Function

Muscle satellite cells (MuSCs) are muscle-resident stem cells that are responsible for myofiber regeneration. Although the importance of calcium ions (Ca2+) in muscle physiology has been well established, the mechanism by which Ca2+ mobilization governs MuSC function remains poorly understood. In this study, we aimed to systematically characterize Ca2+ dynamics in MuSCs and to define the mechanisms regulating these signals during muscle regeneration. By employing modified protocols for mouse MuSC isolation and Ca2+ measurement, we observed spontaneous Ca2+ fluctuations in MuSCs isolated from regenerating muscle after cardiotoxin-induced myofiber injury. Our detailed analysis using chemical Ca2+ indicators and a genetically encoded Ca2+ indicator revealed that the frequency and amplitude of Ca2+ fluctuations increased significantly during the activated and proliferative stages of MuSCs in muscle regeneration. This effect was more pronounced in MuSCs isolated from dystrophic and aged mice. Mechanistically, these Ca2+ fluctuations were at least partially mediated by mechanosensitive ion channels, including PIEZO1 and TRPM7, which promote MuSC migration. Collectively, our findings demonstrate that Ca2+ fluctuations through mechanosensitive ion channels act as a key regulator of MuSC activation during muscle regeneration and may provide new insights into the role of Ca2+ influx in muscle biology and the pathogenesis of muscle diseases.

BackgroundCalcitonin gene related peptide (CGRP) hyperpolarizes pulmonary arterial smooth muscle cells (SMCs) and endothelial cells (ECs) through PKA-dependent activation of KATP channels. CGRP can diminish the severity of pulmonary fibrosis (PF), however, the effects on vascular signaling were poorly defined. We hypothesized that hyperpolarization to CGRP would be augmented in a mouse model of PF. MethodsPF was induced in male and female C57BL/6 mice by intratracheal delivery of bleomycin (3 wk), with saline used as control (sham). Pulmonary arteries (PAs; 100-150 {micro}m diameter) were cannulated and pressurized to 16 cmH2O, and endothelial tubes were studied in complementary experiments to eliminate the influence of SMCs. Membrane potential (Vm) was recorded continuously using intracellular microelectrodes. Responses were also evaluated in isolated lungs preconstricted with U46619 ([~]10 mmHg). ResultsPF led to greater indices of PH in males vs. females. Isolated lungs and PAs from male PF mice had enhanced vasodilation and hyperpolarization of Vm to CGRP, although no effect was observed in females. The greater vasodilation and hyperpolarization of SMCs to CGRP in males persisted in endothelium-disrupted PAs and during treatment with L-NAME indicating that ECs are not required for greater responsiveness to CGRP. With no effect on resting Vm, inhibition of KATP channels or PKA significantly attenuated hyperpolarization of SMCs and ECs, attenuated vasodilation to CGRP in PAs, and eliminated differences between groups in males. Direct activation of PKA, but not KATP, evoked greater Vm hyperpolarization and vasodilation in PF vs. sham PAs and lungs. Although no difference in sensory nerves was observed in fibrotic mice, perivascular nerve stimulation evoked greater vasodilation in PAs. ConclusionsIn a mouse model of PF, CGRP-dependent hyperpolarization of pulmonary arterial SMCs and ECs is augmented through increased PKA-dependent activation of KATP channels leading to increased vasodilator sensitivity.

Aminoglycoside (AG) antibiotic safety is limited by ototoxicity, the mitigation of which is vital considering bacterial resistance mediated erosion of our antibiotic arsenal. Previously, we observed tectorial membrane (TM) sequestration of Ca2+. We hypothesized that the TM sequesters other cations, including the AG gentamicin. We proposed to test the effect of TM genetic ablation on ototoxicity and TM-AG sequestration. After intraperitoneal AG-furosemide, TM-lacking Tecta{Delta}ENT/{Delta}ENT mice showed limited outer hair cell loss, unlike wildtype littermates. Spectroscopy measurements of gentamicin-Texas red (GTTR) were made in isolated wildtype and TectaY1870C TMs and guinea pig cochleae following direct or intraperitoneal GTTR administration. TM-GTTR sequestration was observed in all cases, while negatively correlated with TectaY1870C zygosity. In summary, we discovered a novel TM component in the AG ototoxicity pathway. Intact TM structure is necessary for sequestration, and the TM modulates AG ototoxicity. TM-GTTR sequestration following systemic injection indicates that this phenomenon occurs during AG therapy. Single sentence summaryOtotoxic aminoglycosides collect inside the acellular tectorial membrane of the inner ear, likely due to electrostatic interactions, and the structural status of that membrane modulates the toxic effect of those aminoglycosides on sensory hair cells.

