Function

BackgroundIndividuals with spinal cord injury (SCI) experience accelerated atherosclerotic cardiovascular disease that is not fully explained by traditional risk factors. Endothelial dysfunction is a key mechanism in atherosclerosis. We tested the hypothesis that endothelium-dependent vasodilation is impaired in adults with SCI and is due, at least in part, to oxidative stress. MethodsTwenty-four adults (age:19-58 yr) free of overt cardiometabolic disease were studied: 12 non-injured adults (9 M/3 F) and 12 adults with chronic SCI (8 M/4 F; time since injury 1.5 - 25 years). Forearm blood flow was determined (FBF; via strain-gauge plethysmography) in response to intra-arterial infusion of acetylcholine and isoproterenol in the absence and presence of the antioxidant vitamin C as well as the FBF response to sodium nitroprusside. ResultsAdults with SCI demonstrated significantly lower vasodilator response to acetylcholine (from 4.1{+/-}0.6 to 10.7{+/-}2.6 mL/100 mL tissue/min vs 4.1{+/-}1.1 to 15.7{+/-}3.4 mL/100 mL tissue/min) and isoproterenol (4.0{+/-}0.6 to 11.2{+/-}2.2 mL/100 mL tissue/min vs 4.3{+/-}1.0 to 15.0{+/-}2.6 mL/100 mL tissue/min) compared with non-injured adults. FBF response to sodium nitroprusside was not significantly different between the groups. Co-infusion of vitamin C significantly increased the vasodilator response to acetylcholine (~45%) and isoproterenol (~25%) in the adults with SCI to levels comparable with non-injured adults. ConclusionsChronic SCI is associated with endothelial-dependent vasodilator dysfunction. Impaired vasodilation across two distinct endothelial agonists suggests that chronic SCI is associated with endothelial dysfunction not confined to a specific receptor or intracellular signaling pathway. Moreover, oxidative stress is a contributing factor underlying SCI-related endothelial vasodilator dysfunction. NCT06443151 CLINICAL PERSPECTIVEO_LIThe novel finding of this study is that individuals with SCI demonstrate impaired endothelial vasodilator function in absence of traditional cardiovascular risk factors. C_LIO_LIOxidative stress is a contributing factor to SCI-related endothelial vasodilator dysfunction. C_LIO_LIFuture studies are needed to determine the efficacy of therapeutic interventions, either lifestyle or pharmacologic, in improving endothelial function in order to mitigate the elevated ASCVD risk after SCI. C_LI

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely recognized to potentially interfere with skeletal muscle regeneration. However, current knowledge is based almost exclusively on non-aspirin NSAIDs. Aspirin (ASA) differs from other NSAIDs in its ability to irreversibly acetylate cyclooxygenase-2 (COX-2), thereby redirecting its activity toward a lipoxygenase (LOX)-like function that enables the production of unique ASA-triggered specialized pro-resolving lipid mediators (AT-SPMs). Despite this, the potential impact of ASA on musculoskeletal tissue repair remains poorly understood. This study directly compared the effect of ASA against non-ASA NSAIDs on in vitro myogenesis and in vivo skeletal muscle injury and regeneration. Unlike non-ASA NSAIDs, including indomethacin (INDO), celecoxib, and SC-236, which markedly impaired C2C12 myotube formation at concentrations near their pharmacological ranges, ASA only interfered with myogenesis at overtly supraphysiological concentrations. In mice, an oral dose of 3 mg/kg/day INDO following barium chloride-induced muscle injury reduced regenerating myofiber cross-sectional area and impaired the recovery of muscle force-generating capacity. In contrast, a potency-matched oral treatment with 30 mg/kg/day ASA hastened the resolution of cellular inflammation, promoted myonuclear accretion, and improved recovery of absolute muscle strength. The beneficial effects of ASA on inflammatory resolution and muscle strength--but notably not myonuclear accretion--were reversed in mice co-treated with ASA + INDO. These findings demonstrate that, unlike non-ASA NSAIDs, ASA does not impair skeletal muscle regeneration and may promote a favorable early inflammatory environment for repair via unique COX-dependent pro-resolving and COX-independent anabolic mechanisms.

