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BackgroundThe possible therapeutic benefits of using steroids to enhance muscle strength and slow disease progression in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) has been examined previously. In this investigation, it was hypothesised that steroid therapy is associated with morphological changes in the dystrophic muscle.Objectives and methodsTo test this hypothesis, two muscle biopsies were obtained (one biopsy before treatment, and the second 6 months following prednisone therapy) from 24 patients with dystrophies (18 DMD, 6 BMD). The participants were categorised into: control (6 specimens, normal muscle), untreated and treated groups. The muscle was evaluated for ultrastructural changes using transmission electron microscopy (TEM).ResultsIn the untreated group, the muscle fibres were degenerated and of variable sizes. The myofibrils were thin with either complete loss of bands and/or abnormal banding patterns. The Z-lines were irregularly spaced and loosely registered. The mitochondria of the myofibrils were small, few, spherical and irregularly distributed. Numerous dendritic cells (DCs) with euchromatic nuclei, and multiple and long dendrites, were seen among the myofibrils. The collagen fibres among the muscle fibres (endomysium) were numerous and large. The satellite cells had euchromatic nuclei with clumps of heterochromatin. In the treated group, the muscle fibres had a relatively uniform size with occasional fibres showing partial degeneration. The myofibrils had a relatively similar diameter comparable to that of normal muscle .The degenerated areas were small in size with occasional foci showing loss of banding pattern, and abnormal short bands with thick and hazy Z-lines. The mitochondria of the myofibrils were numerous, spherical, small in size and regularly arranged between the myofibrils. Few DCs, with heterochromatic nuclei, and few and short dendrites appeared between the myofibrils. The collagen fibres between the muscle fibres (endomysium) were numerous and large. As compared with the treated group, there was a statistically significant increase (p<0.05) in the numbers of DCs (0.7±0.2 vs 1.6±0.3) and fibroblasts (1.9±0.2 vs 2.9 ±0.3) in the untreated group. Alternatively, there was a statistically significant decrease (p<0.05) in the numbers of satellite cells (1.2±0.2 vs 0.6±0.1).ConclusionThe ability of steroids to induce ultrastructural features of improvement supports the notion that they have beneficial therapeutic role. The clinical ramifications of these observations mandate further studies.

The purpose of this study was to investigate the effect of concurrent strength and endurance training on strength, endurance, endocrine status and muscle fibre properties. A total of 45 male and female subjects were randomly assigned to one of four groups; strength training only (S), endurance training only (E), concurrent strength and endurance training (SE), or a control group (C). Groups S and E trained 3 days a week and the SE group trained 6 days a week for 12 weeks. Tests were made before and after 6 and 12 weeks of training. There was a similar increase in maximal oxygen consumption (VO2max) in both groups E and SE (P < 0.05). Leg press and knee extension one repetition maximum (1 RM) was increased in groups S and SE (P < 0.05) but the gains in knee extension 1 RM were greater for group S compared to all other groups (P < 0.05). Types I and II muscle fibre area increased after 6 and 12 weeks of strength training and after 12 weeks of combined training in type II fibres only (P < 0.05). Groups SE and E had an increase in succinate dehydrogenase activity and group E had a decrease in adenosine triphosphatase after 12 weeks of training (P < 0.05). A significant increase in capillary per fibre ratio was noted after 12 weeks of training in group SE. No changes were observed in testosterone, human growth hormone or sex hormone binding globulin concentrations for any group but there was a greater urinary cortisol concentration in the women of group SE and decrease in the men of group E after 12 weeks of training (P < 0.05). These findings would support the contention that combined strength and endurance training can suppress some of the adaptations to strength training and augment some aspects of capillarization in skeletal muscle.

