Tissue Tregs.

The immune system is responsible for defending an organism against the myriad of microbial invaders it constantly confronts. It has become increasingly clear that the immune system has a second major function: the maintenance of organismal homeostasis. Foxp3(+)CD4(+) regulatory T cells (Tregs) are important contributors to both of these critical activities, defense being the primary purview of Tregs circulating through lymphoid organs, and homeostasis ensured mainly by their counterparts residing in parenchymal tissues. This review focuses on so-called tissue Tregs. We first survey existing information on the phenotype, function, sustaining factors, and human equivalents of the three best-characterized tissue-Treg populations-those operating in visceral adipose tissue, skeletal muscle, and the colonic lamina propria. We then attempt to distill general principles from this body of work-as concerns the provenance, local adaptation, molecular sustenance, and targets of action of tissue Tregs, in particular.

Impairment of an Endothelial NAD+-H2S Signaling Network Is a Reversible Cause of Vascular Aging.

A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-SIRT7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ booster nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.

Ciliary Hedgehog Signaling Restricts Injury-Induced Adipogenesis.

Injured skeletal muscle regenerates, but with age or in muscular dystrophies, muscle is replaced by fat. Upon injury, muscle-resident fibro/adipogenic progenitors (FAPs) proliferated and gave rise to adipocytes. These FAPs dynamically produced primary cilia, structures that transduce intercellular cues such as Hedgehog (Hh) signals. Genetically removing cilia from FAPs inhibited intramuscular adipogenesis, both after injury and in a mouse model of Duchenne muscular dystrophy. Blocking FAP ciliation also enhanced myofiber regeneration after injury and reduced myofiber size decline in the muscular dystrophy model. Hh signaling through FAP cilia regulated the expression of TIMP3, a secreted metalloproteinase inhibitor, that inhibited MMP14 to block adipogenesis. A pharmacological mimetic of TIMP3 blocked the conversion of FAPs into adipocytes, pointing to a strategy to combat fatty degeneration of skeletal muscle. We conclude that ciliary Hh signaling by FAPs orchestrates the regenerative response to skeletal muscle injury.

PGC-1α senses the CBC of pre-mRNA to dictate the fate of promoter-proximally paused RNAPII.

PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes. We show that PGC-1α promotes pausing release in a two-arm mechanism (1) by recruiting the positive transcription elongation factor b (P-TEFb) and (2) by outcompeting the premature transcription termination complex Integrator. Using mice homozygous for five amino acid changes in the CBP80-binding motif (CBM) of PGC-1α that destroy CBM function, we show that efficient differentiation of primary myoblasts to myofibers and timely skeletal muscle regeneration after injury require PGC-1α binding to CBP80. Our findings reveal how PGC-1α activates stress-response gene transcription in a previously unanticipated pre-mRNA quality-control pathway.

The molecular athlete: exercise physiology from mechanisms to medals.

Human skeletal muscle demonstrates remarkable plasticity, adapting to numerous external stimuli including the habitual level of contractile loading. Accordingly, muscle function and exercise capacity encompass a broad spectrum, from inactive individuals with low levels of endurance and strength to elite athletes who produce prodigious performances underpinned by pleiotropic training-induced muscular adaptations. Our current understanding of the signal integration, interpretation, and output coordination of the cellular and molecular mechanisms that govern muscle plasticity across this continuum is incomplete. As such, training methods and their application to elite athletes largely rely on a "trial-and-error" approach, with the experience and practices of successful coaches and athletes often providing the bases for "post hoc" scientific enquiry and research. This review provides a synopsis of the morphological and functional changes along with the molecular mechanisms underlying exercise adaptation to endurance- and resistance-based training. These traits are placed in the context of innate genetic and interindividual differences in exercise capacity and performance, with special consideration given to aging athletes. Collectively, we provide a comprehensive overview of skeletal muscle plasticity in response to different modes of exercise and how such adaptations translate from "molecules to medals."

Mechanisms of mechanical overload-induced skeletal muscle hypertrophy: current understanding and future directions.

Mechanisms underlying mechanical overload-induced skeletal muscle hypertrophy have been extensively researched since the landmark report by Morpurgo (1897) of "work-induced hypertrophy" in dogs that were treadmill trained. Much of the preclinical rodent and human resistance training research to date supports that involved mechanisms include enhanced mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, an expansion in translational capacity through ribosome biogenesis, increased satellite cell abundance and myonuclear accretion, and postexercise elevations in muscle protein synthesis rates. However, several lines of past and emerging evidence suggest that additional mechanisms that feed into or are independent of these processes are also involved. This review first provides a historical account of how mechanistic research into skeletal muscle hypertrophy has progressed. A comprehensive list of mechanisms associated with skeletal muscle hypertrophy is then outlined, and areas of disagreement involving these mechanisms are presented. Finally, future research directions involving many of the discussed mechanisms are proposed.

