Emanuela Longa

Transplantation of Genetically Corrected Human iPSC-Derived Progenitors in Mice with Limb-Girdle Muscular Dystrophy

Genetically corrected mesoangioblasts from human iPSCs derived from limb-girdle muscular dystrophy patients produce muscle fibers expressing the therapeutic gene in a mouse model of the disease. Muscle Progenitors Find Their Way Home Muscular dystrophies are genetic disorders primarily affecting skeletal muscle that result in greatly impaired mobility and, in severe cases, respiratory and cardiac dysfunction. There is no effective treatment, although several new approaches are entering clinical testing including cell therapy. Cell therapy aims to replace lost muscle fibers by transplanting healthy donor muscle progenitor cells or cells from dystrophic patients that have been genetically corrected in vitro. Mesoangioblasts are progenitor cells from blood vessel walls that have shown potential as a cell therapy in animal models of muscular dystrophy. In a new study, Tedesco et al. explore whether genetically corrected mesoangioblasts from patients with limb-girdle muscular dystrophy 2D (LGMD2D) have potential as an autologous cell therapy to treat this disease. The authors quickly found that they could not derive a sufficient number of mesoangioblasts from LGMD2D patients because the muscles of the patients were depleted of these progenitor cells. To overcome this problem, the authors reprogrammed fibroblasts or myoblasts from the LGMD2D patients to obtain human induced pluripotent stem cells (iPSCs) and induced them to differentiate into mesoangioblast-like cells that were then genetically corrected in vitro using a viral vector expressing the defective gene SGCA, which encodes α-sarcoglycan. After intramuscular or intra-arterial injection of these genetically corrected, iPSC-derived mesoangioblasts into mice with LGMD2D (immune-deficient Sgca-null mice), the cells homed to damaged mouse skeletal muscle, engrafted, and formed muscle fibers expressing α-sarcoglycan. Using mouse iPSC-derived mesoangioblasts, the researchers showed that the transplanted engrafted cells imbued muscle with greater strength and enabled the dystrophic mice to run for longer on a treadmill than dystrophic mice that did not receive the cells. This strategy offers the advantage of being able to produce unlimited numbers of genetically corrected progenitor cells, which perhaps could be used in the future as cell therapy for treating LGMD2D and other forms of muscular dystrophy. Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne’s muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan–null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan–null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.

Autophagy impairment in muscle induces neuromuscular junction degeneration and precocious aging.

Summary The cellular basis of age-related tissue deterioration remains largely obscure. The ability to activate compensatory mechanisms in response to environmental stress is an important factor for survival and maintenance of cellular functions. Autophagy is activated both under short and prolonged stress and is required to clear the cell of dysfunctional organelles and altered proteins. We report that specific autophagy inhibition in muscle has a major impact on neuromuscular synaptic function and, consequently, on muscle strength, ultimately affecting the lifespan of animals. Inhibition of autophagy also exacerbates aging phenotypes in muscle, such as mitochondrial dysfunction, oxidative stress, and profound weakness. Mitochondrial dysfunction and oxidative stress directly affect acto-myosin interaction and force generation but show a limited effect on stability of neuromuscular synapses. These results demonstrate that age-related deterioration of synaptic structure and function is exacerbated by defective autophagy.

In vivo generation of a mature and functional artificial skeletal muscle

Extensive loss of skeletal muscle tissue results in mutilations and severe loss of function. In vitro‐generated artificial muscles undergo necrosis when transplanted in vivo before host angiogenesis may provide oxygen for fibre survival. Here, we report a novel strategy based upon the use of mouse or human mesoangioblasts encapsulated inside PEG‐fibrinogen hydrogel. Once engineered to express placental‐derived growth factor, mesoangioblasts attract host vessels and nerves, contributing to in vivo survival and maturation of newly formed myofibres. When the graft was implanted underneath the skin on the surface of the tibialis anterior, mature and aligned myofibres formed within several weeks as a complete and functional extra muscle. Moreover, replacing the ablated tibialis anterior with PEG‐fibrinogen‐embedded mesoangioblasts also resulted in an artificial muscle very similar to a normal tibialis anterior. This strategy opens the possibility for patient‐specific muscle creation for a large number of pathological conditions involving muscle tissue wasting.