Mass is a fundamental aspect of muscle contractile function, yet the inertial effects of inactive muscle mass is generally neglected in modeling and not quantified in studies on small muscles or isolated fibers. However, during submaximal contractions, inactive muscle tissue may take longer to be accelerated by active fibers, and may be subject to prolonged deceleration, both of which may potentially reduce force development and work output. We sought to test if inactive tissue mass imposes an inertial penalty on muscle performance, using in situ sinusoidal work-loop experiments on rat plantaris muscles. Regional fascicle dynamics, measured across supramaximal and submaximal levels of activation, showed that decreasing activation significantly reduced fascicle strain and increased both shortening and lengthening latency. Contrary to our predictions, however, reductions in work, beyond those explained by decreased fascicle strain, were negligible. Normalized work did not decline disproportionately relative to force, suggesting no clear inertial penalty on work at this muscle size. Our findings suggest that while inactive muscle mass influences the dynamics of submaximal contractions, its impact on work during submaximal contractions at small muscle sizes is limited.

Regulation of water permeability in the collecting duct is important for osmoregulatory acclimation in teleost fish. In hyperosmotic environments such as seawater (SW), the teleost kidney functions as a site of divalent ion excretion. The collecting ducts reabsorb Na+, Cl-, and water, thereby reducing urine volume and producing small amounts of isotonic urine with high concentrations of divalent ions. In hypoosmotic environments such as freshwater (FW) or low-salinity brackish water (BW), the kidney produces large volumes of hypotonic urine and serves as a site of water excretion; under these conditions, the collecting ducts reabsorb Na+ and Cl- but not water. To identify aquaporins (Aqps) involved in regulating water permeability in the collecting ducts of teleosts, we analyzed renal Aqp expression in a euryhaline marine fish, the Japanese pufferfish (Takifugu rubripes), which possesses 16 Aqp genes in its genome, seven of which (Aqp1aa, 1ab, 3a, 4a, 7, 8bb, and 11a) are expressed in the kidney. Quantitative RT-PCR analysis showed that Aqp1aa and Aqp4a were highly expressed in collecting duct tissues, and that Aqp1aa expression was markedly reduced in fish acclimated to BW. Immunohistochemistry revealed apical localization of Aqp1aa and basolateral localization of Aqp4 in collecting duct cells, with apical Aqp1aa downregulated in BW. These results suggest that Aqp1aa and Aqp4 mediate water reabsorption in SW and that downregulation of Aqp1aa contributes to hypotonic urine production in BW. NEW & NOTEWORTHYRegulation of water permeability in the collecting duct is important for osmoregulation in teleost fish. Expression analyses of aquaporins (Aqps) in the marine pufferfish Takifugu rubripes showed that Aqp1aa and Aqp4a are highly expressed in the collecting duct and localized to the apical and basolateral membranes, respectively. Renal Aqp1aa expression was markedly reduced in fish acclimated to hypoosmotic brackish water. These results indicate that collecting duct water permeability is regulated by Aqp1aa expression.

