Calcium and strontium contractile activation properties of single skinned skeletal muscle fibres from elderly women 66-90 years of age

The single skinned muscle fibre technique was used to investigate Ca2+- and Sr2+- activation properties of skeletal muscle fibres from elderly women (66-90 years). Muscle biopsies were obtained from the vastus lateralis muscle. Three populations of muscle fibres were identified according to their specific Sr2+- activation properties: slow-twitch (type I) fast-twitch (type II) and hybrid (type I/II) fibres. All three fibre types were sampled from the biopsies of 66 to 72 years old women, but the muscle biopsies of women older than 80 years yielded only slow-twitch (type I) fibres. The proportion of hybrid fibres in the vastus lateralis muscle of women of circa 70 years of age (24%) was several-fold greater than in the same muscle of adults (<10%), suggesting that muscle remodelling occurs around this age. There were no differences between the Ca2+- and Sr2+- activation properties of slow-twitch fibres from the two groups of elderly women, but there were differences compared with muscle fibres from adults with respect to sensitivity to Ca2+, steepness of the activation curves, and characteristics of the fibre-type dependent phenomenon of spontaneous force oscillations (SOMO) occurring at sub-maximal levels of activation. The maximal Ca2+ activated specific force from all the fibres collected from the seven old women use in the present study was significantly lower by 20% than in the same muscle of adults. Taken together these results show there are qualitative and quantitative changes in the activation properties of the contractile apparatus of muscle fibres from the vastus lateralis muscle of women with advancing age, and that these changes need to be considered when explaining observed changes in womens mobility with aging.

Loss of α-actinin-3 confers protection from eccentric contraction damage in fast-twitch EDL muscles from aged mdx dystrophic mice by reducing pathological fibre branching

ABSTRACTThe common null polymorphism (R577X) in the ACTN3 gene is present in over 1.5 billion people worldwide and results in the absence of the protein α-actinin-3 from the Z-discs of fast-twitch skeletal muscle fibres. We have previously reported that this polymorphism is a modifier of dystrophin deficient Duchenne Muscular Dystrophy. To investigate the mechanism underlying this we use a double knockout (dk)Actn3KO/mdx (dKO) mouse model which lacks both dystrophin and sarcomere α-actinin-3. We used dKO mice and mdx dystrophic mice at 12 months (aged) to investigate the correlation between morphological changes to the fast-twitch dKO EDL and the reduction in force deficit produced by an in vitro eccentric contraction protocol. In the aged dKO mouse we found a marked reduction in fibre branching complexity that correlated with protection from eccentric contraction induced force deficit. Complex branches in the aged dKO EDL fibres (28%) were substantially reduced compared to aged mdx EDL fibres (68%) and this correlates with a graded force loss over three eccentric contractions for dKO muscles (∼35% after first contraction, ∼66% overall) compared to an abrupt drop in mdx upon the first eccentric contraction (∼73% after first contraction, ∼89% after three contractions). In dKO protection from eccentric contraction damage was linked with a doubling of SERCA1 pump density the EDL. We propose that the increased oxidative metabolism of fast-twitch glycolytic fibres characteristic of the null polymorphism (R577X) and increase in SR Ca2+ pump proteins reduces muscle fibre branching and decreases susceptibility to eccentric injury in the dystrophinopathies.

Skeletal Muscles: Insight into Embryonic Development, Satellite Cells, Histology, Ultrastructure, Innervation, Contraction and Relaxation, Causes, Pathophysiology, and Treatment of Volumetric Muscle I