Human skeletal muscle fibre contractile properties and proteomic profile: adaptations to 3 weeks of unilateral lower limb suspension and active recovery

It is generally assumed that muscle fibres go through atrophy following disuse with a loss of specific force and an increase in unloaded shortening velocity. However, the underlying mechanisms remain to be clarified. Most studies have focused on events taking place during the development of disuse, whereas the subsequent recovery phase, which is equally important, has received little attention. Our findings support the hypotheses that the specific force of muscle fibres decreased following unilateral lower limb suspension (ULLS) and returned to normal after 3 weeks of active recovery as a result of a loss and recovery of myosin and actin content. Furthermore, muscle fibres went through extensive qualitative changes in muscle protein pattern following ULLS, and these were reversed by active recovery. Resistance training was very effective in restoring both muscle mass and qualitative muscle changes, indicating that long‐term ULLS did not prevent the positive effect of exercise on human muscle.

Quantitative and qualitative adaptations of muscle fibers to glucocorticoids.

Introduction: The aim of this study was to understand the effects of short‐term glucocorticoid administration in healthy subjects. Methods: Five healthy men received dexamethasone (8 mg/day) for 7 days. Vastus lateralis muscle biopsy and knee extension torque measurement were performed before and after administration. A large number of individual muscle fibers were dissected from the biopsy samples (pre‐administration: n = 165, post‐administration: n = 177). Results: Maximal knee extension torque increased after administration (∼13%), whereas both type 1 and type 2A fibers had decreased cross‐sectional area (type 1: ∼11%, type 2A: ∼17%), myosin loss (type 1: ∼18%, type 2A: ∼32%), and loss of specific force (type 1: ∼24%, type 2A: ∼33%), which were preferential for fast fibers. Conclusion: Short‐term dexamethasone administration in healthy subjects elicits quantitative and qualitative adaptations of muscle fibers that precede (and may predict) the clinical appearance of myopathy in glucocorticoid‐treated subjects. Muscle Nerve 52: 631–639, 2015

Structure and function of human muscle fibres and muscle proteome in physically active older men

Loss of muscle mass and strength in the growing population of elderly people is a major health concern for modern societies. This condition, termed sarcopenia, is a major cause of falls and of the subsequent increase in morbidity and mortality. Despite numerous studies on the impact of ageing on individual muscle fibres, the contribution of single muscle fibre adaptations to ageing‐induced atrophy and functional impairment is still unsettled. The level of physical function and disuse is often associated with ageing. We studied relatively healthy older adults in order to understand the effects of ageing per se without the confounding impact of impaired physical function. We found that in healthy ageing, structural and functional alterations of muscle fibres occur. Protein post‐translational modifications, oxidation and phosphorylation contribute to such alterations more than loss of myosin and other muscle protein content.

Actomyosin kinetics of pure fast and slow rat myosin isoforms studied by in vitro motility assay approach

