The endothelial Dll4-muscular Notch2 axis regulates skeletal muscle mass.

Adult skeletal muscle is a highly plastic tissue that readily reduces or gains its mass in response to mechanical and metabolic stimulation; however, the upstream mechanisms that control muscle mass remain unclear. Notch signalling is highly conserved, and regulates many cellular events, including proliferation and differentiation of various types of tissue stem cell via cell-cell contact. Here we reveal that multinucleated myofibres express Notch2, which plays a crucial role in disuse- or diabetes-induced muscle atrophy. Mechanistically, in both atrophic conditions, the microvascular endothelium upregulates and releases the Notch ligand, Dll4, which then activates muscular Notch2 without direct cell-cell contact. Inhibition of the Dll4-Notch2 axis substantively prevents these muscle atrophy and promotes mechanical overloading-induced muscle hypertrophy in mice. Our results illuminate a tissue-specific function of the endothelium in controlling tissue plasticity and highlight the endothelial Dll4-muscular Notch2 axis as a central upstream mechanism that regulates catabolic signals from mechanical and metabolic stimulation, providing a therapeutic target for muscle-wasting diseases.

Hoxa10 mediates positional memory to govern stem cell function in adult skeletal muscle.

Muscle stem cells (satellite cells) are distributed throughout the body and have heterogeneous properties among muscles. However, functional topographical genes in satellite cells of adult muscle remain unidentified. Here, we show that expression of Homeobox-A (Hox-A) cluster genes accompanied with DNA hypermethylation of the Hox-A locus was robustly maintained in both somite-derived muscles and their associated satellite cells in adult mice, which recapitulates their embryonic origin. Somite-derived satellite cells were clearly separated from cells derived from cranial mesoderm in Hoxa10 expression. Hoxa10 inactivation led to genomic instability and mitotic catastrophe in somite-derived satellite cells in mice and human. Satellite cell-specific Hoxa10 ablation in mice resulted in a decline in the regenerative ability of somite-derived muscles, which were unobserved in cranial mesoderm-derived muscles. Thus, our results show that Hox gene expression profiles instill the embryonic history in satellite cells as positional memory, potentially modulating region-specific pathophysiology in adult muscles.

Muscle fibre distribution in forelimb, hindlimb and trunk muscles in three bat species: The little Japanese horseshoe, greater horseshoe and Egyptian fruit.

This study characterised muscle fibres in trunk, forelimb and hindlimb muscles of three bat species: little Japanese horseshoe (Rhinolophus cornutus), greater horseshoe (Rhinolophus ferrumequinum) and Egyptian fruit (Rousettus aegyptiacus). Twenty-seven muscles from trunk, forelimb and hindlimb were dissected, weighed and analysed by immunohistochemistry and sodium didecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and determined their cross-sectional areas (CSA). Results showed that Type IIa and Type IIa/x made the highest proportion of total muscle mass. Moderate proportion was formed by Type IIb. Type I and IIx appeared at very low levels in all bats. Type IIb was the only fibre type detected in patagial muscles in wing membrane of greater horseshoe while other fibre types were not observed. Type I muscle fibres were very few and appeared infrequently in fifteen muscles of Egyptian fruit and in only one muscle in each, greater horseshoe and little Japanese horseshoe. Type IIx was also detected in three muscles in greater horseshoe and only one muscle in Egyptian fruit but none in little Japanese horseshoe. The highest average CSA µm2 was detected in Type IIb and values were 734.2µm2 for LHB; 1537.9µm2 for GHB and 1,720.9µm2 for EFB. Lowest and undetermined values were observed for Type I and IIx. These data demonstrate that Type IIa, IIa/x and IIb form significant proportion of adult bat muscle mass and Type IIb is the largest fibre type. The distribution pattern is suggestive of specialised functions of the fibres in relation to orientation and speed of bats during flight.

Characteristics of Muscle Fiber-Type Distribution in Moles.

Moles are a strictly fossorial Soricomorpha species and possess a suite of specialized adaptations to subterranean life. However, the contractile function of skeletal muscles in moles remains unclear. We compared muscle fiber-type distribution in two mole species (the large Japanese mole and lesser Japanese mole) with that in four other Soricomorpha species that are semi-fossorial, terrestrial, or semi-aquatic (the Japanese shrew-mole, house shrew, Japanese white-toothed shrew, and Japanese water shrew). For a single species, the fiber-type distribution in up to 38 muscles was assessed using immunohistochemical staining and/or gel electrophoresis. We found that slow and fatigue-resistant Type I fibers were absent in almost all muscles of all species studied. Although, the two methods of determining the fiber type did not give identical results, they both revealed that fast Type IIb fibers were absent in mole muscles. The fiber-type distribution was similar among different anatomical regions in the moles. This study demonstrated that the skeletal muscles of moles have a homogenous fiber-type distribution compared with that in Soricomorpha species that are not strictly fossorial. Mole muscles are composed of Type IIa fibers alone or a combination of Type IIa and relatively fast Type IIx fibers. The homogenous fiber-type distribution in mole muscles may be an adaptation to structurally simple subterranean environments, where there is no need to support body weight with the limbs, or to move at high speeds to pursue prey or to escape from predators. Anat Rec, 302:1010-1023, 2019. © 2018 Wiley Periodicals, Inc.

Preconditioning Contractions Suppress Muscle Pain Markers after Damaging Eccentric Contractions.