Regulating Striated Muscle Contraction: Through Thick and Thin.

Force generation in striated muscle is primarily controlled by structural changes in the actin-containing thin filaments triggered by an increase in intracellular calcium concentration. However, recent studies have elucidated a new class of regulatory mechanisms, based on the myosin-containing thick filament, that control the strength and speed of contraction by modulating the availability of myosin motors for the interaction with actin. This review summarizes the mechanisms of thin and thick filament activation that regulate the contractility of skeletal and cardiac muscle. A novel dual-filament paradigm of muscle regulation is emerging, in which the dynamics of force generation depends on the coordinated activation of thin and thick filaments. We highlight the interfilament signaling pathways based on titin and myosin-binding protein-C that couple thin and thick filament regulatory mechanisms. This dual-filament regulation mediates the length-dependent activation of cardiac muscle that underlies the control of the cardiac output in each heartbeat.

Muscle-derived Dpp regulates feeding initiation via endocrine modulation of brain dopamine biosynthesis.

In animals, the brain regulates feeding behavior in response to local energy demands of peripheral tissues, which secrete orexigenic and anorexigenic hormones. Although skeletal muscle is a key peripheral tissue, it remains unknown whether muscle-secreted hormones regulate feeding. In Drosophila, we found that decapentaplegic (dpp), the homolog of human bone morphogenetic proteins BMP2 and BMP4, is a muscle-secreted factor (a myokine) that is induced by nutrient sensing and that circulates and signals to the brain. Muscle-restricted dpp RNAi promotes foraging and feeding initiation, whereas dpp overexpression reduces it. This regulation of feeding by muscle-derived Dpp stems from modulation of brain tyrosine hydroxylase (TH) expression and dopamine biosynthesis. Consistently, Dpp receptor signaling in dopaminergic neurons regulates TH expression and feeding initiation via the downstream transcriptional repressor Schnurri. Moreover, pharmacologic modulation of TH activity rescues the changes in feeding initiation due to modulation of dpp expression in muscle. These findings indicate that muscle-to-brain endocrine signaling mediated by the myokine Dpp regulates feeding behavior.

Discordant skeletal muscle gene and protein responses to exercise.

The ability of skeletal muscle to adapt to repeated contractile stimuli is one of the most intriguing aspects of physiology. The molecular bases underpinning these adaptations involve increased protein activity and/or expression, mediated by an array of pre- and post-transcriptional processes, as well as translational and post-translational control. A longstanding dogma assumes a direct relationship between exercise-induced increases in mRNA levels and subsequent changes in the abundance of the proteins they encode. Drawing on the results of recent studies, we dissect and question the common assumption of a direct relationship between changes in the skeletal muscle transcriptome and proteome induced by repeated muscle contractions (e.g., exercise).

A multi-epigenomic map of endurance exercise training.

The Molecular Transducers of Physical Activity Consortium (MoTrPAC) aims to comprehensively map molecular alterations in response to acute exercise and chronic training. In one of a recent series of papers from MoTrPAC, Nair et al. provide the first multi-epigenomic and transcriptomic integration across eight tissues in both sexes following adaptation to endurance exercise training (EET).

High-Dimensional Single-Cell Cartography Reveals Novel Skeletal Muscle-Resident Cell Populations.

Adult tissue repair and regeneration require stem-progenitor cells that can self-renew and generate differentiated progeny. Skeletal muscle regenerative capacity relies on muscle satellite cells (MuSCs) and their interplay with different cell types within the niche. However, our understanding of skeletal muscle tissue cellular composition is limited. Here, using a combined approach of single-cell RNA sequencing and mass cytometry, we precisely mapped 10 different mononuclear cell types in adult mouse muscle. We also characterized gene signatures and determined key discriminating markers of each cell type. We identified two previously understudied cell populations in the interstitial compartment. One expresses the transcription factor scleraxis and generated tenocytes in vitro. The second expresses markers of smooth muscle and mesenchymal cells (SMMCs) and, while distinct from MuSCs, exhibited myogenic potential and promoted MuSC engraftment following transplantation. The blueprint presented here yields crucial insights into muscle-resident cell-type identities and can be exploited to study muscle diseases.

Neuronal innervation regulates the secretion of neurotrophic myokines and exosomes from skeletal muscle.