Effects of previous strength training can be long-lived, even after prolonged subsequent inactivity, and retraining is facilitated by a previous training episode. Traditionally, such \"muscle memory\" has been attributed to neural factors in the absence of any identified local memory mechanism in the muscle tissue. We have used in vivo imaging techniques to study live myonuclei belonging to distinct muscle fibers and observe that new myonuclei are added before any major increase in size during overload. The old and newly acquired nuclei are retained during severe atrophy caused by subsequent denervation lasting for a considerable period of the animal's lifespan. The myonuclei seem to be protected from the high apoptotic activity found in inactive muscle tissue. A hypertrophy episode leading to a lasting elevated number of myonuclei retarded disuse atrophy, and the nuclei could serve as a cell biological substrate for such memory. Because the ability to create myonuclei is imparied in the elderly, individuals may benefit from strength training at an early age, and because anabolic steroids facilitate more myonuclei, nuclear permanency may also have implications for exclusion periods after a doping offense.

As sarcomeres produce the force necessary for contraction, assessment of sarcomere order is paramount in evaluation of cardiac and skeletal myocytes. The uniaxial force produced by sarcomeres is ideally perpendicular to their z-lines, which couple parallel myofibrils and give cardiac and skeletal myocytes their distinct striated appearance. Accordingly, sarcomere structure is often evaluated by staining for z-line proteins such as α-actinin. However, due to limitations of current analysis methods, which require manual or semi-manual handling of images, the mechanism by which sarcomere and by extension z-line architecture can impact contraction and which characteristics of z-line architecture should be used to assess striated myocytes has not been fully explored. Challenges such as isolating z-lines from regions of off-target staining that occur along immature stress fibers and cell boundaries and choosing metrics to summarize overall z-line architecture have gone largely unaddressed in previous work. While an expert can qualitatively appraise tissues, these challenges leave researchers without robust, repeatable tools to assess z-line architecture across different labs and experiments. Additionally, the criteria used by experts to evaluate sarcomeric architecture have not been well-defined. We address these challenges by providing metrics that summarize different aspects of z-line architecture that correspond to expert tissue quality assessment and demonstrate their efficacy through an examination of engineered tissues and single cells. In doing so, we have elucidated a mechanism by which highly elongated cardiomyocytes become inefficient at producing force. Unlike previous manual or semi-manual methods, characterization of z-line architecture using the metrics discussed and implemented in this work can quantitatively evaluate engineered tissues and contribute to a robust understanding of the development and mechanics of striated muscles.

In contrast to processive molecular motors, skeletal myosins form a large motor ensemble for contraction of muscles against high loads. Despite numerous information on the molecular properties of skeletal myosin, its ensemble effects on collective force generation have not been rigorously clarified. Here we show 4 nm stepwise actin displacements generated by synthetic myofilaments beyond a load of 30 pN, implying that steps cannot be driven exclusively by single myosins, but potentially by coordinated force generations among multiple myosins. The simulation model shows that stepwise actin displacements are primarily caused by coordinated force generation among myosin molecules. Moreover, the probability of coordinated force generation can be enhanced against high loads by utilizing three factors: strain-dependent kinetics between force-generating states; multiple power stroke steps; and high ATP concentrations. Compared with other molecular motors, our findings reveal how the properties of skeletal myosin are tuned to perform cooperative force generation for efficient muscle contraction. Skeletal muscle myosin forms large ensembles to generate force against high loads. Using optical tweezers and simulation Kaya et al . provide experimental evidence for cooperative force generation, and describe how the molecular properties of skeletal myosins are tuned for coordinated power strokes.

To engineer tissue-like structures, cells must organize themselves into three-dimensional (3D) networks that mimic the native tissue microarchitecture. Microfabricated hydrogel substrates provide a potentially useful platform for directing cells into biomimetic tissue architecture in vitro . Here, we present microgrooved methacrylated gelatin hydrogels as a suitable platform to build muscle-like fibrous structures in a facile and highly reproducible fashion. Microgrooved hydrogel substrates with two different ridge sizes (50 and 100 μm) were fabricated to assess the effect of the distance between engineered myofibers on the orientation of the bridging C2C12 myoblasts and the formation of the resulting multinucleated myotubes. It was shown that although the ridge size did not significantly affect the C2C12 myoblast alignment, the wider-ridged micropatterned hydrogels generated more myotubes that were not aligned to the groove direction as compared to those on the smaller-ridge micropatterns. We also demonstrated that electrical stimulation improved the myoblast alignment and increased the diameter of the resulting myotubes. By using the microstructured methacrylated gelatin substrates, we built free-standing 3D muscle sheets, which contracted when electrically stimulated. Given their robust contractility and biomimetic microarchitecture, engineered tissues may find use in tissue engineering, biological studies, high-throughput drug screening, and biorobotics.