Study questionDoes the human placenta utilize the creatine phosphagen system for energy homeostasis during development? Summary answerComponents of the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system are dynamically expressed by the trophoblast and mesenchymal compartments throughout gestation wherein creatine kinase is required for cellular ATP metabolism, cell cycle, and proliferation of trophoblast cells. What is known alreadyThe Cr-CK-PCr system maintains ATP homeostasis in tissues with high energy demand and is required for proliferation, migration, and invasion of tumor cells. The term human placenta can synthesize and transport creatine locally. Early placental development involves trophoblast proliferation, an event requiring ATP, but the role of the creatine phosphagen system during early placental development remains unknown. Study design, size, durationWe performed immunohistochemistry (IHC) and immunofluorescence (IF) for different components (biosynthesis, transport, utilization) of the Cr-Ck-PCr system in human placentae (n=3/group) across gestation including first trimester, second trimester, and term. Using primary human trophoblast stem cells (hTSCs) and trophoblast organoids (TO), we determined the role of the creatine phosphagen system in trophoblast growth by functional inhibition of creatine kinase. Participants/materials, setting, methodsIHC/IF were performed in human placentae across gestation for proteins involved in biosynthesis (AGAT and GAMT), transport (SLC6A8, SLC22A15, and SLC6A13) and utilization (CKB and CKMT1) of creatine to determine the presence of the creatine phosphagen system locally in the placenta. For delineating the functional importance of this system in placental development, cyclocreatine (cCr), a creatine analogue, was used for functional inhibition of CK. Primary hTSCs were culture in medium containing 0 (control), 1, 10, 20 mM cCr for 48 hours followed by analysis of cell growth (cell count), cell cycle (EdU incorporation assay), apoptosis (Annexin V/PI flow cytometry), energy metabolism (Sea horse mito-stress and glycolytic stress tests), and gene expression (qPCR). Primary TO were also treated with 20mM cCr for 6 days in vitro to determine the role of Cr-CK-PCr system in placental development. Main results and the role of chanceAGAT localized to the fetal villous mesenchyme, while GAMT was broadly expressed in the trophoblast and fetal mesenchyme compartments across gestation. CKB localized primarily to fetal mesenchyme with strongest expression at term. CKMT1 was broadly expressed in all trophoblast subtypes. SLC6A8 was abundant in early syncytiotrophoblast but absent at term, where its expression shifted to fetal blood vessels. SLC22A15 was expressed in the endothelial cells of fetal capillaries across gestation. In primary hTSCs, cyclocreatine (20mM) treatment reduced proliferation (P<0.001), decreased expression of trophoblast epithelial marker EGFR (P<0.05), induced G0/G1 and G2/M arrests (P<0.0001), enhanced early and late apoptosis (P<0.0001), and downregulated GPX8 expression (P<0.05). Seahorse analysis revealed marked reductions (P<0.01) in mitochondrial (basal, maximal, and ATP-linked) and glycolytic (rate, capacity, and reserve) function compared to controls. In primary human TO, cyclocreatine treatment reduced the growth of organoids (P<0.05) as well the expression of EGFR (P<0.05). Large scale dataN/A Limitations, reasons for cautionFurther experiments assessing apoptosis, cellular stress and redox imbalance may provide more mechanistic role of the creatine phosphagen system in trophoblast metabolism and function. Since the functional role of the Cr-CK-PCr system was investigated in vitro, findings of this study should be taken with caution for implications of in vivo placental development. Nevertheless, reproducible results of reduced growth of trophoblast cells using both 2D and 3D cultures is highly suggestive of the importance of the creatine phosphagen system in early placental development. Wider implications of the findingsThis study provides foundational knowledge that the placenta contains the creatine phosphagen system, known for ATP homeostasis, and that this system ensures proper cell division, survival and placental development. Dysregulation of components of Cr-CK-PCr system in placenta has been observed in pregnancy disorders such as preeclampsia and fetal growth restriction warranting continued investigation into mechanisms and potential remediation using creatine supplementation. Stem cells share similar metabolic features so findings of this study can be implicated in other stem cells models as well. Study funding/competing interest(s)This work was supported by CIRM EDUC4-12804 Interdisciplinary Stem Cell Training Grant and a Lalor Foundation Postdoctoral Fellowship awarded to NS, and by the California Institute for Regenerative Medicine (DISC0-13757) and the National Institute of Child Health and Human Development (R01-HD096260) award to FS. The authors have no competing interest to declare.

Pulmonary arterial hypertension is a progressive, fatal disease driven by pathologic vascular remodeling including arterial medial hypertrophy, occlusive neointimal lesion formation, and venous muscularization. Current vasodilatory therapies improve hemodynamics but do not reverse established remodeling. Imatinib mesylate, a tyrosine kinase inhibitor targeting the PDGF-PDGFR signaling axis, has been proposed as an anti-remodeling therapy for pulmonary arterial hypertension and has demonstrated hemodynamic benefit in both preclinical models and clinical trials. However, prior preclinical models lack the neointimal lesions characteristic of human disease, effects on venous remodeling have not been examined, and direct histologic assessment in human trials is precluded by the invasiveness of serial lung biopsy. Here, leveraging the house dust mite mouse model of pulmonary hypertension, which recapitulates medial thickening, neointimal lesion formation, and venous muscularization, we rigorously evaluate the anti-remodeling and hemodynamic effects of imatinib during two defined remodeling stages: neointimal lesion growth and neointimal lesion maintenance. Imatinib treatment significantly reduced right ventricular systolic pressure at both stages. Despite this hemodynamic improvement, quantitative vessel-level analysis of over 1,700 arteries and 1,200 veins revealed no significant effect of imatinib on arterial medial thickness, neointimal lesion growth, neointimal lesion maintenance, or venous muscularization across any vessel size class. These findings dissociate imatinibs hemodynamic benefit from structural vascular remodeling and suggest that imatinib functions primarily as a pulmonary vasodilator rather than an anti-remodeling agent.

