Mustn1 is a smooth muscle cell-secreted microprotein that modulates skeletal muscle extracellular matrix composition.

OBJECTIVE: Skeletal muscle plasticity and remodeling are critical for adapting tissue function to use, disuse, and regeneration. The aim of this study was to identify genes and molecular pathways that regulate the transition from atrophy to compensatory hypertrophy or recovery from injury. Here, we have used a mouse model of hindlimb unloading and reloading, which causes skeletal muscle atrophy, and compensatory regeneration and hypertrophy, respectively. METHODS: We analyzed mouse skeletal muscle at the transition from hindlimb unloading to reloading for changes in transcriptome and extracellular fluid proteome. We then used qRT-PCR, immunohistochemistry, and bulk and single-cell RNA sequencing data to determine Mustn1 gene and protein expression, including changes in gene expression in mouse and human skeletal muscle with different challenges such as exercise and muscle injury. We generated Mustn1-deficient genetic mouse models and characterized them in vivo and ex vivo with regard to muscle function and whole-body metabolism. We isolated smooth muscle cells and functionally characterized them, and performed transcriptomics and proteomics analysis of skeletal muscle and aorta of Mustn1-deficient mice. RESULTS: We show that Mustn1 (Musculoskeletal embryonic nuclear protein 1, also known as Mustang) is highly expressed in skeletal muscle during the early stages of hindlimb reloading. Mustn1 expression is transiently elevated in mouse and human skeletal muscle in response to intense exercise, resistance exercise, or injury. We find that Mustn1 expression is highest in smooth muscle-rich tissues, followed by skeletal muscle fibers. Muscle from heterozygous Mustn1-deficient mice exhibit differences in gene expression related to extracellular matrix and cell adhesion, compared to wild-type littermates. Mustn1-deficient mice have normal muscle and aorta function and whole-body glucose metabolism. We show that Mustn1 is secreted from smooth muscle cells, and that it is present in arterioles of the muscle microvasculature and in muscle extracellular fluid, particularly during the hindlimb reloading phase. Proteomics analysis of muscle from Mustn1-deficient mice confirms differences in extracellular matrix composition, and female mice display higher collagen content after chemically induced muscle injury compared to wild-type littermates. CONCLUSIONS: We show that, in addition to its previously reported intracellular localization, Mustn1 is a microprotein secreted from smooth muscle cells into the muscle extracellular space. We explore its role in muscle ECM deposition and remodeling in homeostasis and upon muscle injury. The role of Mustn1 in fibrosis and immune infiltration upon muscle injury and dystrophies remains to be investigated, as does its potential for therapeutic interventions.

Muscle-secreted neurturin couples myofiber oxidative metabolism and slow motor neuron identity.

Endurance exercise promotes skeletal muscle vascularization, oxidative metabolism, fiber-type switching, and neuromuscular junction integrity. Importantly, the metabolic and contractile properties of the muscle fiber must be coupled to the identity of the innervating motor neuron (MN). Here, we show that muscle-derived neurturin (NRTN) acts on muscle fibers and MNs to couple their characteristics. Using a muscle-specific NRTN transgenic mouse (HSA-NRTN) and RNA sequencing of MN somas, we observed that retrograde NRTN signaling promotes a shift toward a slow MN identity. In muscle, NRTN increased capillary density and oxidative capacity and induced a transcriptional reprograming favoring fatty acid metabolism over glycolysis. This combination of effects on muscle and MNs makes HSA-NRTN mice lean with remarkable exercise performance and motor coordination. Interestingly, HSA-NRTN mice largely recapitulate the phenotype of mice with muscle-specific expression of its upstream regulator PGC-1ɑ1. This work identifies NRTN as a myokine that couples muscle oxidative capacity to slow MN identity.

LIM and cysteine-rich domains 1 (LMCD1) regulates skeletal muscle hypertrophy, calcium handling, and force.