Myokines and exosomes, originating from skeletal muscle, are shown to play a significant role in maintaining brain homeostasis. While exercise has been reported to promote muscle secretion, little is known about the effects of neuronal innervation and activity on the yield and molecular composition of biologically active molecules from muscle. As neuromuscular diseases and disabilities associated with denervation impact muscle metabolism, we hypothesize that neuronal innervation and firing may play a pivotal role in regulating secretion activities of skeletal muscles. We examined this hypothesis using an engineered neuromuscular tissue model consisting of skeletal muscles innervated by motor neurons. The innervated muscles displayed elevated expression of mRNAs encoding neurotrophic myokines, such as interleukin-6, brain-derived neurotrophic factor, and FDNC5, as well as the mRNA of peroxisome-proliferator-activated receptor γ coactivator 1α, a key regulator of muscle metabolism. Upon glutamate stimulation, the innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs than neuron-free muscles. Consequently, biological factors secreted by innervated muscles enhanced branching, axonal transport, and, ultimately, spontaneous network activities of primary hippocampal neurons in vitro. Overall, these results reveal the importance of neuronal innervation in modulating muscle-derived factors that promote neuronal function and suggest that the engineered neuromuscular tissue model holds significant promise as a platform for producing neurotrophic molecules.

Ammonia transporter RhBG initiates downstream signaling and functional responses by activating NFκB.

Transceptors, solute transporters that facilitate intracellular entry of molecules and also initiate intracellular signaling events, have been primarily studied in lower-order species. Ammonia, a cytotoxic endogenous metabolite, is converted to urea in hepatocytes for urinary excretion in mammals. During hyperammonemia, when hepatic metabolism is impaired, nonureagenic ammonia disposal occurs primarily in skeletal muscle. Increased ammonia uptake in skeletal muscle is mediated by a membrane-bound, 12 transmembrane domain solute transporter, Rhesus blood group-associated B glycoprotein (RhBG). We show that in addition to its transport function, RhBG interacts with myeloid differentiation primary response-88 (MyD88) to initiate an intracellular signaling cascade that culminates in activation of NFκB. We also show that ammonia-induced MyD88 signaling is independent of the canonical toll-like receptor-initiated mechanism of MyD88-dependent NFκB activation. In silico, in vitro, and in situ experiments show that the conserved cytosolic J-domain of the RhBG protein interacts with the Toll-interleukin-1 receptor (TIR) domain of MyD88. In skeletal muscle from human patients, human-induced pluripotent stem cell-derived myotubes, and myobundles show an interaction of RhBG-MyD88 during hyperammonemia. Using complementary experimental and multiomics analyses in murine myotubes and mice with muscle-specific RhBG or MyD88 deletion, we show that the RhBG-MyD88 interaction is essential for the activation of NFkB but not ammonia transport. Our studies show a paradigm of substrate-dependent regulation of transceptor function with the potential for modulation of cellular responses in mammalian systems by decoupling transport and signaling functions of transceptors.

Endoplasmic Reticulum-Plasma Membrane Junctions as Sites of Depolarization-Induced Ca2+ Signaling in Excitable Cells.

Membrane contact sites between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are found in all eukaryotic cells. In excitable cells they play unique roles in organizing diverse forms of Ca2+ signaling as triggered by membrane depolarization. ER-PM junctions underlie crucial physiological processes such as excitation-contraction coupling, smooth muscle contraction and relaxation, and various forms of activity-dependent signaling and plasticity in neurons. In many cases the structure and molecular composition of ER-PM junctions in excitable cells comprise important regulatory feedback loops linking depolarization-induced Ca2+ signaling at these sites to the regulation of membrane potential. Here, we describe recent findings on physiological roles and molecular composition of native ER-PM junctions in excitable cells. We focus on recent studies that provide new insights into canonical forms of depolarization-induced Ca2+ signaling occurring at junctional triads and dyads of striated muscle, as well as the diversity of ER-PM junctions in these cells and in smooth muscle and neurons.

Twist2 amplification in rhabdomyosarcoma represses myogenesis and promotes oncogenesis by redirecting MyoD DNA binding.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of TWIST2 in fusion-negative RMS. Knockdown of TWIST2 in RMS cells results in up-regulation of MYOGENIN and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.

Retention of stress susceptibility in the mdx mouse model of Duchenne muscular dystrophy after PGC-1α overexpression or ablation of IDO1 or CD38.