Background: Skeletal muscles are attached to bone and are responsible for the axial and appendicular movement of the skeleton and for maintenance of body position and posture. Objectives: The present review aimed to high light on embryonic development of skeletal muscles, histological and ultrastructure, innervation, contraction and relaxation, causes, pathophysiology, and treatment of volumetric muscle injury. The heterogeneity of the muscle fibers is the base of the flexibility which allows the same muscle to be used for various tasks from continuous low-intensity activity, to repeated submaximal contractions, and to fast and strong maximal contractions. The formation of skeletal muscle begins during the fourth week of embryonic development as specialized mesodermal cells, termed myoblasts. As growth of the muscle fibers continues, aggregation into bundles occurs, and by birth, myoblast activity has ceased. Satellite cells (SCs), have single nuclei and act as regenerative cells. Satellite cells are the resident stem cells of skeletal muscle; they are considered to be self-renewing and serve to generate a population of differentiation-competent myoblasts that will participate as needed in muscle growth, repair, and regeneration. Based on various structural and functional characteristics, skeletal muscle fibres are classified into three types: Type I fibres, Type II-B fibres, and type II-A fibres. Skeletal muscle fibres vary in colour depending on their content of myoglobin. Each myofibril exhibits a repeating pattern of cross-striations which is a product of the highly ordered arrangement of the contractile proteins within it. The parallel myofibrils are arranged with their cross-striations in the register, giving rise to the regular striations seen with light microscopy in longitudinal sections of skeletal muscle. Each skeletal muscle receives at least two types of nerve fibers: motor and sensory. Striated muscles and myotendinous junctions contain sensory receptors that are encapsulated proprioceptors. The process of contraction, usually triggered by neural impulses, obeys the all-or-none law. During muscle contraction, the thin filaments slide past the thick filaments, as proposed by Huxley's sliding filament theory. In response to a muscle injury, SCs are activated and start to proliferate; at this stage, they are often referred to as either myogenic precursor cells (MPC) or myoblasts. In vitro, evidence has been presented that satellite cells can be pushed towards the adipogenic and osteogenic lineages, but contamination of such cultures from non-myogenic cells is sometimes hard to dismiss as the underlying cause of this observed multipotency. There are, however, other populations of progenitors isolated from skeletal muscle, including endothelial cells and muscle-derived stem cells (MDSCs), blood-vessel-associated mesoangioblasts, muscle side-population cells, CD133+ve cells, myoendothelial cells, and pericytes. Volumetric muscle loss (VML) is defined as the traumatic or surgical loss of skeletal muscle with resultant functional impairment. It represents a challenging clinical problem for both military and civilian medicine. VML results in severe cosmetic deformities and debilitating functional loss. In response to damage, skeletal muscle goes through a well-defined series of events including; degeneration (1 to 3days), inflammation, and regeneration (3 to 4 weeks), fibrosis, and extracellular matrix remodeling (3 to 6 months).. Mammalian skeletal muscle has an impressive ability to regenerate itself in response to injury. During muscle tissue repair following damage, the degree of damage and the interactions between muscle and the infiltrating inflammatory cells appear to affect the successful outcome of the muscle repair process. The transplantation of stem cells into aberrant or injured tissue has long been a central goal of regenerative medicine and tissue engineering. Conclusion: It can be concluded that the formation of skeletal muscle begins during the fourth week of embryonic development as specialized mesodermal cells, termed myoblasts, by birth myoblast activity has ceased. Satellite cells are considered to be self-renewing, and serve to generate a population of differentiation-competent myoblasts. Skeletal muscle fibres are classified into three types. The process of contraction, usually triggered by neural impulses, obeys the all-or-none law. VML results in severe cosmetic deformities and debilitating functional loss. Mammalian skeletal muscle has an impressive ability to regenerate itself in response to injury. The transplantation of stem cells into aberrant or injured tissue has long been a central goal of regenerative medicine and tissue engineering.