Diabetes is associated with endothelial dysfunction, impaired wound healing, and increased thrombotic risk, yet the impact of diabetes on endothelial secretory organelles remains poorly understood. Weibel-Palade bodies (WPBs) are specialised endothelial granules that store and release von Willebrand factor (VWF) and other vasoactive cargo essential for haemostasis, inflammation, and vascular repair. Here, we investigated how diabetic environments influence WPB biogenesis and VWF structure under physiologically relevant flow conditions. Acute exposure of endothelial cells to constant or fluctuating high glucose concentrations, designed to model diabetic glycaemic conditions, did not alter WPB number or morphology under either static or high laminar shear stress conditions. In contrast, primary endothelial cells derived from a diabetic donor exhibited reduced Akt and eNOS signalling, significantly fewer WPBs, reduced intracellular VWF content, and shorter stimulus-evoked VWF strings compared with non-diabetic endothelial cells. Although total cellular VWF levels were reduced, high molecular weight (HMW) VWF content within endothelial lysates was not significantly altered. Plasma from diabetic patients demonstrated elevated circulating VWF levels together with marked inter-patient heterogeneity in VWF multimer composition. These findings suggest that chronic diabetes-associated endothelial dysfunction, rather than hyperglycaemia alone, alters WPB biology and VWF handling. We propose that dysregulated basal endothelial secretion may deplete endothelial VWF stores, limiting appropriate stimulus-coupled WPB release during vascular injury and contributing to defective vascular repair in diabetes.

The heart maintains systemic perfusion through the coordinated function of its four chambers: the left and right atria and ventricles. Each chamber has distinct structural, functional, and molecular properties tailored to its role in circulation, which may result in chamber-specific differences in myofilament structure and regulation between atria and ventricles. To test this hypothesis, we employed muscle mechanics and X-ray diffraction to investigate functional and structural differences in porcine left atrial (LA) and left ventricular (LV) tissue. Here, we report the first X-ray diffraction study of atrial tissue, demonstrating that under resting conditions, myosin filaments in LA adopted a more ON-like, structurally distinct configuration compared with those in LV. Under contracting conditions, LV generated greater force and exhibited higher sinusoidal stiffness than LA across multiple calcium concentrations. LA showed faster kTR than in LV, with no calcium-dependence, in contrast to the calcium-dependence of kTR seen in LV. Structurally, the distinct myosin head configuration seen in the relaxed LA persisted during contraction. Furthermore, using the troponin inhibitor MYK-7660 to inhibit active contraction, we showed that, unlike LV, LA showed no direct calcium-dependent thick filament activation, reconciling discrepancies between fast rat and slow porcine ventricular myocardium regarding calciums role in thick filament regulation. Altogether, our study reveals that LA myosin filaments adopt a molecular architecture and regulatory mechanism distinct from their LV counterparts, suggesting that myosin filament structure and regulation have evolved differently to meet the unique functional demands of each cardiac chamber. Moreover, atrial disease is often associated with cardiomyopathy-related genetic variants, highlighting the atrial myocardium as an important therapeutic target and understanding atrial-specific regulatory mechanisms provides new insights into therapeutic strategies for atrial diseases.

Since NO can modulate mesenchymal cell function, we posit that NO can modulate gene expression associated with excitation-contraction coupling. Our study shows that treating asthma-derived HASMCs with a low dose of NO plus sGC stimulator BAY-41, in most cases sensitized smooth muscle sGC towards activation via an elevated sGC heterodimer and in some cases also improved sGC{beta}1, catalase, Cyb5r3 or Trx1 expression (n=24 non-asthma and n=25 asthma). Interestingly we found that majority of asthma HASMCs showed a marked downregulation of G6PD expression inducing a low GSH/GSSG ratio in asthma, and these findings were replicated in murine lungs of allergic asthma (OVA and CFA/HDM). Studies with HEK/COS-7 cells showed G6PD synergizing with hsp90 in enabling sGC heme-maturation. G6PD overexpression in HASMCs enhanced the sGC heterodimerization while silencing of endogenous G6PD abrogated it. Complementation of these cellular results with whole animal models of G6PD deficiency or overexpression provided verification to our findings. Mouse lung tissue from the humanized variant of G6PD deficiency, V68M (G6PD A-deficiency) showed significant downregulation in the sGC heterodimer, with a concomitant reduction in its NO heme-dependent activity, thereby showing that G6PD deficiency lowers sGC heme. Conversely, G6PD overexpressing mouse lung tissue displayed an elevated sGC heterodimer and also showed a robust G6PD-sGC{beta}1 interaction, suggesting G6PD to be involved in the heme-maturation of sGC{beta}1. While G6PD maintains the cell redox by generating NADPH, its new role in regulating sGC maturation links sGC dysfunction in asthma to G6PD deficiency and may potentially uncover new targets for asthma treatment.