An in vitro motility assay approach was used to investigate the mechanisms of the functional differences between myosin isoforms, by studying the effect of MgATP and MgADP on actin sliding velocity (Vf) of pure slow and fast rat skeletal myosin at different temperatures. The value of Vf depended on [MgATP] according to Michaelis–Menten kinetics, with an apparent constant (Km) of 54.2, 64.4 and 200 μm for the fast isoform and 18.6, 36.5 and 45.5 μm for the slow isoform at 20, 25 and 35°C, respectively. The presence of 2 mm MgADP decreased Vf and yielded an inhibition constant (Ki) of 377, 463 and 533 μm for the fast isoform at 20, 25 and 35°C, respectively, and 120 and 355 μm for the slow isoform at 25 and 35°C, respectively. The analysis of Km and Ki suggested that slow and fast isoforms differ in the kinetics limiting Vf. Moreover, the higher sensitivity of the fast myosin isoform to a drop in [MgATP] is consistent with the higher fatigability of fast fibres than slow fibres. From the Michaelis–Menten relation in the absence of MgADP, we calculated the rate of actomyosin dissociation by MgATP (k+ATP) and the rate of MgADP release (k‐ADP). We found values of k+ATP of 4.8 × 106, 6.5 × 106 and 6.6 × 106m−1 s−1 for the fast isoform and 3.3 × 106, 2.9 × 106 and 6.7 × 106m−1 s−1 for the slow isoform and values of k‐ADP of 263, 420 and 1320 s−1 for the fast isoform and 62, 107 and 306 s−1 for the slow isoform at 20, 25 and 35°C, respectively. The results suggest that k‐ADP could be the major determinant of functional differences between the fast and slow myosin isoforms at physiological temperatures.

An integrated approach in a case of facioscapulohumeral dystrophy

BackgroundMuscle fatigue, weakness and atrophy are basilar clinical features that accompany facioscapulohumeral dystrophy (FSHD) the third most common muscular dystrophy.No therapy is available for FSHD.Case presentationWe describe the effects of 6mo exercise therapy and nutritional supplementation in a 43-year-old woman severely affected by FSHD.ConclusionA mixed exercise program combined with nutritional supplementation can be safely used with beneficial effects in selected patients with FSHD.

Sarcolab pilot study into skeletal muscle’s adaptation to long-term spaceflight

Spaceflight causes muscle wasting. The Sarcolab pilot study investigated two astronauts with regards to plantar flexor muscle size, architecture, and function, and to the underlying molecular adaptations in order to further the understanding of muscular responses to spaceflight and exercise countermeasures. Two crew members (A and B) spent 6 months in space. Crew member A trained less vigorously than B. Postflight, A showed substantial decrements in plantar flexor volume, muscle architecture, in strength and in fiber contractility, which was strongly mitigated in B. The difference between these crew members closely reflected FAK-Y397 abundance, a molecular marker of muscle’s loading history. Moreover, crew member A showed downregulation of contractile proteins and enzymes of anaerobic metabolism, as well as of systemic markers of energy and protein metabolism. However, both crew members exhibited decrements in muscular aerobic metabolism and phosphate high energy transfer. We conclude that countermeasures can be effective, particularly when resistive forces are of sufficient magnitude. However, to fully prevent space-related muscular deterioration, intersubject variability must be understood, and intensive exercise countermeasures programs seem mandatory. Finally, proteomic and metabolomic analyses suggest that exercise benefits in space may go beyond mere maintenance of muscle mass, but rather extend to the level of organismic metabolism.Muscles: Onboard exercise limits muscle loss in spacePhysical activity with resistive forces helps preserve muscle volume, architecture and strength in space. A team led by Jörn Rittweger from the German Aerospace Center in Cologne studied two crew members who spent six months on board the International Space Station. During the Sarcolab pilot study, one of these astronauts performed less exercise than the other. After returning to Earth, the one who trained less showed more substantial deterioration of the plantar flexor muscle in the foot—a difference detectable also at the molecular level, with lower levels of proteins involved in anaerobic and aerobic muscle metabolism. The findings highlight the need to vigorously exercise in space to limit muscle weakness. Doing so does not seem to fully prevent space-related problems, though, as evidenced by signs of muscle wasting even in the astronaut who trained regularly.

Author Correction: Sarcolab pilot study into skeletal muscle’s adaptation to longterm spaceflight

The original version of this Article contained an error in the spelling of the author Claudio Franceschi, which was incorrectly given as Claudio Francheschi. This has now been corrected in both the PDF and HTML versions of the Article.