donor myoblasts seen in untreated host mice. BMR restored the massive and rapid loss (approximately 25% surviving) of donor myoblasts at 1 hour after MTT. Local pre-irradiation also resulted in increased donor myoblast numbers (approximately 35-40%) compared with untreated controls (approximately 10%) at 3 weeks after MTT. Preirradiation of host muscle with 10 Gy did not significantly stimulate proliferation of the injected donor myoblasts. Serum protein levels of TNFalpha, IL-1beta, IL-6 and IL-12 fluctuated following irradiation treatments. These combined results strongly reinforce a major role for host immune cells in the rapid death of injected cultured donor myoblasts.
In this study, the effects of phospholipase A2 (PLA2) inhibitors on excitation-contraction coupling (ECC) and sarcoplasmic reticulum (SR) function were examined in skinned extensor digitorum longus (EDL) muscle fibers of the rat. The nonspecific PLA2 inhibitor indomethacin (200 microM) significantly increased the peak (approximately 2-fold, P = 0.02) and the width (approximately 6-fold, P = 0.008) of depolarization-induced force responses (DIFRs) elicited in the fibers (n = 4). Exposure of the skinned EDL fibers to indomethacin (200 microM) (n = 7) and another PLA2 inhibitor quinacrine (200 microM) (n = 5) resulted in the return of large DIFRs after use-dependent rundown. However, aristolochic acid (100 microM), an inhibitor of secretory PLA2, failed to return DIFRs after rundown. Indomethacin did not protect against the loss of DIFRs induced by exposure to elevated myofibrillar [Ca2+]. Indomethacin (200 microM) produced a small but significant increase in the Ca2+ sensitivity of the contractile apparatus of skinned EDL fibers and the maximum force production. Indomethacin (200 microM) also had significant effects on SR function, increasing SR Ca2+ loading in the skinned fibers (117.2 +/- 3.0% of controls, P = 0.0008, n = 8) and inducing intracellular Ca2+ release in isolated intact flexor digitorum brevis (FDB) fibers (n = 7) and C2C12 myotubes (n = 6). These data suggest that intracellular PLA2 may be an important modulator of ECC in skeletal muscle.
BACKGROUND: The rapid and massive death of cultured donor myoblasts after injection in vivo is a major problem for clinical myoblast transfer therapy (MTT). This study shows blood-borne factors are responsible and that ablating the host natural killer (NK) cell response greatly enhances the survival of such donor myoblasts. METHODS: Cultured male donor myoblasts were injected into muscles of female host mice and surviving donor male DNA (myoblasts) quantified using a Y-chromosome specific (Y1) probe. Survival of donor myoblasts transfected with m144, a murine major histocompatibility complex (MHC) class I homologue that protects against NK attack, was quantified. In addition, donor myoblast survival was investigated in host mice following initial (before MTT) and sustained (repeatedly for 3 weeks after MTT) depletion of host NK1.1+ and CD4+/CD8+ cells using specific monoclonal antibodies (either alone or in combination) for up to 3 weeks after MTT, as well as in beige (deficient in NK activity) and in perforin-deficient mdx host mice. RESULTS: A major role for blood-borne factors (especially cells) was confirmed by MTT experiments in irradiated and perfused host mice. Substantially enhanced myoblast survival was seen with donor myoblasts modified by transfection with the m144 molecule or following antibody depletion of host NK1.1+ cells and in beige host mice. Other studies support some role for CD8+ but not CD4+ cells. CONCLUSIONS: These combined data support a central role for host NK cells in the rapid initial death of donor myoblasts. The demonstrated role of NK cells provides strategies to enhance the efficacy of clinical myoblast transplantation.