American journal of physiology. Cell physiology最新文献

Endothelial cells (ECs) play a critical role in managing vascular homeostasis and neovascularization. EC functions vary significantly depending on their anatomic locations, especially for ECs forming macrovascular versus microvascular vessels. ECs possess heterogeneous signaling pathways, energy metabolism, and cellular behaviors that enable them to handle both physiological and hyperglycemic conditions. These variations can impact the efficacy of pharmacotherapy and influence the likelihood of unexpected side effects. In this study, we compared human aortic ECs (HAECs) and human dermal microvascular ECs (HDMVECs) to observe the functional and proteomic differences potentially contributing to EC heterogeneity. Compared with HAECs, HDMVECs exhibited faster proliferation, but lower migration and permeability. Under high glucose (HG), migration was worsened for both cell types, whereas proliferation was unaffected, and permeability increased for HDMVECs. Using proteomic analysis, we identified 126 proteins whose abundance was significantly different between HAECs and HDMVECs. Database for Annotation, Visualization, and Integrated Discovery (DAVID) analysis revealed their biological processes, cellular compartments, molecular functions, and pathways. Under high glucose, WARS1 increased whereas SOD2 decreased. Reversing WARS1 or SOD2 expression levels improved HDMVEC migration and permeability functions. The combined treatment of WARS1 knockdown and SOD2 overexpression fully restored EC migration and reduced permeability to levels comparable with those of their counterparts under normal glucose (NG) conditions. Furthermore, in the cutaneous wound in type 2 diabetic mice, the combination therapy of Wars1 knockdown and SOD2 overexpression accelerated the wound closure and augmented wound angiogenesis. Our studies provide novel molecular insights into EC heterogeneity and identified WARS1 and SOD2 as potential targets for dermal angiogenesis during tissue repair.NEW & NOTEWORTHY Our research highlights the functional and proteomic differences between human aortic endothelial cells (HAECs) and human dermal microvascular endothelial cells (HDMVECs). Importantly, we identified WARS1 as a novel target in HDMVECs. Together with SOD2, correcting the abnormalities of these two molecules, HDMVECs' migration and permeability can be fine-tuned under high glucose (HG) conditions. Furthermore, WARS1 knockdown and SOD2 overexpression accelerated wound healing in type 2 diabetic mice, highlighting the therapeutic potential of targeting WARS1 and SOD2 to address delayed wound healing in diabetes.

Serotonin (5-hydroxytryptamine, 5-HT) is a highly conserved signaling molecule present across diverse taxa, including plants, invertebrates, and vertebrates. In mammals, the majority of peripheral serotonin is synthesized in the gastrointestinal tract by enteric neurons and enterochromaffin cells via tryptophan hydroxylases. Its biosynthesis and release are influenced by dietary components and microbial metabolites, particularly short-chain fatty acids produced by the gut microbiota. Once released into the periphery, serotonin exerts pleiotropic effects, regulating intestinal motility and secretion, modulating vascular tone, and influencing blood pressure through both direct actions and vagal sensory pathways engaging central and autonomic circuits. Dysregulation of colonic serotonin production or signaling has been implicated in metabolic, neuropsychiatric, and cardiovascular disorders, including postprandial blood pressure abnormalities and hypertension. Emerging evidence highlights a bidirectional relationship between gut microbes and host serotonergic pathways, suggesting that microbiota-targeted interventions may hold therapeutic potential for cardiometabolic regulation. Advancing our understanding of gut serotonergic signaling, particularly the interplay between host and microbial factors, could inform the development of innovative strategies to treat hypertension and related conditions.

Oxygen (O₂) regulates a multitude of cell functions, and many pathological states are linked to its delivery. We present an automated system for implementing rapid changes in dissolved gas composition in the inflow of a perifusion system that facilitates multiple assessments of tissue function. Features of the system include the ability to assess the effects of changes in both aqueous and dissolved components of the inflow media, and to collect fractions while measuring O₂ consumption rate (OCR) (in the face of changing dissolved O₂), facilitating the subsequent measurement of multiple classes of secreted compounds including metabolites, hormones, neurotransmitters, cell signals, and cytokines. We quantified OCR and lactate secretion rate (LSR) from retinal pigment epithelial (RPE) and INS-1 cells, and from primary tissues (retina, liver, and islets). Higher concentrations of extracellular O₂ were required for tissues than cells monolayers. Consistent with this observation, we found that OCR was not maximal at 21% O₂ for any tissue type we tested. That suggests 21% is too low to adequately provide O₂ for tissues in vitro. However, we found that at high levels of O₂, OCR in some tissues/cells rapidly decrease. LSR was reciprocally regulated relative to the O₂ dependency of OCR, except in tissue where high O₂ inhibits OCR. In summary, we describe a system that can control the concentration of extracellular O₂ and other gases. This instrument will allow researchers to investigate rapid effects of dissolved O₂ on metabolic activities of tissues and cells at O₂ concentrations optimal for the biological specimen.NEW & NOTEWORTHY Oxygen regulates a multitude of cell functions, and many diseases are linked to its delivery. In vitro experiments are critical for elucidating intracellular mechanisms. Whereas standard cell culture incubators allow exposure to varying oxygen concentrations over hours and days, systems enabling rapid and precise control of dissolved oxygen are not available. We present a system for implementing rapid changes in dissolved gas composition with a perifusion system that facilitates multiple assessments of tissue function.