BACKGROUNDVascular endothelial cells (ECs) experience two different blood flow patterns: laminar and disturbed flows. Their responses to laminar flow contribute to vascular homeostasis, whereas their responses to disturbed flow result in EC dysfunction and vascular diseases. However, it remains unclear how ECs differentially sense laminar and disturbed flows and trigger signalings that elicit different EC responses. We aimed to investigate EC flow-sensing and signaling mechanisms, focusing on the role of the plasma membrane and mitochondria. METHODSWe exposed cultured human aortic ECs to laminar flow and disturbed flow in flow-loading devices and used real-time imaging with optical probes to examine changes in the lipid order of the plasma and mitochondria membranes and the mitochondrial adenosine triphosphate (ATP) production and hydrogen peroxide (H2O2) release. RESULTSThe lipid order of EC plasma membranes immediately decreased in response to laminar flow, while it increased in response to disturbed flow. Laminar flow also decreased the lipid order of mitochondrial membranes and increased mitochondrial ATP production. In contrast, disturbed flow increased the lipid order of mitochondrial membranes and increased the release of H2O2 from mitochondria. Addition of cholesterol to the cells increased the lipid order of both membranes and abrogated the laminar flow-induced ATP production, while treatment of the cells with a cholesterol-depleting reagent, methyl-{beta} cyclodextrin, decreased the lipid order of both membranes and abolished the disturbed flow-induced H2O2 release, indicating that the changes in the membrane lipid order are closely linked to the flow-induced changes in the mitochondrial functions. CONCLUSIONSECs differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes in opposite directions, which result in distinct changes in the mitochondrial functions, namely, increased ATP production for laminar flow and increased H2O2 release for disturbed flow, leading to ATP- and H2O2-mediated signalings, respectively.

Regular exercise induces adaptations in skeletal muscle and other organ systems to improve physical performance and overall health. Exercise results in phosphorylation of 5 AMP-activated protein kinase (AMPK) at threonine 172 (T172) of the 2 subunit; however, the role of this activation in cellular and functional adaptations has not been elucidated. To this end, we subjected non-activatable Ampk2(T172A) knock-in (KI) adult mice and wild-type (WT) littermates to 4 weeks of voluntary wheel running (VWR). Exercise training led to significant improvements in endurance capacity, maximal oxygen consumption ([Formula]O2max), and glucose tolerance, as well as skeletal muscle IIb-to-IIa fiber type shift in both WT and KI mice. Contrastingly, VWR resulted in increased mitochondrial OxPhos protein expression, mitochondrial volume density, and capillary density in skeletal muscle of WT but not KI mice. Exercise-induced improvements of mitochondrial respiration and conductance revealed by high-resolution respirometry of isolated mitochondria were blunted in KI mice. Therefore, for the first time, we reveal that AMPK2 T172 activation is required for exercise training-induced mitochondrial biogenesis, improvement of mitochondrial respiratory function, and angiogenesis in skeletal muscle, but that these adaptations are not solely responsible for improved [Formula]O2max and exercise endurance capacity. Significance StatementExercise is the most effective lifestyle intervention for promoting health and preventing chronic diseases through adaptive changes in skeletal muscle and many other tissues/organs. AMPK is an energy sensor and signaling regulator for exercise-induced skeletal muscle adaptation, yet its functional role and the impact on exercise capacity have been studied in mouse genetic models wherein protein stoichiometry is disrupted. Using non-activatable Ampk2(T172A) knock-in mice, we ascertained that AMPK2 activation via T172 phosphorylation is required for endurance training-induced mitochondrial and angiogenic adaptations in skeletal muscle. Importantly, these adaptations are not required for improved exercise capacity, challenging the prevailing concept that increased mitochondrial content and function and microvasculature are the sole driving factors for the performance gains with endurance training.