A hallmark of damaged skeletal muscle fibers is displaced myonuclei that are no longer peripherally positioned. Displaced myonuclei are dogmatically thought to be derived exclusively from muscle stem cell (satellite cell) fusion. Using a surgical resection muscle injury model and in vivo recombination-independent resident myonuclear labeling, we detail the prevalence, time course, and origin of displaced myonuclei in response to a non-chemically-mediated muscle trauma. We found that: 1) non-satellite cell-derived (resident) displaced myonuclei emerge seven days after surgical injury in similar proportion to exogenous (satellite cell-derived) displaced myonuclei in intact muscle fibers, with a biased prevalence in myosin heavy chain IIB muscle fibers, 2) muscle fibers with multiple ([&ge;]2) displaced resident myonuclei was an unexpected but noteworthy feature of muscle fibers 7 days after injury, 3) embryonic myosin-expressing fibers at seven days post-surgery expectedly contain predominantly satellite-cell derived displaced myonuclei, but a subset have displaced resident myonuclei, and 4) satellite cell numbers in intact muscle do not increase until 7 days post-surgery. These data may help inform whether to target satellite cell-initiated processes, myonuclear-initiated processes, or both to facilitate muscle fiber injury repair. This information could lead to more effective therapeutic strategies for treating muscle trauma.

The adult skeletal muscle regenerates robustly upon injury, but this regenerative capacity rapidly declines with age. In this study, we identify the lanthionine synthetase C-Like (LanCL) proteins, mammalian homologs of the bacterial peptide cyclase LanC, as positive regulators of muscle regeneration in middle-aged mice. In a barium chloride-induced injury model, we found the protein levels of LanCL1 and LanCL2 to increase during an early phase of regeneration in middle-aged (12-month-old) but not young adult (4-month-old) mice. Utilizing a mouse line lacking all three LanCL proteins (LanCL triple KO or LTKO), we examined a potential role of LanCL in injury-induced muscle regeneration. Consistent with an age-dependent function of LanCL, we observed a delayed regeneration of the tibialis anterior (TA) muscle after injury, as reflected by reduced sizes of regenerating myofibers in middle-aged (but not young) LTKO compared to age-matched WT mice. Although the pool size of quiescent satellite cells (Pax7+) was comparable between 12-month-old LTKO and WT muscles without injury, the number of Pax7+ cells was significantly higher in regenerating LTKO muscles at day 5 after injury, accompanied by drastically decreased numbers of MyoD+ and MyoG+ cells, as well as increased numbers of proliferating cells. In addition, we detected elevated expression of pro-inflammatory cytokines in regenerating LTKO muscles, while the number of macrophages was similar comparing LTKO and WT muscles. Taken together, our observations suggest that in aging muscles LanCLs are important for proper timing of inflammation resolution and regeneration upon injury. New & NoteworthyPhysiological roles of the mammalian homologs of bacterial LanC, LanCLs, are poorly understood. Our work uncovers a function of LanCLs in post-injury regeneration of aging skeletal muscles. Middle-aged LanCL triple KO mice displayed a delay in satellite cell differentiation and regenerative myofiber formation, as well as persistent inflammatory cytokine expression, suggesting that LanCLs may have an age-dependent role in modulating inflammation in the injured muscles to facilitate regeneration.