BACKGROUND: Skeletal muscle mass and strength are crucial determinants of health. Muscle mass loss is associated with weakness, fatigue, and insulin resistance. In fact, it is predicted that controlling muscle atrophy can reduce morbidity and mortality associated with diseases such as cancer cachexia and sarcopenia. METHODS: We analyzed gene expression data from muscle of mice or human patients with diverse muscle pathologies and identified LMCD1 as a gene strongly associated with skeletal muscle function. We transiently expressed or silenced LMCD1 in mouse gastrocnemius muscle or in mouse primary muscle cells and determined muscle/cell size, targeted gene expression, kinase activity with kinase arrays, protein immunoblotting, and protein synthesis levels. To evaluate force, calcium handling, and fatigue, we transduced the flexor digitorum brevis muscle with a LMCD1-expressing adenovirus and measured specific force and sarcoplasmic reticulum Ca2+ release in individual fibers. Finally, to explore the relationship between LMCD1 and calcineurin, we ectopically expressed Lmcd1 in the gastrocnemius muscle and treated those mice with cyclosporine A (calcineurin inhibitor). In addition, we used a luciferase reporter construct containing the myoregulin gene promoter to confirm the role of a LMCD1-calcineurin-myoregulin axis in skeletal muscle mass control and calcium handling. RESULTS: Here, we identify LIM and cysteine-rich domains 1 (LMCD1) as a positive regulator of muscle mass, that increases muscle protein synthesis and fiber size. LMCD1 expression in vivo was sufficient to increase specific force with lower requirement for calcium handling and to reduce muscle fatigue. Conversely, silencing LMCD1 expression impairs calcium handling and force, and induces muscle fatigue without overt atrophy. The actions of LMCD1 were dependent on calcineurin, as its inhibition using cyclosporine A reverted the observed hypertrophic phenotype. Finally, we determined that LMCD1 represses the expression of myoregulin, a known negative regulator of muscle performance. Interestingly, we observed that skeletal muscle LMCD1 expression is reduced in patients with skeletal muscle disease. CONCLUSIONS: Our gain- and loss-of-function studies show that LMCD1 controls protein synthesis, muscle fiber size, specific force, Ca2+ handling, and fatigue resistance. This work uncovers a novel role for LMCD1 in the regulation of skeletal muscle mass and function with potential therapeutic implications.

Kynurenic Acid and Gpr35 Regulate Adipose Tissue Energy Homeostasis and Inflammation.

The role of tryptophan-kynurenine metabolism in psychiatric disease is well established, but remains less explored in peripheral tissues. Exercise training activates kynurenine biotransformation in skeletal muscle, which protects from neuroinflammation and leads to peripheral kynurenic acid accumulation. Here we show that kynurenic acid increases energy utilization by activating G protein-coupled receptor Gpr35, which stimulates lipid metabolism, thermogenic, and anti-inflammatory gene expression in adipose tissue. This suppresses weight gain in animals fed a high-fat diet and improves glucose tolerance. Kynurenic acid and Gpr35 enhance Pgc-1α1 expression and cellular respiration, and increase the levels of Rgs14 in adipocytes, which leads to enhanced beta-adrenergic receptor signaling. Conversely, genetic deletion of Gpr35 causes progressive weight gain and glucose intolerance, and sensitizes to the effects of high-fat diets. Finally, exercise-induced adipose tissue browning is compromised in Gpr35 knockout animals. This work uncovers kynurenine metabolism as a pathway with therapeutic potential to control energy homeostasis.

Neurturin is a PGC-1α1-controlled myokine that promotes motor neuron recruitment and neuromuscular junction formation.

OBJECTIVE: We examined whether skeletal muscle overexpression of PGC-1α1 or PGC-1α4 affected myokine secretion and neuromuscular junction (NMJ) formation. METHODS: A microfluidic device was used to model endocrine signaling and NMJ formation between primary mouse myoblast-derived myotubes and embryonic stem cell-derived motor neurons. Differences in hydrostatic pressure allowed for fluidic isolation of either cell type or unidirectional signaling in the fluid phase. Myotubes were transduced to overexpress PGC-1α1 or PGC-1α4, and myokine secretion was quantified using a proximity extension assay. Morphological and functional changes in NMJs were measured by fluorescent microscopy and by monitoring muscle contraction upon motor neuron stimulation. RESULTS: Skeletal muscle transduction with PGC-1α1, but not PGC-1α4, increased NMJ formation and size. PGC-1α1 increased muscle secretion of neurturin, which was sufficient and necessary for the effects of muscle PGC-1α1 on NMJ formation. CONCLUSIONS: Our findings indicate that neurturin is a mediator of PGC-1α1-dependent retrograde signaling from muscle to motor neurons.