We tested the hypothesis that the ubiquitous store-operated Ca2+ entry (SOCE) pathway contributes to histone-induced endothelial Ca2+ events. We also considered an alternate hypothesis: cationic electrostatic interactions between histones and negatively charged phospholipids deform endothelial membranes and thereby allow extracellular Ca2+ entry. A role for SOCE in histone responses was ruled out by genetic ablation of the ORAI1/2/3 channel trio; yet, histone effects were blocked by application of the multivalent cation gadolinium Gd3+. Using live cell video microscopy of endothelial cells labeled with membrane dye FM1-43, we recorded plasma membrane movements including vesiculation, blebbing, and ruffling of lamellipodia over 60 minutes following histone exposure. These cell membrane theatrics were markedly different from the uniform pattern of exocytosis and subsequent blebbing produced by calcium overload with ionomycin. The membrane permeabilization produced by histones, and not ionomycin, was transient and a subset of cells recovered membrane integrity within 1 hour. Removal of extracellular Ca2+ prevented histone-induced intracellular Ca2+ overload while surprisingly exacerbating plasma membrane deformation. Conversely, decreasing the density of the negative charge surface by adding calcium or or increasing extracellular Ca2+ levels effectively screened common membrane phospholipids from interactions with labeled histones and prevented endothelial damage in cells exposed to histones. Collectively these results indicate that low extracellular Ca2+ levels enhance interactions between histones and endothelial cell membrane phospholipids to increase cytotoxicity. Importantly, this supports the concept of aggressive Ca2+ repletion during resuscitation to prevent hypocalcemia, stabilize endothelial cell membranes and improve cardiovascular recovery from shock. SignificanceIn acute critical illness, the rapid collapse of vascular endothelial functions drives aberrant blood clotting and organ failure through mechanisms that are not understood. Emerging evidence that early administration of donor plasma improves survival of trauma patients has transformed the massive transfusion protocols used in surgical settings, but the sodium citrate included in transfused blood products to prevent coagulation often produces significant and severe hypocalcemia. Here, we demonstrate that cytotoxic trauma factors that are elevated in the blood during resuscitation interact electrostatically with endothelial cell phospholipids, and that low Ca2+ exacerbates toxicity by increasing this interaction. Using high speed video imaging, we demonstrate fast endothelial cell membrane movements in response to injury, including protrusion and ruffling of lamellipodia, release and reuptake of extracellular vesicles, and blebbing. These findings provide important insights into the nature of shock-induced endotheliopathy and highlight the potential cardiovascular risk associated with chelation-induced hypocalcemia during resuscitation.

Respiration and blood pressure (BP) are regulated to maintain optimal delivery of O2 to every cell in the body. Arterial hypoxemia is sensed by the carotid body (CB), which initiates sympathetic reflex arcs to the diaphragm to increase ventilation, and to the adrenal medulla (AM) to increase catecholamine secretion and thereby increase BP. However, the underlying molecular mechanisms have not been fully delineated. Here, we report that the relative activities of hypoxia-inducible factor-1 (HIF-1) and HIF-2 determine the set point for the CB and AM, with respect to their maintenance of BP and respiration. In Hif2a+/- mice, which are heterozygous for a knockout allele at the locus encoding HIF-2, expression of HIF-1 and NADPH oxidase 2 was increased in the CB and AM, resulting in an oxidized intracellular redox state with augmented sensitivity to hypoxia, increased BP, and respiratory abnormalities, which were all normalized by treatment with a HIF-1 inhibitor or a superoxide anion scavenger. By contrast, in Hif1a+/- mice, which are heterozygous for a knockout allele at the locus encoding HIF-1, the expression of HIF-2 and superoxide dismutase 2 was increased in the CB and AM, resulting in a reduced intracellular redox state with impaired CB and ventilatory responses to chronic hypoxia, which were normalized by treatment with a HIF-2 inhibitor. None of the abnormalities that were observed in Hif1a+/- or Hif2a+/- mice were observed in Hif1a+/-; Hif2a+/- double- heterozygous mice. Our results demonstrate that redox balance in the CB and AM, which is determined by mutual antagonism between HIF- isoforms, establishes the set point for responses of the CB and AM to hypoxia, and is required for the maintenance of normal BP and respiration.

