Heparan Sulfate Chain-Conjugated Laminin-E8 Fragments Advance Paraxial Mesodermal Differentiation Followed by High Myogenic Induction from hiPSCs.

Human-induced pluripotent stem cells (hiPSCs) have great therapeutic potential. The cell source differentiated from hiPSCs requires xeno-free and robust methods for lineage-specific differentiation. Here, a system is described for differentiating hiPSCs on new generation laminin fragments (NGLFs), a recombinant form of a laminin E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. Using NGLFs, hiPSCs are highly promoted to direct differentiation into a paraxial mesoderm state with high-efficiency muscle lineage generation. HS conjugation to the C-terminus of Laminin E8 fragments brings fibroblast growth factors (FGFs) bound to the HS close to the cell surface of hiPSCs, thereby facilitating stronger FGF signaling pathways stimulation and initiating HOX gene expression, which triggers the paraxial mesoderm differentiation of hiPSCs. This highly efficient differentiation system can provide a roadmap for paraxial mesoderm development and an infinite source of myocytes and muscle stem cells for disease modeling and regenerative medicine.

Regeneration of neuromuscular synapses after acute and chronic denervation by inhibiting the gerozyme 15-prostaglandin dehydrogenase.

To date, there are no approved treatments for the diminished strength and paralysis that result from the loss of peripheral nerve function due to trauma, heritable neuromuscular diseases, or aging. Here, we showed that denervation resulting from transection of the sciatic nerve triggered a marked increase in the prostaglandin-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in skeletal muscle in mice, providing evidence that injury drives early expression of this aging-associated enzyme or gerozyme. Treating mice with a small-molecule inhibitor of 15-PGDH promoted regeneration of motor axons and formation of neuromuscular synapses leading to an acceleration in recovery of force after an acute nerve crush injury. In aged mice with chronic denervation of muscles, treatment with the 15-PGDH inhibitor increased motor neuron viability and restored neuromuscular junctions and function. These presynaptic changes synergized with previously reported muscle tissue remodeling to result in a marked increase in the strength of aged muscles. We further found that 15-PGDH aggregates defined the target fibers that are histopathologic hallmarks of human neurogenic myopathies, suggesting that the gerozyme may be involved in their etiology. Our data suggest that inhibition of 15-PGDH may constitute a therapeutic strategy to physiologically boost prostaglandin E2, restore neuromuscular connectivity, and promote recovery of strength after acute or chronic denervation due to injury, disease, or aging.

Physical Fitness Tests as Predictors of High-Intensity Running Performance in Rugby.

Understanding the physical fitness elements that influence high-intensity running ability during rugby matches is crucial for optimizing player performance and developing effective training strategies. In this study, we aimed to investigate the relationships between various physical fitness components and high-intensity running ability in rugby. For this purpose, 60 Japanese university rugby players were randomized into four groups and two matches were played. The participants were monitored in two matches, and their running abilities were assessed using GPS sensors. The running time was divided into three running velocity categories: distance run at ≤5.4 km/h (low-intensity running); distance run at 5.5~17.9 km/h (medium-intensity running), and distance run at ≥18.0 km/h ≤(high-intensity running) and backs and forwards were evaluated separately. To determine which physical fitness test is more predictive of performance, we decided to correlate several physical test performances with the running time intensities during the matches. Independently of the position, the high-intensity running time correlated with the repeated sprint ability (RSA) and the 40 m sprint speed. The results suggest that RSA measured in the field is the most important high-intensity running ability predictor during a match for both positions.

Evaluation of hiPSC-Derived Muscle Progenitor Cell Transplantation in a Mouse Duchenne Muscular Dystrophy Model.

For cell therapy toward Duchenne muscle dystrophy (DMD), muscle progenitor cells derived from human-induced pluripotent stem cell (hiPSC-MuPCs) are recognized as a good candidate, and currently, cell transplantation of hiPSC-MuPCs is being tested with several DMD animal models. In this article, we describe an efficient method to dissociate, purify by cell sorting, transplant, and evaluate the transplantation efficacy of hiPSC-MuPCs.