The chaperone co-inducer BGP-15 alleviates ventilation-induced diaphragm dysfunction.

Ventilation-induced diaphragm dysfunction (VIDD) is a marked decline in diaphragm function in response to mechanical ventilation, which has negative consequences for individual patients' quality of life and for the health care system, but specific treatment strategies are still lacking. We used an experimental intensive care unit (ICU) model, allowing time-resolved studies of diaphragm structure and function in response to long-term mechanical ventilation and the effects of a pharmacological intervention (the chaperone co-inducer BGP-15). The marked loss of diaphragm muscle fiber function in response to mechanical ventilation was caused by posttranslational modifications (PTMs) of myosin. In a rat model, 10 days of BGP-15 treatment greatly improved diaphragm muscle fiber function (by about 100%), although it did not reverse diaphragm atrophy. The treatment also provided protection from myosin PTMs associated with HSP72 induction and PARP-1 inhibition, resulting in improvement of mitochondrial function and content. Thus, BGP-15 may offer an intervention strategy for reducing VIDD in mechanically ventilated ICU patients.

Peroxisome Proliferator-activated Receptor γ Coactivator-1 α Isoforms Selectively Regulate Multiple Splicing Events on Target Genes.

Endurance and resistance exercise training induces specific and profound changes in the skeletal muscle transcriptome. Peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) coactivators are not only among the genes differentially induced by distinct training methods, but they also participate in the ensuing signaling cascades that allow skeletal muscle to adapt to each type of exercise. Although endurance training preferentially induces PGC-1α1 expression, resistance exercise activates the expression of PGC-1α2, -α3, and -α4. These three alternative PGC-1α isoforms lack the arginine/serine-rich (RS) and RNA recognition motifs characteristic of PGC-1α1. Discrete functions for PGC-1α1 and -α4 have been described, but the biological role of PGC-1α2 and -α3 remains elusive. Here we show that different PGC-1α variants can affect target gene splicing through diverse mechanisms, including alternative promoter usage. By analyzing the exon structure of the target transcripts for each PGC-1α isoform, we were able to identify a large number of previously unknown PGC-1α2 and -α3 target genes and pathways in skeletal muscle. In particular, PGC-1α2 seems to mediate a decrease in the levels of cholesterol synthesis genes. Our results suggest that the conservation of the N-terminal activation and repression domains (and not the RS/RNA recognition motif) is what determines the gene programs and splicing options modulated by each PGC-1α isoform. By using skeletal muscle-specific transgenic mice for PGC-1α1 and -α4, we could validate, in vivo, splicing events observed in in vitro studies. These results show that alternative PGC-1α variants can affect target gene expression both quantitatively and qualitatively and identify novel biological pathways under the control of this system of coactivators.

Mechano-signalling pathways in an experimental intensive critical illness myopathy model.