Inexperienced vigorous exercise, including eccentric contraction (ECC), causes muscle pain and damage. Similar prior light exercise suppresses the development of muscle pain (repeated-bout effect), but the molecular mechanisms behind this are not sufficiently understood. In this study, the influence of a nondamaging preconditioning ECC load (Precon) on muscle pain-related molecules and satellite cell-activating factors was investigated at the mRNA expression level. Nine-week-old male Wistar rats (n=36) were divided into 2 groups: a group receiving only a damaging ECC (100 contractions) load (non-Precon) and a group receiving a nondamaging ECC (10 contractions) load 2 days before receiving the damaging ECC load (Precon). ECC was loaded on the left leg, and the right leg was regarded as the intact control (CTL). The medial head of the gastrocnemius muscle from all rats was excised 2 or 4 days after the damaging ECC loading, and the relative mRNA expression levels of muscle pain- and satellite cell-related molecules were quantitated using real-time RT PCR. Precon suppressed increases in MHC-embryonic and MHC-neonatal mRNA expressions. Enhancement of HGF, Pax7, MyoD, and myogenin mRNA expression was also suppressed, suggesting that Precon decreased the degree of muscle damage and no muscle regeneration or satellite cell activation occurred. Similarly, increases in mRNA expression of muscle pain-related molecules (BKB2 receptor, COX-2, and mPGEC-1) were also suppressed. This study clearly demonstrated that at the mRNA level, prior light ECC suppressed muscle damage induced by later damaging ECC and promoted recovery from muscle pain.

Influence of hypoxic stimulation on angiogenesis and satellite cells in mouse skeletal muscle.

We clarified in our previous study that hypoxic training promotes angiogenesis in skeletal muscle, but the mechanism of angiogenesis in skeletal muscle remains unknown. In this study, we investigated the influence of differences in hypoxia exposure on angiogenesis in skeletal muscles at differing ages and metabolic characteristics at which the production of reactive oxygen species and nitric oxide may differ. Ten-week-old (young) and 20-month-old (old) mice were separated into control (N), continuous hypoxia (H), and intermittent hypoxia (IH) groups. The H group was exposed to 16% O2 hypoxia for 5 days and the IH group was exposed to 16% O2 hypoxia at one-hour intervals during the light period for 5 days. After completion of hypoxia exposure, the soleus and gastrocnemius muscles were immediately excised, and mRNA expression of angiogenesis- and satellite cell-related genes was investigated using real-time RT-PCR. In addition, muscle fiber type composition, muscle fiber area, number of satellite cells, and capillary density were measured immunohistochemically. In the young soleus muscle, the muscle fiber area was decreased in the H group, and mRNA expression of satellite cell activation-related MyoD, MHCe, and BDNF was significantly increased. On the other hand, in the old soleus muscle, nNOS and VEGF-A mRNA expression, and the capillary density were significantly increased in the H group. In the superficial portion of the gastrocnemius, mRNA expression of FGF2, an angiogenic factor secreted by satellite cells, was significantly increased in the young IH group. In addition, a positive correlation between VEGF-A mRNA expression and nNOS mRNA expression in the soleus muscle and eNOS mRNA expression in the superficial portion of the gastrocnemius was noted. These data demonstrated that age, hypoxia exposure method and muscle metabolic characteristics are related, which results in significant differences in angiogenesis.

Effect of High-Intensity Training in Normobaric Hypoxia on Thoroughbred Skeletal Muscle.

Hypoxic training is believed to increase endurance capacity in association with hypoxia inducible factor-1α (HIF-1α), a modulator of vascular endothelial growth factor-A (VEGF-A), and to influence activation of satellite cells (SCs). However, the effect of hypoxic training on SC activation and its relation to angiogenesis has not been thoroughly investigated. Eight Thoroughbred horses were subjected to normoxic (FIO2 = 21%) or hypoxic (FIO2 = 15%) training for 3 days/week (100% [Formula: see text]) for 4 weeks. Incremental exercise tests (IET) were conducted on a treadmill under normoxia and the maximal oxygen consumption ([Formula: see text]) and running distance were measured before and after each training session. Muscle biopsy samples were obtained from the gluteus medius muscle at 6 scheduled times before, during, and one week after IET for immunohistochemical analysis and real-time RT-PCR analysis. Running distance and [Formula: see text], measured during IET, increased significantly after hypoxic training compared with normoxic training. Capillary density and mRNA expression related to SC activation (e.g., myogenin and hepatocyte growth factor) and angiogenesis (VEGF-A) increased only after hypoxic training. These results suggest that increases in mRNA expression after training enhance and prolong SC activation and angiogenesis and that nitric oxide plays an important role in these hypoxia-induced training effects.

Characteristics of Skeletal Muscle Fibers of SOD1 Knockout Mice.

Cu/Zn superoxide dismutase (SOD1) knockout (KO) mice are known as an aging model in some aspects, but the damage and regeneration process of each fiber type have not been sufficiently studied. In this study, we investigated the damage and satellite cell state of the gastrocnemius muscle in SOD1 KO mice (6 months old) using immunohistochemical staining and real-time RT-PCR. The proportion of central nuclei-containing Type IIx/b fibers in the deep and superficial portions of the gastrocnemius muscle was significantly higher in SOD1 KO than control mice. The number of satellite cells per muscle fiber decreased in all muscle fiber types in the deep portion of the gastrocnemius muscle in SOD1 KO mice. In addition, the mRNA expression levels of Pax7 and myogenin, which are expressed in satellite cells in the activation, proliferation, and differentiation states, significantly increased in the gastrocnemius muscle of SOD1 KO mice. Furthermore, mRNA of myosin heavy chain-embryonic, which is expressed in the early phase of muscle regeneration, significantly increased in SOD1 KO mice. It was suggested that muscle is damaged by reactive oxygen species produced in the mitochondrial intermembrane space in Type IIxb fibers, accelerating the proliferation and differentiation of satellite cells through growth factors in SOD1 KO mice.