Duchenne muscular dystrophy (DMD) is a lethal degenerative muscle wasting disease caused by the loss of the structural protein dystrophin with secondary pathological manifestations including metabolic dysfunction, mood and behavioral disorders. In the mildly affected mdx mouse model of DMD, brief scruff stress causes inactivity, while more severe subordination stress results in lethality. Here, we investigated the kynurenine pathway of tryptophan degradation and the nicotinamide adenine dinucleotide (NAD+) metabolic pathway in mdx mice and their involvement as possible mediators of mdx stress-related pathology. We identified downregulation of the kynurenic acid shunt, a neuroprotective branch of the kynurenine pathway, in mdx skeletal muscle associated with attenuated peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) transcriptional regulatory activity. Restoring the kynurenic acid shunt by skeletal muscle-specific PGC-1α overexpression in mdx mice did not prevent scruff -induced inactivity, nor did abrogating extrahepatic kynurenine pathway activity by genetic deletion of the pathway rate-limiting enzyme, indoleamine oxygenase 1. We further show that reduced NAD+ production in mdx skeletal muscle after subordination stress exposure corresponded with elevated levels of NAD+ catabolites produced by ectoenzyme cluster of differentiation 38 (CD38) that have been implicated in lethal mdx response to pharmacological ß-adrenergic receptor agonism. However, genetic CD38 ablation did not prevent mdx scruff-induced inactivity. Our data do not support a direct contribution by the kynurenine pathway or CD38 metabolic dysfunction to the exaggerated stress response of mdx mice.

Morphological and functional alterations of neuromuscular synapses in a mouse model of ACTA1 congenital myopathy.

Mutations in skeletal muscle α-actin (Acta1) cause myopathies. In a mouse model of congenital myopathy, heterozygous Acta1 (H40Y) knock-in (Acta1+/Ki) mice exhibit features of human nemaline myopathy, including premature lethality, severe muscle weakness, reduced mobility, and the presence of nemaline rods in muscle fibers. In this study, we investigated the impact of Acta1 (H40Y) mutation on the neuromuscular junction (NMJ). We found that the NMJs were markedly fragmented in Acta1+/Ki mice. Electrophysiological analysis revealed a decrease in amplitude but increase in frequency of miniature end-plate potential (mEPP) at the NMJs in Acta1+/Ki mice, compared with those in wild type (Acta1+/+) mice. Evoked end-plate potential (EPP) remained similar at the NMJs in Acta1+/Ki and Acta1+/+ mice, but quantal content was increased at the NMJs in Acta1+/Ki, compared with Acta1+/+ mice, suggesting a homeostatic compensation at the NMJs in Acta1+/Ki mice to maintain normal levels of neurotransmitter release. Furthermore, short-term synaptic plasticity of the NMJs was compromised in Acta1+/Ki mice. Together, these results demonstrate that skeletal Acta1 H40Y mutation, albeit muscle-origin, leads to both morphological and functional defects at the NMJ.

A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolone's activities. Using an RNA-sequencing, bioinformatics, and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone-treated and untreated Smn-/-; SMN2 SMA and Smn+/-; SMN2 healthy mice, we identified molecular targets linked to prednisolone's ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolone's potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.

CGG repeat expansion in LRP12 in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of motor neurons. Although repeat expansion in C9orf72 is its most common cause, the pathogenesis of ALS isn't fully clear. In this study, we show that repeat expansion in LRP12, a causative variant of oculopharyngodistal myopathy type 1 (OPDM1), is a cause of ALS. We identify CGG repeat expansion in LRP12 in five families and two simplex individuals. These ALS individuals (LRP12-ALS) have 61-100 repeats, which contrasts with most OPDM individuals with repeat expansion in LRP12 (LRP12-OPDM), who have 100-200 repeats. Phosphorylated TDP-43 is present in the cytoplasm of iPS cell-derived motor neurons (iPSMNs) in LRP12-ALS, a finding that reproduces the pathological hallmark of ALS. RNA foci are more prominent in muscle and iPSMNs in LRP12-ALS than in LRP12-OPDM. Muscleblind-like 1 aggregates are observed only in OPDM muscle. In conclusion, CGG repeat expansions in LRP12 cause ALS and OPDM, depending on the length of the repeat. Our findings provide insight into the repeat length-dependent switching of phenotypes.

Native collagen VI delays early muscle stem cell differentiation.

Adult muscle stem cells (MuSCs) are critical for muscle homeostasis and regeneration, and their behavior relies on a finely regulated niche made of specific extracellular matrix (ECM) components and soluble factors. Among ECM proteins, collagen VI (Col6) influences the mechanical properties of the niche and, in turn, MuSC self-renewal capabilities. Here, we investigated whether Col6 can exert a direct function as a biochemical signal for regulating the stemness and differentiation of murine MuSCs and myoblasts. Native Col6, but not its pepsin-resistant fragment, counteracts the early differentiation of myogenic cells by reducing the expression of differentiation marker genes and preserving stemness features, with inhibition of the canonical Wnt pathway. Our data indicate that extracellular Col6 acts as a soluble ligand in delaying early myogenic differentiation by regulating intracellular signals involved in adult myogenesis.