Single-cell RNA-seq reveals heterogeneity in hiPSC-derived muscle progenitors and E2F family as a key regulator of proliferation.

Human pluripotent stem cell-derived muscle progenitor cells (hiPSC-MuPCs) resemble fetal-stage muscle progenitor cells and possess in vivo regeneration capacity. However, the heterogeneity of hiPSC-MuPCs is unknown, which could impact the regenerative potential of these cells. Here, we established an hiPSC-MuPC atlas by performing single-cell RNA sequencing of hiPSC-MuPC cultures. Bioinformatic analysis revealed four cell clusters for hiPSC-MuPCs: myocytes, committed, cycling, and noncycling progenitors Using FGFR4 as a marker for noncycling progenitors and cycling cells and CD36 as a marker for committed and myocyte cells, we found that FGFR4+ cells possess a higher regenerative capacity than CD36+ cells. We also identified the family of E2F transcription factors are key regulators of hiPSC-MuPC proliferation. Our study provides insights on the purification of hiPSC-MuPCs with higher regenerative potential and increases the understanding of the transcriptional regulation of hiPSC-MuPCs.

Characterization of hiPSC-Derived Muscle Progenitors Reveals Distinctive Markers for Myogenic Cell Purification Toward Cell Therapy.

The transplantation of muscle progenitor cells (MuPCs) differentiated from human induced pluripotent stem cells (hiPSCs) is a promising approach for treating skeletal muscle diseases such as Duchenne muscular dystrophy (DMD). However, proper purification of the MuPCs before transplantation is essential for clinical application. Here, by using MYF5 hiPSC reporter lines, we identified two markers for myogenic cell purification: CDH13, which purified most of the myogenic cells, and FGFR4, which purified a subset of MuPCs. Cells purified with each of the markers showed high efficiency for regeneration after transplantation and contributed to the restoration of dystrophin expression in DMD-immunodeficient model mice. Moreover, we found that MYF5 regulates CDH13 expression by binding to the promoter regions. These findings suggest that FGFR4 and CDH13 are strong candidates for the purification of hiPSC-derived MuPCs for therapeutical application.

Corrigendum: Lactate Metabolism and Satellite Cell Fate.

[This corrects the article DOI: 10.3389/fphys.2020.610983.].

Lactate Metabolism and Satellite Cell Fate.

Lactate is one of the metabolic products of glycolysis. It is widely accepted as an important energy source for many cell types and more recently has been proposed to actively participate in cell-cell communication. Satellite cells (SCs), which are adult skeletal muscle stem cells, are the main players of the skeletal muscle regeneration process. Recent studies have proposed a metabolic switch to increase glycolysis in activated SCs. Moreover, lactate has been shown to affect SCs and myoblasts in vivo and in vitro. In this short review, we describe how metabolic variations relate with SC fate (quiescence, activation, proliferation, migration, differentiation, fusion, and self-renewal), as well as discuss possible relationships between lactate as a metabolite and as a signaling molecule affecting SC fate.

Induced Fetal Human Muscle Stem Cells with High Therapeutic Potential in a Mouse Muscular Dystrophy Model.

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle-wasting disease caused by DYSTROPHIN deficiency. Cell therapy using muscle stem cells (MuSCs) is a potential cure. Here, we report a differentiation method to generate fetal MuSCs from human induced pluripotent stem cells (iPSCs) by monitoring MYF5 expression. Gene expression profiling indicated that MYF5-positive cells in the late stage of differentiation have fetal MuSC characteristics, while MYF5-positive cells in the early stage of differentiation have early myogenic progenitor characteristics. Moreover, late-stage MYF5-positive cells demonstrated good muscle regeneration potential and produced DYSTROPHIN in vivo after transplantation into DMD model mice, resulting in muscle function recovery. The engrafted cells also generated PAX7-positive MuSC-like cells under the basal lamina of DYSTROPHIN-positive fibers. These findings suggest that MYF5-positive fetal MuSCs induced in the late stage of iPSC differentiation have cell therapy potential for DMD.