OP0247 IN MYOSITIS MUSCLE FIBRE PLAYS A DIRECT AND CRITICAL ROLE IN THERAPEUTIC RESPONSE TO GLUCOCORTICOIDS

Background:Myositis are rare autoimmune diseases, affecting more women than men, characterized by chronic inflammation of skeletal muscle causing muscle weakness, decreased quality of life and increased mortality.Glucocorticoids (GC) are potent anti-inflammatory drugs, and are the first line treatment of myositis. They improve muscle strength of myositis patients (therapeutic effect), yet muscle recovery is generally only partial. Moreover, GC have an iatrogenic effect on skeletal muscle fibre leading to steroid myopathy. Thus myositis care has to be improved. Despite the autoimmune terrain of myositis, our team has recently shown that muscle fibres themselves develop immuno-metabolic modifications that participate to muscle weakness and perpetuation of the disease1. GC effects are mediated by the glucocorticoid receptor (GR), which is expressed in various cell types including immune cells and myofibres, but the cells mediating therapeutic responses remain to be determined.Objectives:Unravel the mechanisms underlying the therapeutic effect of GC in myositis, particularly elucidate the role of skeletal muscle fibres.Methods:Experimental myositis was induced in eight to ten week-old C57BL/6J female mice by a single intradermal injection of part of skeletal muscle fast-type C protein along with Freund’s adjuvant and an intraperitoneal (IP) injection of pertussis toxin, as previously described2. Prednisone (PDN) was administered 14 days (D) after the immunization at 1 mg/kg/day for 7 days by gavage. Mice were euthanized 21 days after myositis induction. Muscle strength was assessed by grip test at D 0, before the 1st PDN administration (D 14) and the day before sacrifice (D 20). To investigate whether the PDN effects are mediated by myofibre, we generated transgenic mice carrying two LoxP sites within the GR gene in muscle, expressing the tamoxifen-inducible Cre-ERT2 recombinase selectively in skeletal muscle fibre (HSA-CreERT2/GR L2/L2). Tamoxifen (1 mg/day for 5 days by IP injection) was administered 9 days after immunization to induce GR ablation selectively in skeletal muscle fibres (GR(i)skm-/- mice). Similar treatments were applied to GR L2/L2 that do not express Cre-ER(T2), and served as controls.We compared 4 groups of myositis mice, GR L2/L2 treated by PDN (n=9) or vehicle (n=9) and GR(i)skm-/- treated by PDN (n=10) or vehicle (n=10), by grip test and at the histological level (hematoxylin-eosin (HE) and Gomori trichrome (GT) staining). Moreover, LC3 expression was studied by RTqPCR and western blot.Results:Muscle strength was decreased in both GR L2/L2 and GR(i)skm-/- myositis mice from D 14 to D 20. GR L2/L2 myositis mice recovered muscle strength after PDN treatment; no significant difference compared to D 0 was detected. In contrast, PDN did not improve muscle strength in GR(i)skm-/- myositis mice (Figure 1).HE and GT staining did not reveal quantitative differences in inflammatory infiltrate. Necrotic and degenerative fibres were detected in the 4 groups. At RTqPCR, LC3, an autophagy marker, was upregulated in PDN-treated GR L2/L2 myositis mice compared to untreated GR L2/L2 myositis mice; moreover it was 2-fold more expressed in PDN-treated GR L2/L2 myositis mice compared to PDN-treated GR(i)skm-/- mice.Conclusion:GR in skeletal muscle fibre is crucial to mediate the therapeutic response to GC in a murine model of myositis. Autophagy is one of the candidate pathways controlled by myofibre GR underlying this effect.References:[1]Meyer A et al. IFN-β-induced reactive oxygen species and mitochondrial damage contribute to muscle impairment and inflammation maintenance in dermatomyositis. Acta Neuropathol. 2017 Oct;134(4):655-666.[2]Sugihara T et al. A new murine model to define the critical pathologic and therapeutic mediators of polymyositis. Arthritis Rheum. 2007 Apr;56(4):1304-14.Disclosure of Interests:None declared

Cytoskeleton-extracellular matrix interaction is required to maintain mitochondrial Ca2+ control in mouse skeletal muscle fibre

Cells rapidly lose their physiological phenotype upon isolation from their native microenvironment. Here, we investigated the role of the extracellular matrix (ECM) for mitochondrial morphology and Ca2+ handling in adult mouse skeletal muscle fibres. Adult skeletal muscle fibres were isolated from mouse toe muscle either by collagenase-induced dissociation of the ECM or by mechanical dissection that leaves the proximate ECM intact. Experiments were generally performed four hours after cell isolation. At this time, the expression of genes encoding for structural proteins was lower in enzymatically dissociated than in mechanically dissected fibres. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria in enzymatically dissociated than in mechanically dissected fibres. The increase in free cytosolic [Ca2+] during repeated tetanic stimulation was similar in the two groups of fibres, but this was accompanied by an excessive mitochondrial Ca2+ uptake only in enzymatically dissociated muscle fibres. The aberrant mitochondrial Ca2+ uptake was partially prevented by the mitochondrial Ca2+ uniporter inhibitor Ru360 and by cyclosporine A and NV556, which inhibit the mitochondrial matrix protein PPIF (also called cyclophilin D). Importantly, inhibition of PPIF with NV556 significantly improved survival of mice with mitochondrial myopathy in which muscle mitochondria take up excessive amounts of Ca2+ also with intact ECM. In conclusion, skeletal muscle fibres isolated by collagenase-induced dissociation of the ECM display aberrant mitochondrial Ca2+ uptake, which involves a PPIF-dependent mitochondrial Ca2+ influx resembling that observed in mitochondrial myopathies.