AimMaternal iron deficiency (ID) during pregnancy induces cardiovascular adaptations, including reduced blood pressure and improved cardiac efficiency in hypertensive pregnancy. Iron is essential for mitochondrial function, particularly oxidative phosphorylation, where it serves as a cofactor within electron transfer complexes. Given the high metabolic demands of the maternal heart and irons central role in mitochondrial metabolism, we examined how maternal ID affects cardiac mitochondrial ultrastructure, respiration, dynamics, and redox status in pregnant spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Methods and ResultsFemale SHR and WKY rats were fed iron-replete or iron-restricted diets before and throughout gestation. On gestational day 21, cardiac mitochondrial ultrastructure was assessed by transmission electron microscopy (TEM), respiration by high-resolution respirometry, and the expression of proteins involved in fusion, fission, autophagy, and apoptosis markers by immunoblotting. Antioxidant gene expression was quantified by RT-qPCR. Data were analyzed by two-way ANOVA with Holm-Sidaks post hoc test. Maternal iron restriction reduced hemoglobin levels in both strains. TEM revealed enlarged, morphologically heterogeneous mitochondria with reduced and disrupted cristae architecture in ID dams of both strains. Iron restriction reduced succinate-supported respiration and tended to reduce NADH-supported respiration, in both strains. SHR dams exhibited reduced fusion signalling, reflected by a lower L-OPA1:S-OPA1 ratio. MFN1 expression was reduced by ID in both strains, whereas MFN2 expression was lower in SHR and further reduced by ID. In contrast, DRP1 phosphorylation increased selectively in ID-WKY dams. Iron restriction increased LC3-II:I ratio and BNIP3 in SHR, and increased PINK1 in both strains, while Parkin and p62 were unchanged. Antioxidant gene expression increased in ID-SHR but decreased in ID-WKY dams. Despite these alterations, markers of oxidative damage and apoptosis were unchanged by iron restriction. ConclusionMaternal ID induces marked remodeling of myocardial mitochondrial ultrastructure and selectively constrains iron-dependent respiration in hypertensive pregnancy without overt oxidative damage or apoptosis. These mitochondrial alterations occur alongside previously observed reductions in blood pressure and improved cardiac efficiency, suggesting favorable hemodynamic adaptations may coexist with underlying bioenergetic constraints in the maternal heart. Translational PerspectiveMaternal iron deficiency anemia (IDA) may alter the course of hypertensive pregnancy in ways not evident from hemodynamic indices alone. Here, IDA was associated with abnormal myocardial mitochondrial ultrastructure, selective reductions in respiratory capacity and stress response pathways, despite previously observed improvements in blood pressure and cardiac efficiency. These findings suggest that favourable hemodynamic changes may reflect reduced metabolic demand rather than enhanced bioenergetic capacity. If confirmed in human pregnancy, management of ID in women with underlying hypertension may need closer attention to cardiac metabolic health, as cardiovascular adaptions could coexist with myocardial stress and may vary with anemia severity and duration.

Sympathetic neuronal (SN) activity critically regulates the development and function of peripheral organs and tissues. Activity-dependent plasticity has been shown to modulate SN output, suggesting that compensatory forms of plasticity could contribute to maintaining stability of sympathetic circuits. Early SN hyperactivity drives the development of hypertension in humans and in the spontaneously hypertensive rat (SHR). In this study we used chemogenetic and pharmacological approaches, and took advantage of the enhanced activity of SHR SNs, to examine how long-term changes in activity impact synaptic properties in neonatal SN cultures. We showed that bidirectional changes in SN activity result in compensatory shifts in synaptic density that counteract long-term activity manipulations. These changes were mediated by satellite glial cells (SGCs), a non-neuronal cell in the sympathetic ganglia that has been shown to influence cholinergic synaptic sites during development. In the absence of SGCs there was no induction of homeostatic plasticity. Further, direct chemogenetic activation of SGCs was sufficient to drive compensatory plasticity, while glial inhibition blocked SN plasticity. We found that SGCs respond to cholinergic signaling by downregulating the expression of the synaptic regulators NGF and TNF, suggesting that neurons and glia interact to stabilize sympathetic output during long-term changes in circuit activity. Finally, we investigated whether these plasticity mechanisms are present in neonatal SHR SNs. We demonstrated that SHR SNs have an attenuated response to glia, both during synapse formation and activity-dependent plasticity. Taken together, this work outlines a novel homeostatic activity-dependent plasticity mechanism in the peripheral nervous system.