KEY POINTS: Using an experimental rat intensive care unit (ICU) model, not limited by early mortality, we have previously shown that passive mechanical loading attenuates the loss of muscle mass and force-generation capacity associated with the ICU intervention. Mitochondrial dynamics have recently been shown to play a more important role in muscle atrophy than previously recognized. In this study we demonstrate that mitochondrial dynamics, as well as mitophagy, is affected by mechanosensing at the transcriptional level, and muscle changes induced by unloading are counteracted by passive mechanical loading. The recently discovered ubiquitin ligases Fbxo31 and SMART are induced by mechanical silencing, an induction that similarly is prevented by passive mechanical loading. ABSTRACT: The complete loss of mechanical stimuli of skeletal muscles, i.e. loss of external strain related to weight bearing and internal strain related to activation of contractile proteins, in mechanically ventilated, deeply sedated and/or pharmacologically paralysed intensive care unit (ICU) patients is an important factor triggering the critical illness myopathy (CIM). Using a unique experimental ICU rat model, mimicking basic ICU conditions, we have recently shown that mechanical silencing is a dominant factor triggering the preferential loss of myosin, muscle atrophy and decreased specific force in fast- and slow-twitch muscles and muscle fibres. The aim of this study is to gain improved understanding of the gene signature and molecular pathways regulating the process of mechanical activation of skeletal muscle that are affected by the ICU condition. We have focused on pathways controlling myofibrillar protein synthesis and degradation, mitochondrial homeostasis and apoptosis. We demonstrate that genes regulating mitochondrial dynamics, as well as mitophagy are induced by mechanical silencing and that these effects are counteracted by passive mechanical loading. In addition, the recently identified ubiquitin ligases Fbxo31 and SMART are induced by mechanical silencing, an induction that is reversed by passive mechanical loading. Thus, mechano-cell signalling events are identified which may play an important role for the improved clinical outcomes reported in response to the early mobilization and physical therapy in immobilized ICU patients.

Neuregulin improves response to glucose tolerance test in control and diabetic rats.

Neuregulin (NRG) is an EGF-related growth factor that binds to the tyrosine kinase receptors ErbB3 and ErbB4, thus inducing tissue development and muscle glucose utilization during contraction. Here, we analyzed whether NRG has systemic effects regulating glycemia in control and type 2 diabetic rats. To this end, recombinant NRG (rNRG) was injected into Zucker diabetic fatty (ZDF) rats and their respective lean littermates 15 min before a glucose tolerance test (GTT) was performed. rNRG enhanced glucose tolerance without promoting the activation of the insulin receptor (IR) or insulin receptor substrates (IRS) in muscle and liver. However, in control rats, rNRG induced the phosphorylation of protein kinase B (PKB) and glycogen synthase kinase-3 (GSK-3) in liver but not in muscle. In liver, rNRG increased ErbB3 tyrosine phosphorylation and its binding to phosphatidylinositol 3-kinase (PI3K), thus indicating that rNRG activates the ErbB3/PI3K/PKB signaling pathway. rNRG increased glycogen content in liver but not in muscle. rNRG also increased the content of fructose-2,6-bisphosphate (Fru-2,6-P2), an activator of hepatic glycolysis, and lactate in liver but not in muscle. Increases in lactate were abrogated by wortmannin, a PI3K inhibitor, in incubated hepatocytes. The liver of ZDF rats showed a reduced content of ErbB3 receptors, entailing a minor stimulation of the rNRG-induced PKB/GSK-3 cascade and resulting in unaltered hepatic glycogen content. Nonetheless, rNRG increased hepatic Fru-2,6-P2 and augmented lactate both in liver and in plasma of diabetic rats. As a whole, rNRG improved response to the GTT in both control and diabetic rats by enhancing hepatic glucose utilization.

Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise.

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

The hitchhiker's guide to PGC-1α isoform structure and biological functions.

Proteins of the peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1 (PGC-1) family of transcriptional coactivators coordinate physiological adaptations in many tissues, usually in response to demands for higher nutrient and energy supply. Of the founding members of the family, PGC-1α (also known as PPARGC1A) is the most highly regulated gene, using multiple promoters and alternative splicing to produce a growing number of coactivator variants. PGC-1α promoters are selectively active in distinct tissues in response to specific stimuli. To date, more than ten novel PGC-1α isoforms have been reported to be expressed from a novel promoter (PGC-1α-b, PGC-1α-c), to undergo alternative splicing (NT-PGC-1α) or both (PGC-1α2, PGC-1α3, PGC-1α4). The resulting proteins display differential regulation and tissue distribution and, most importantly, exert specific biological functions. In this review we discuss the structural and functional characteristics of the novel PGC-1α isoforms, aiming to provide an integrative view of this constantly expanding system of transcriptional coactivators.

Bioenergetic cues shift FXR splicing towards FXRα2 to modulate hepatic lipolysis and fatty acid metabolism.