Spaceflight and partial-gravity environments impose profound challenges to the human musculoskeletal system, driving rapid muscle atrophy, progressive bone loss, and tendon maladaptation. At the molecular level, unloading suppresses anabolic signaling, enhances proteolysis, and induces mitochondrial stress, whereas bone and tendon exhibit reduced extracellular matrix turnover and impaired mechanotransduction. Recent space-omics and cross-species studies, including rodent and Caenorhabditis elegans models, reveal that these catabolic responses are evolutionarily conserved and involve systemic pathways mediated by myokines, osteokines, and tendon-derived signals. Current countermeasure strategies primarily consist of structured exercise regimens with limited pharmacologic support. Although these strategies mitigate some loss, they fail to fully preserve musculoskeletal integrity, particularly tendon properties and microarchitectural bone quality. Key gaps remain in the development of tendon-specific interventions, integrated pharmacologic and exercise regimens, nutrition and dietary protocols, and methods for partial-gravity adaptation and safe re-entry. Leveraging real-time monitoring, individualized exercise programs, and systemic biomarker discovery through space omics presents major opportunities for next-generation, personalized countermeasures. This mini-review synthesizes current knowledge of musculoskeletal responses with a particular focus on tendon maladaptation and interorgan cross talk to spaceflight and partial gravity, highlights countermeasure efficacy and limitations, and identifies critical gaps that must be addressed to ensure astronaut health and performance during future missions. Insights from these studies also provide translational relevance for disuse atrophy, osteoporosis, and tendon injuries on Earth.

The N-degron pathway contributes to proteolysis by targeting N-terminal residues of destabilized proteins via E3 ligases that contain a UBR-box domain. Emerging evidence suggests the UBR-box family of E3 ubiquitin ligases (UBR1-7) is involved in the positive regulation of skeletal muscle mass. The purpose of this study was to explore the role of UBR-box E3 ubiquitin ligases under enhanced protein synthesis and skeletal muscle growth conditions. Cohorts of adult male mice were electroporated with constitutively active Akt (Akt-CA) or UBR5 RNAi constructs with a rapamycin diet intervention for 7 and 30 days, respectively. In addition, the UBR-box family was studied during the regrowth phase after nerve crush-induced inactivity. Skeletal muscle growth with Akt-CA or regrowth following inactivity increased protein abundance of UBR1, UBR2, UBR4, UBR5, and UBR7. This occurred with corresponding increases in Akt-mTORC1/S6K and MAPK/p90RSK signaling and protein synthesis. The increases in UBR-box E3s, ubiquitination, and proteasomal activity occurred independently of mTORC1 activity and were associated with increases in markers related to autophagy, ER-stress, and protein quality control pathways. Finally, while UBR5 knockdown (KD) evokes atrophy, it occurs together with hyperactivation of mTORC1 and protein synthesis. In UBR5 KD muscles, we identified an increase in protein abundance for UBR2, UBR4, and UBR7, which may highlight a compensatory response to maintain proteome integrity. Future studies will seek to understand the role of UBR-box E3s toward protein quality control in skeletal muscle plasticity.NEW & NOTEWORTHY Novel UBR-box E3 ubiquitin ligases are responsive to heightened protein synthesis and alterations in skeletal muscle mass and fiber size, to maintain proteome integrity.