BackgroundPeripheral artery disease is a major manifestation of atherosclerotic cardiovascular disease (ASCVD) that affects both men and women. In women, menopause increases the ASCVD risk. However, preclinical ASCVD research has historically been conducted predominantly in males, with relatively few studies focused on females and even fewer incorporating menopause models that more closely reflect human ASCVD pathobiology. Herein, we tested whether the chemical 4-vinylcyclohexene diepoxide (4-VCD)-induced ovarian failure or ovariectomy (OVX) would drive atherosclerotic development and worsen ischemic limb pathophysiology. MethodsFemale C57BL/6J mice were injected with adeno-associated virus-mediated encoding a gain-of-function mutant PCSK9 and fed an atherogenic diet for 23 weeks. Based on the baseline body weight, mice were randomly assigned to normally cycling controls (CON), 4-VCD, or OVX groups. Three weeks after the conformation of ovarian failure (4-VCD) or surgical ovarian removal (OVX), hindlimb ischemia (HLI) was induced via femoral artery ligation, and limb perfusion recovery and limb muscle performance were assessed. ResultsBoth 4-VCD treatment and OVX reduced uterus mass, without impacting body weight or composition, or circulating cholesterol levels compared to CON mice. Despite the similar metabolic and cholesterol profiles, atherosclerotic lesion areas were 1.5-1.7-fold greater in 4-VCD and OVX mice than CON mice. Perfusion recovery following HLI and plantar flexor muscle function in the ischemic limb were similar across groups, though muscle oxygenation was reduced in 4-VCD and OVX groups. ConclusionsOvarian failure and removal exacerbated atherosclerotic development but had minimal impacts on perfusion recovery and limb function following HLI. These findings confirm the inclusion of menopausal models, whether through ovarian failure or OVX, should be carefully considered to improve translatability of preclinical ASCVD studies, especially for womens health. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSWe demonstrate that both gradual ovarian failure (4-VCD) and surgical ovariectomy exacerbate atherosclerotic plaque development in a clinically relevant AAV-PCSK9 model, despite similar circulating lipid levels. In contrast, loss of ovarian function did not impair limb perfusion recovery or muscle functional outcomes following hindlimb ischemia, revealing a dissociation between atherosclerotic burden and limb functional recovery in experimental peripheral artery disease (PAD). What are the Clinical Implications?These findings provide new insight into why menopause increases atherosclerotic cardiovascular disease (ASCVD) risk while not necessarily demonstrating proportional impairments in limb recovery following ischemia. The data suggest that menopause-associated factors accelerate large-vessel atherosclerosis independent of circulating lipids, highlighting the need for targeted therapies beyond lipid lowering in postmenopausal women. Moreover, the dissociation between plaque burden and ischemic limb function underscores the importance of assessing functional outcomes in PAD independently of vascular imaging. Finally, these findings suggest that the incorporation of menopause-relevant models in preclinical research should be considered within the context of the specific biological endpoints and translational goals being evaluated.

Sympathetic preganglionic neurons (SPNs) distribute signals widely across paravertebral ganglia, yet the reliability of spike propagation along their predominantly unmyelinated axons remains poorly defined. We examined temperature- and activity-dependent modulation of SPN axonal conduction using an ex vivo adult mouse thoracic sympathetic chain preparation. Population compound action potentials (CAPs) were evoked by supramaximal stimulation of T10 ventral roots and recorded from branching axons in interganglionic compared to unbranching axons in the splanchnic nerve. At physiological temperature (36{degrees}C), scaled CAP magnitude was reduced by [~]50% relative to 22{degrees}C, with preferential loss of slower-conducting axonal components. These reductions are consistent with substantial temperature-dependent decreases in effective axonal recruitment, likely reflecting conduction failure in a large fraction of SPNs. Losses were more pronounced in interganglionic pathways, suggesting increased vulnerability in branching projections. To assess activity-dependent effects, stimuli were delivered at 1, 5, and 20 Hz with focus on 5 and 20 Hz stimulus trains (20s duration). The overall time-course of train-evoked depression was similar across temperatures; however, the underlying axonal populations differed. At 22{degrees}C, slower-conducting axons exhibited marked frequency-dependent depression, whereas at 36{degrees}C the remaining faster-conducting axons displayed facilitation, particularly at 20 Hz. Slower-conducting responses also showed post-train potentiation at physiological temperature. These findings indicate that SPN axonal conduction is not uniformly reliable and is strongly modulated by temperature and activation history. Preferential vulnerability of slow-conducting, likely small-diameter and branching axons identifies axonal conduction as a physiologically regulated site of gain control in sympathetic output.

Mechanisms that promote organ injury after trauma and hemorrhagic shock (T/HS) remain poorly defined. Endothelial heparan sulfates with a 3-O-sulfate (3-OS) modification, controlled by the HS3ST1 gene, have anticoagulant and anti-inflammatory properties through their interaction with antithrombin. Our objective was to determine whether HS3ST1 deficiency drives organ injury and poor outcomes after T/HS. Hs3st1-/- and wild-type (WT) mice were subjected to T/HS followed by resuscitation with lactated ringers (LR) or fresh frozen plasma (FFP). While no differences were observed between WT and Hs3st1-/- LR resuscitated mice, lung injury and leukocyte infiltrates were significantly increased in FFP resuscitated Hs3st1-/-compared to WT mice. In vitro, leukocyte slow rolling and adherence was increased in HS3ST1 KO compared to WT cells. Among 472 T/HS patients, of which 31 (7%) were homozygous for the rs16881446 variant allele (GG), the number of ventilator free days was lower, and mortality was significantly higher in AG and GG patients. The rs16881446 genotype was independently associated with mortality. In conclusion, HS3ST1 deficiency mitigates organ protection from FFP resuscitation, partly through mediating EC:leukocyte engagement, and predicts mortality after T/HS. These findings identify a novel therapeutic target and prognostic tool that can be leveraged towards improved risk stratification after trauma.