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber’s periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber’s maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca 2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis.

Megaconial Congenital Muscular Dystrophy (CMD) is a rare autosomal recessive disorder characterized by enlarged mitochondria located mainly at the periphery of muscle fibers and caused by mutations in the Choline Kinase Beta ( CHKB ) gene. Although the pathogenesis of this disease is not well understood, there is accumulating evidence for the presence of mitochondrial dysfunction. In this study, we aimed to investigate whether imbalanced mitochondrial dynamics affects mitochondrial function and bioenergetic efficiency in skeletal muscle cells of Megaconial CMD. Immunofluorescence, confocal and transmission electron microscopy studies revealed impaired mitochondrial network, morphology, and localization in primary skeletal muscle cells of Megaconial CMD. The organelle disruption was specific only to skeletal muscle cells grown in culture. The expression levels of mitochondrial fission proteins (DRP1, MFF, FIS1) were found to be decreased significantly in both primary skeletal muscle cells and tissue sections of Megaconial CMD by Western blotting and/or immunofluorescence analysis. The metabolomic and fluxomic analysis, which were performed in Megaconial CMD for the first time, revealed decreased levels of phosphonucleotides, Krebs cycle intermediates, ATP, and altered energy metabolism pathways. Our results indicate that reduced mitochondrial fission and altered mitochondrial energy metabolism contribute to mitochondrial dysmorphology and dysfunction in the pathogenesis of Megaconial CMD.

Volumetric muscle loss (VML) is a traumatic and functionally debilitating muscle injury with limited treatment options. Developmental regenerative therapies for the repair of VML typically comprise an ECM scaffold. In this study, we tested if the complete reliance on host cell migration to a devitalized muscle scaffold without myogenic cells is sufficient for de novo muscle fiber regeneration. Devitalized (muscle ECM with no living cells) and, as a positive control, vital minced muscle grafts were transplanted to a VML defect in the tibialis anterior muscle of Lewis rats. Eight weeks post-injury, devitalized grafts did not appreciably promote de novo muscle fiber regeneration within the defect area, and instead remodeled into a fibrotic tissue mass. In contrast, transplantation of vital minced muscle grafts promoted de novo muscle fiber regeneration. Notably, pax7+ cells were absent in remote regions of the defect site repaired with devitalized scaffolds. At 2 weeks post-injury, the devitalized grafts were unable to promote an anti-inflammatory phenotype, while vital grafts appeared to progress to a pro-regenerative inflammatory response. The putative macrophage phenotypes observed in vivo were supported in vitro, in which soluble factors released from vital grafts promoted an M2-like macrophage polarization, whereas devitalized grafts failed to do so. These observations indicate that although the remaining muscle mass serves as a source of myogenic cells in close proximity to the defect site, a devitalized scaffold without myogenic cells is inadequate to appreciably promote de novo muscle fiber regeneration throughout the VML defect.

The Z-disc, appearing as a fine dense line forming sarcomere boundaries in striated muscles, when studied in detail reveals crosslinked filament arrays that transmit tension and house myriads of proteins with diverse functions. At the Z-disc the barbed ends of the antiparallel actin filaments from adjoining sarcomeres interdigitate and are crosslinked primarily by layers of α-actinin. The Z-disc is therefore the site of polarity reversal of the actin filaments, as needed to interact with the bipolar myosin filaments in successive sarcomeres. The layers of α-actinin determine the Z-disc width: fast fibres have narrow (~30-50 nm) Z-discs and slow and cardiac fibres have wide (~100 nm) Z-discs. Comprehensive reviews on the roles of the numerous proteins located at the Z-disc in signalling and disease have been published; the aim here is different, namely to review the advances in structural aspects of the Z-disc.