OBJECTIVE: Farnesoid X receptor (FXR) plays a prominent role in hepatic lipid metabolism. The FXR gene encodes four proteins with structural differences suggestive of discrete biological functions about which little is known. METHODS: We expressed each FXR variant in primary hepatocytes and evaluated global gene expression, lipid profile, and metabolic fluxes. Gene delivery of FXR variants to Fxr(-/-) mouse liver was performed to evaluate their role in vivo. The effects of fasting and physical exercise on hepatic Fxr splicing were determined. RESULTS: We show that FXR splice isoforms regulate largely different gene sets and have specific effects on hepatic metabolism. FXRα2 (but not α1) activates a broad transcriptional program in hepatocytes conducive to lipolysis, fatty acid oxidation, and ketogenesis. Consequently, FXRα2 decreases cellular lipid accumulation and improves cellular insulin signaling to AKT. FXRα2 expression in Fxr(-/-) mouse liver activates a similar gene program and robustly decreases hepatic triglyceride levels. On the other hand, FXRα1 reduces hepatic triglyceride content to a lesser extent and does so through regulation of lipogenic gene expression. Bioenergetic cues, such as fasting and exercise, dynamically regulate Fxr splicing in mouse liver to increase Fxrα2 expression. CONCLUSIONS: Our results show that the main FXR variants in human liver (α1 and α2) reduce hepatic lipid accumulation through distinct mechanisms and to different degrees. Taking this novel mechanism into account could greatly improve the pharmacological targeting and therapeutic efficacy of FXR agonists.

G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 (PGC-1α4) is a protein isoform derived by alternative splicing of the PGC1α mRNA and has been shown to promote muscle hypertrophy. We show here that G protein-coupled receptor 56 (GPR56) is a transcriptional target of PGC-1α4 and is induced in humans by resistance exercise. Furthermore, the anabolic effects of PGC-1α4 in cultured murine muscle cells are dependent on GPR56 signaling, because knockdown of GPR56 attenuates PGC-1α4-induced muscle hypertrophy in vitro. Forced expression of GPR56 results in myotube hypertrophy through the expression of insulin-like growth factor 1, which is dependent on Gα12/13 signaling. A murine model of overload-induced muscle hypertrophy is associated with increased expression of both GPR56 and its ligand collagen type III, whereas genetic ablation of GPR56 expression attenuates overload-induced muscle hypertrophy and associated anabolic signaling. These data illustrate a signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise.

Skeletal muscle PGC-1α1 modulates kynurenine metabolism and mediates resilience to stress-induced depression.

Depression is a debilitating condition with a profound impact on quality of life for millions of people worldwide. Physical exercise is used as a treatment strategy for many patients, but the mechanisms that underlie its beneficial effects remain unknown. Here, we describe a mechanism by which skeletal muscle PGC-1α1 induced by exercise training changes kynurenine metabolism and protects from stress-induced depression. Activation of the PGC-1α1-PPARα/δ pathway increases skeletal muscle expression of kynurenine aminotransferases, thus enhancing the conversion of kynurenine into kynurenic acid, a metabolite unable to cross the blood-brain barrier. Reducing plasma kynurenine protects the brain from stress-induced changes associated with depression and renders skeletal muscle-specific PGC-1α1 transgenic mice resistant to depression induced by chronic mild stress or direct kynurenine administration. This study opens therapeutic avenues for the treatment of depression by targeting the PGC-1α1-PPAR axis in skeletal muscle, without the need to cross the blood-brain barrier.

Emerging role of neuregulin as a modulator of muscle metabolism.

Neuregulin was described initially as a neurotrophic factor involved in the formation of the neuromuscular junction in skeletal muscle. However, in recent years, neuregulin has been reported to be a myokine that exerts relevant effects on myogenesis and the regulation of muscle metabolism. In this new context, the rapid and chronic metabolic effects of neuregulin appear to be related to muscle contraction. Indeed, the effects of neuregulin resemble those of exercise, which are accompanied by an improvement in insulin sensitivity. In this review, we challenge the classical role assigned to neuregulin in muscle and propound the emerging concept of its involvement in the regulation of energetic metabolism and insulin responsiveness.