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.

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.

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.

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.

Pro-inflammatory response and adverse drug reactions: mechanisms of action of ximelagatran on chemokine and cytokine activation in a monocyte in vitro model.

There is a lack of suitable human in vitro systems which can predict drug hepatotoxicity that in many cases involves inflammatory responses mediated by macrophages. In the present investigation we used an in vitro model based on human THP-1 cells to evaluate the inflammatory cytokine/chemokine activation properties of ximelagatran, a drug previously shown to cause elevation of liver transferases in a subset of patients. Treatment of the cells with ximelagatran caused an intracellular accumulation of the metabolites hydroxymelagatran and melagatran. A decreased viability and increased release of the pro-inflammatory cytokines and chemokines IL-8, VEGF and MCP-1 was seen. Ximelagatran exposure caused activation of ERK1/2 and JNK as evident from determination of the phosphorylation status. In accordance, the release of IL-8 was attenuated by inhibitors of the ERK- and JNK-pathways. It is concluded that human monocytes might constitute a valuable additional in vitro model for monitoring the basis for cytotoxic action of drugs.

GYKI-47261, a new AMPA [2-amino-3-(3-hydroxymethylisoxazole-4-yl)propionic acid] antagonist, is a CYP2E1 inducer.

CYP2E1-inducing capacity of xenobiotics was determined in cultured hepatocytes on the basis of enzyme activities (chlorzoxazone 6-hydroxylation and 7-ethoxycoumarin O-dealkylation) and protein levels. Hepatocytes in culture showed rapid loss of CYP2E1 enzyme during 72 h. CYP2E1 inducers (ethanol, dimethyl sulfoxide, acetone, isopropanol, pyrazole, and imidazole) were able to prevent the fast decrease of the activities and protein levels of CYP2E1 enzyme. Imidazole was found to be the most effective inducer in rat hepatocytes, and it was selected as a reference in subsequent experiments. The effect of GYKI-47261 [6-(4-aminophenyl)-8-chloro-2-methyl-11H-imidazo[1,2c] [2,3]benzodiazepine], a new AMPA [2-amino-3-(3-hydroxymethylisoxazole-4-yl)propionic acid] antagonist drug-candidate, was also tested in the in vitro system. On the basis of enzyme activities and CYP2E1 protein content of rat hepatocytes, GYKI-47261 was considered as a potent CYP2E1 inducer. Furthermore, it was more effective than imidazole, since 10 microM GYKI-47261 produced the maximal induction, whereas 500 microM imidazole brought about the maximal response. Human hepatocytes were more sensitive to GYKI-47261 than were rat cells. In rat hepatocytes, 10 microM caused maximal increase, whereas 0.01 microM produced the highest induction in human cells. Elevation of CYP2E1 gene transcription as the mechanism of induction caused by GYKI-47261 can be excluded. It seems to act mainly on stabilization of CYP2E1 enzyme protein, whereas the role of stabilization of CYP2E1 mRNA can be considered negligible. Although the imidazole part of GYKI-47261 can explain its CYP2E1-inducing capacity, the other part of the molecule must contribute to the final inducing potency.

The neuroprotective agents chlomethiazole and SB203580 inhibit IL-1beta signalling but not its biosynthesis in rat cortical glial cells.

Chlomethiazole and pyridinyl imidazole compounds, exemplified by SB203580, are structurally distinct p38 mitogen-activated protein kinase inhibitors with neuroprotective properties in models of cerebral ischaemia. We have examined their effects in interleukin-1beta (IL-1beta) synthesis, release and signalling in rat cortical glial cells, given the important role of IL-1beta in cerebral ischaemia. We analysed (i) IL-1beta mRNA expression by northern blot, (ii) IL-1beta protein precursor levels within the cells by western blot, and (iii) the levels of the mature IL-1beta protein secreted into the medium by enzyme-linked immunosorbent assay (ELISA) after treatment of rat cortical glial cells with lipopolysaccharide. While the induction of IL-1beta expression by lipopolysaccharide or by IL-1beta itself was very sensitive to nuclear factor kappa B (NF-kappaB) inhibitors, chlomethiazole or SB203580 were nearly without effect, indicating a differential regulation as compared to peripheral cells, e.g. monocytes. In contrast, chlomethiazole and SB203580 potently inhibited the IL-1beta-induced expression of c-fos and inducible nitric oxide synthase, as monitored by northern blot and quantitative RT-PCR, respectively. Because IL-1beta-induced expression of c-fos and inducible nitric oxide synthase is believed to directly contribute to the pathology of cerebral ischaemic injury, the results suggest a direct mechanism for the neuroprotective effects of chlomethiazole and SB203580, and further establish the anti-inflammatory properties of chlomethiazole.