Chronic, low-grade inflammation is increasingly recognized as a fundamental driver of noncommunicable diseases-including obesity, metabolic dysfunction-associated steatotic liver disease (MASLD), and neurodegeneration-yet the initiating events remain incompletely understood. Accumulating evidence implicates gut barrier dysfunction and bacterial translocation as pivotal mechanisms linking environmental and metabolic stressors to systemic inflammation. Mechanistically, obesity-associated depletion of typically beneficial taxa (e.g., Faecalibacterium, Roseburia, Akkermansia muciniphila) and enrichment of proinflammatory Enterobacteriaceae reduce expression of tight junction proteins-including, occludin, claudins, and zonula occludens-1 (ZO-1)-and increase the vascular permeability marker, plasmalemma vesicle-associated protein (PV-1). Combined with diminished secretion of host defense peptides (e.g., Reg3γ, lysozyme) and mucus thinning, these changes facilitate LPS-driven activation of Toll-like receptor (TLR)4 and downstream cytokines. We integrate preclinical and clinical data demonstrating how these processes propagate systemic inflammation via the gut-liver and gut-vascular axes, contributing to MASLD, insulin resistance, and vascular dysfunction. Finally, we highlight emerging interventions aimed at restoring barrier integrity-ranging from short-chain fatty acid (SCFA) supplementation and Glucagon-like peptide-2 (GLP-2) receptor agonists to host defense peptide-based therapies-and discuss methodological advances for assessing gut permeability in vivo. Understanding the gut as a dynamic interface between host and environment, and its crucial role in mediating inflammation, will be pivotal for the development of effective interventions targeting the global epidemic of obesity-related disease.

Epicardial adipose tissue (EAT) regulates lipid metabolism and immune cell recruitment in coronary arteries. Increased EAT contributes to coronary artery disease (CAD), but exercise prevents CAD. We hypothesized that exercise, irrespective of CAD presence, would produce EAT with increased M2 macrophages and upregulation of anti-inflammatory cytokine transcripts. Female Yucatan pigs (n = 7) were sedentary or exercised, and the left circumflex coronary artery was occluded or remained nonoccluded (2 × 2 design). Bulk and single-nuclei transcriptomic sequencing performed on EAT identified immune, endothelial, smooth muscle, adipocytes, adipocyte progenitor cells (APSCs), and neuronal cells, with adipocytes and APSCs predominant. Nonoccluded (N) sedentary (Sed) EAT had the most M1 macrophages and CD8⁺ T cells. Sed EAT had the most cells expressing tumor necrosis factor (TNF) superfamily genes. Exercise (Ex) upregulated peroxisome proliferator-activated receptor (PPAR) γ (G) expression and enriched PPAR signaling, which suppresses activation, in macrophages and T cells, particularly in occluded (O) Ex EAT. By contrast, N_Ex EAT had few CD8⁺ T cells with low PPARG expression. Adipocytes and immune cells in O_Sed EAT had the most communication via growth factors and adhesion molecules. Exercise mitigates EAT inflammation via modulation of immune cell subpopulations, decreased TNF superfamily, and increased PPARG gene expression, and decreased communication between adipocytes and immune cells. However, the effect of exercise on the EAT immune environment is modulated by coronary artery occlusion status. Future studies of the impact of exercise and coronary artery occlusion on EAT would benefit from using a progressive nutritionally induced model of CAD.NEW & NOTEWORTHY A sedentary lifestyle increases the number of inflammatory M1 macrophages and CD8⁺ T cells, their expression of tumor necrosis factor genes, and the number of communications between these immune cells and adipocytes in epicardial adipose tissue (EAT). The expression of peroxisome proliferator receptor and genes in control of cell activation in macrophages and T cells in nonoccluded and occluded EAT increases in response to exercise.

Development of myogenic cells, called satellite cells, is determined by transcription factors that regulate their quiescence (e.g., Pax7), activation (e.g., MyoD), and terminal differentiation (e.g., myogenin). Demethylation of lysine 3 on histone 27 (H3K27) activates expression of Myod and Myog. In this investigation, we investigated the effects of a satellite cell-targeted, hemizygous mutation of the H3K27 demethylase Jmjd3 in healthy muscle. Using sequencing of chromatin fragments precipitated from Jmjd3 mutant and control satellite cells using anti-H3K27me3, we found that the Pax7 promoter was the only chromatin that experienced significantly increased H3K27 methylation in mutant cells. However, RNA sequencing showed that 143 genes were downregulated in mutant cells, including Myod, a direct target of Pax7, and at least 72 other genes that contained E-boxes targeted by MyoD. Gene ontology analysis showed enrichment of genes involved in myogenesis in the downregulated genes. Reduced expression of Pax7, Myod, and Myog was confirmed by quantitative PCR (qPCR), Western blots, and immunohistochemistry. Mutant muscles also had smaller diameter fibers, fewer myonuclei, and diminished myogenic cell fusion, indicating impaired growth and differentiation. These findings show that Jmjd3 affects demethylation of H3K27 at the Pax7 promoter, and increased H3K27 methylation reduces expression of Pax7 and its target genes, disrupting muscle growth.NEW & NOTEWORTHY This investigation reveals a new mechanism that regulates the development of muscle. Mutating Jmjd3 produced epigenetic modifications to the transcription factor Pax7, reducing its expression and impairing muscle growth.