N-acetyl-L-cysteine Prevents Lactate-Mediated PGC1-alpha Expression in C2C12 Myotubes.

BACKGROUND: Exercise induces many physiological adaptations. Recently, it has been proposed that some of these adaptations are induced by exercise-mediated lactate production. In this study, we aimed to investigate in vitro the effect of lactate in cultured myotubes and whether antioxidants could inhibit the effect. METHODS: Differentiated myotubes were cultured at different concentrations of L-lactate (0, 10, 30, 50 mM) in the absence or presence of an antioxidant, N-acetyl-L-cysteine (Nac). The temporal effect of lactate exposure in myotubes was also explored. RESULTS: Two hours of exposure to 50 mM L-lactate and six hours of exposure to 30 or 50 mM L-lactate caused a significant increase in PGC1-alpha (peroxisome proliferator-activated receptor γ coactivator-1α) expression in the myotubes. This up-regulation was suppressed by 2 mM Nac. Intermittent and continuous lactate exposure caused similar PGC1-alpha up-regulation. These results suggest that the increase in PGC1-alpha expression is mediated by reactive oxygen species (ROS) production from lactate metabolism and that both continuous and intermittent exposure to L-lactate can cause the up-regulation.

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization.

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different exercise modalities. During some exercise modalities (e.g., high-intensity interval exercise), blood metabolites and hormonal levels change in short periods of time. In the present study, we describe a method to catheterize the antecubital vein, which allows the collection of several blood samples during exercise. Insulin and venous lactate concentrations were measured during high-intensity exercise by the application of the described method. The exercise consisted of three 30 s bouts of high-intensity exercise separated by 4 min of recovery. After the last recovery period, a Wingate test was performed. Blood samples from the antecubital vein were obtained before and after each 30 s bout and before and after the Wingate test. As a result, it was possible to evaluate the plasma insulin and venous blood lactate variations during the exercise.

How Different Respiratory Rate Patterns affect Cardiorespiratory Variables and Performance.

This study aims to elucidate how respiratory rate (RR) patterns may affect respiratory gas exchange variables and performance during incremental intensity- exercise. 10 healthy young men (mean ± SD, age: 20.7 ± 0.5 years, height: 174.3 ± 5.7 cm, and body mass: 72.6 ± 10.4 kg) performed three incremental tests on a cycle ergometer at three different RR (60, 45 and 30 breaths per min) in each trial. During the tests, tidal volume (TV), minute ventilation (VE), fractional content of oxygen (FeO2), fractional content of carbon dioxide (FeCO2), oxygen uptake (VO2), expiratory carbon dioxide (VCO2), equivalent of oxygen (EqO2), VE/VCO2, and respiratory exchange ratio (RER) were determinate breath-by-breath. Additionally, exercise time (as a performance marker) was measured. Statistical analyses for the results were carried out to determine significant differences between the three trials. VCO2, VO2, and exercise time did not show statistical differences in the three trials. Therefore, we concluded that RR affects some respiratory gas exchange variables but does not influence the VO2max and endurance performance.

Lactate as a Signaling Molecule That Regulates Exercise-Induced Adaptations.

Lactate (or its protonated form: lactic acid) has been studied by many exercise scientists. The lactate paradigm has been in constant change since lactate was first discovered in 1780. For many years, it was unfairly seen as primarily responsible for muscular fatigue during exercise and a waste product of glycolysis. The status of lactate has slowly changed to an energy source, and in the last two decades new evidence suggests that lactate may play a much bigger role than was previously believed: many adaptations to exercise may be mediated in some way by lactate. The mechanisms behind these adaptations are yet to be understood. The aim of this review is to present the state of lactate science, focusing on how this molecule may mediate exercise-induced adaptations.