We investigated effects of three aerobic exercise interventions, varying in amount and intensity with durations of 8-9-months on small RNA (smRNA) expression and regulatory pathways in skeletal muscle and plasma from 120 participants. Using untargeted smRNA sequencing focused on miRNAs and piRNAs, adjusting for demographics and bodyweight, we identified 124 muscle smRNAs altered by exercise amount and 15 by intensity, and 47 plasma smRNAs altered by intensity and one by amount. These smRNAs were enriched in metabolic, transcriptional, translational, and cell cycle pathways. Exercise-induced changes in several smRNAs-six from muscle and five from plasma-and exercise-induced reduction in body weight, aligned with improvement in insulin sensitivity (p<0.05). These findings demonstrate tissue-specific regulation of smRNAs by exercise and identify potential candidates for exercise mimetics to modulate muscle insulin sensitivity.

Iron absorption in the small intestine has classically been described by the duodenal DMT1/FPN1 pathway for inorganic non-heme iron, yet emerging evidence suggests that chemically distinct iron forms may use region-specific routes. Nicotianamine (NA), a plant-derived metal chelator, can form NA-iron (NA-Fe) complexes and has been proposed to support intestinal iron absorption through amino acid transporter pathways. However, direct comparisons of transepithelial transfer of inorganic iron and NA-Fe across defined small intestinal regions under controlled epithelial conditions remain limited. Here, we established region-specific 2D epithelial monolayers derived from duodenal and proximal jejunal crypt organoids from male ICR mice cultured on Transwell inserts. Transcriptomic profiling indicated partial retention of regional identity, and barrier integrity was confirmed by junctional marker localization, transepithelial electrical resistance, and low paracellular permeability. We then examined expression and polarized localization of candidate transporters for inorganic iron (Dmt1/Fpn1) and NA-Fe (Pat1/Lat2). Finally, we quantified transepithelial transport using apical loading of isotope-labeled iron (55Fe) or NA-55Fe and measured radioactivity appearing in the basolateral compartment as the primary readout of transepithelial flux. Basolateral appearance of inorganic 55Fe was comparable between duodenum- and proximal jejunum-derived monolayers, whereas NA-55Fe exhibited significantly greater basolateral appearance in proximal jejunum-derived monolayers. These findings demonstrate that organoid derived, region-specific monolayers provide a tractable epithelial platform to evaluate iron form-dependent, region-specific transepithelial transfer and to enable further mechanistic dissection of NA-Fe transport. NEW & NOTEWORTHYNon-heme iron absorption may depend on iron chemical form and intestinal region, but direct epithelial comparisons are scarce. We established duodenum and proximal jejunum derived murine intestinal organoid monolayers on Transwells and quantified transepithelial flux using isotope-labeled iron. Inorganic 55Fe showed no clear regional difference, whereas NA-55Fe displayed greater basolateral appearance in proximal jejunum-derived monolayers. This platform enables mechanistic studies of NA-iron complex transport.

PurposeThe para-cycling classification system aims to minimize the impact of impairments on competition outcomes with the help of scientific evidence. This study investigated the impact of unilateral and bilateral ankle immobility on cycling performance, quantified by the maximal average mechanical power output (AMPO) over one revolution relative to that without ankle immobility. MethodsTen well-trained non-disabled cyclists performed all-out 6-second sprints on a cycle ergometer at 120 rpm under three conditions: without ankle foot orthoses (AFOs), with 1 AFO and with 2 AFOs immobilizing the ankle joint(s). Mechanical power output, pedal forces, cycling kinematics and surface-electromyography were measured. Maximal AMPO; ankle, knee and hip joint AMPO; and the amount of muscle excitation were calculated. ResultsWith 1 AFO and 2 AFOs, respectively, maximal AMPO was 96% (p<0.05) and 91% (p<0.001) of that without AFOs (1188 W). The decrease in maximal AMPO with ankle immobilization was less than the decrease in ankle joint AMPO (126 W decrease with 2 AFOs; p<0.001), due to an increase in hip joint AMPO (69 W increase with 2 AFOs; p<0.05). The amount of muscle excitation was not significantly different across conditions. ConclusionsThese findings provide a first quantitative and mechanistic indication of the impact of ankle immobility on cycling performance, which may offer valuable evidence to support the development of an evidence-based para-cycling classification system.