Mechanical loading drives structural and functional improvements in muscle and tendon, protecting against injury at their interface, at the myotendinous junction (MTJ), and within the tendon matrix. However, the early cellular and molecular events that initiate these adaptations in humans remain poorly understood. To investigate this, we applied single-nucleus RNA sequencing and in situ hybridization to map the acute transcriptional response of the human muscle-tendon unit to a single bout of eccentric resistance exercise, with a focus on extracellular matrix (ECM) regulation. We identified four transcriptionally distinct fibroblast subtypes expressing key ECM components, including COL1A1 and DCN. Three of these subtypes were localized to the tendon and responded to exercise: two were spatially restricted to the collagen fascicles or the MTJ, while the third, enriched in the interfascicular matrix (IFM), exhibited the strongest response. This IFM population, marked by PDGFRA, upregulated PRG4 and VCAN, ECM genes linked to tissue lubrication and resilience. In parallel, exercise induced dynamic ECM regulation in myonuclei, particularly in a distinct subset of type II myonuclei at the MTJ, that expanded in number and robustly upregulated COL22A1, a collagen essential for MTJ integrity. Together, these findings uncover a spatially organized, cell type-specific program of ECM remodeling in response to mechanical load, offering new insight into the early molecular events of human muscle-tendon adaptation.NEW & NOTEWORTHY We provide the first high-resolution, in vivo single-nucleus map of the acute human muscle-tendon response to mechanical loading. Within 4 hours of resistance exercise, spatially distinct tendon fibroblasts and myonuclei activate coordinated extracellular matrix programs. Interfascicular fibroblasts upregulate PRG4 and VCAN in the tendon, while myonuclei at the myotendinous junction induce COL22A1 and LAMA2. These findings reveal a new principle of human mechanobiology, where exercise rapidly engages niche-specific programs to initiate extracellular matrix adaptation.

Mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1) can result in muscle diseases, termed RyR1-related myopathies (RyR1-RM). Examples include malignant hyperthermia (MH), central core disease (CCD), and centronuclear myopathy (CNM). The muscles involved often have more (and mispositioned) nuclei than normal. A subset of the corresponding mutant proteins shows an overactive or leaky sarcoplasmic reticulum (SR) channel behavior that depletes the SR Ca²⁺ content and increases the level of cytosolic Ca²⁺. In addition, two remarkable effects of these RyR1 variants have been reported in cultured myogenic cells: enhanced expression of interleukin-6 (IL-6) and stimulation of myoblast fusion (myonuclei accretion). Here, we have investigated whether the latter effect is due to a possible IL-6-dependent autocrine loop. Toward this goal, we analyzed the impact of the overactive Y523S mutant compared with the wild-type RyR1 after expression in C2C12 cells. The results show that this mutation indeed drastically promotes myoblast fusion up to ∼300%. Moreover, this action depends on the sequential activation of SR Ca²⁺ release, store-operated Ca²⁺ channels, reactive oxygen species (ROS, cytosolic and mitochondrial), calpain, and calcineurin. In addition, a neutralizing antibody directed against IL-6 and a p38 inhibitor completely suppressed the stimulation of myoblast fusion. Furthermore, in RyR1-expressing cells, myotube formation was promoted by either exogenous IL-6 or conditioned medium obtained from the Y523S-expressing cells. These findings suggest an autocrine mechanism involving the interplay between Ca²⁺, ROS, IL-6, and p38 signaling pathways in controlling myonuclei density, which could be essential to explain the pathogenesis of RyR1-RM.NEW & NOTEWORTHY Overactive RyR1 mutant proteins are associated with muscle disease; interestingly, they increase the number of myonuclei when expressed in C2C12 cells. We discovered that this alteration depends on a Ca²⁺/ROS loop, which recruits calpain and calcineurin to stimulate the production of IL-6 and the subsequent autocrine activation of p38. Thus, disease-causing RyR1 mutations require an IL-6 autocrine system to alter myonuclear density. This novel mechanism could be critical to understanding the pathogenesis of congenital myopathies.