Ankle clonus is a sustained, involuntary, rhythmic muscle contraction frequently observed in humans with spinal cord injury (SCI). Although its pathophysiology remains incompletely understood, converging evidence suggests a role for brainstem systems in its generation. Following SCI, brainstem neuromodulatory inputs partially compensate for the loss of descending motor pathways by regulating motoneuron excitability during involuntary contractions, suggesting their involvement in the generation of clonus. To test this hypothesis, motoneuron excitability in response to Ia synaptic input was quantified using the soleus H reflex and maximal motor response (H/M ratio), and brainstem involvement was probed using the long lasting component of the cutaneous reflex (LLR) in the tibialis anterior and soleus muscles, as well as the StartReact response-an involuntary release of a movement triggered by a startling stimulus thought to engage the reticulospinal tract. We studied individuals with chronic SCI, both with and without ankle clonus, using standardized clinical tests across two days. Participants with clonus showed elevated H/M ratios, indicating increased motoneuron excitability, whereas those without clonus exhibited lower values than controls. Additionally, individuals with clonus exhibited longer LLR duration and greater LLR magnitude in both muscles, along with shorter reaction times to startle stimuli, consistent with enhanced monoaminergic and reticulospinal contributions. Notably, LLR duration was positively correlated with both StartReact response and H/M ratio. Together, these findings support a role for descending brainstem systems-particularly monoaminergic and reticulospinal pathways-in the maintenance of clonus in chronic SCI.

Sedentary behavior promotes chronic low-grade inflammation in adipose tissue, contributing to metabolic dysfunction and insulin resistance. High-intensity interval training (HIIT) is a time-efficient exercise strategy with potent anti-inflammatory and metabolic benefits; however, its effects on adipose tissue inflammatory signaling and microRNA (miRNA) regulation remain incompletely understood. This study investigated the effects of eight weeks of HIIT on inflammatory and epigenetic markers in interscapular white adipose tissue (iWAT) of male Wistar rats. Fourteen rats were randomly assigned to either a sedentary (SED; n = 7) or HIIT (n = 7) group. The HIIT protocol consisted of treadmill running five days per week for eight weeks. Body weight and iWAT mass were assessed, and molecular adaptations were evaluated at multiple regulatory levels using RT-qPCR for mRNA targets (NLRP3, TNF-, PPAR-{gamma}, and IL-10) and miRNAs (miR-21 and miR-30d-5p), while protein levels of NLRP3 and PPAR-{gamma} were assessed using Western blotting. Compared with the SED group, HIIT significantly reduced body weight (p < 0.001) and iWAT mass (p = 0.002). Furthermore, HIIT downregulated the expression of pro-inflammatory mediators, including NLRP3 (gene: p = 0.001; protein: p < 0.001) and TNF- (p = 0.025), while upregulating anti-inflammatory regulators PPAR-{gamma} (gene: p = 0.026; protein: p = 0.020) and IL-10 (p = 0.010). In parallel, inflammation-associated miRNAs, including miR-21 (p = 0.004) and miR-30d-5p (p = 0.002), were markedly downregulated. These coordinated transcriptional, post-transcriptional, and translational adaptations suggest that HIIT attenuates adipose tissue inflammation and promotes a favorable immunometabolic phenotype through integrated molecular and epigenetic mechanisms.

BackgroundChronic obstructive pulmonary disease (COPD) patients often experience skeletal muscle dysfunction that may result from a complex combination of mitochondrial dysfunction, metabolic reprogramming and fibre type transitions. Among other factors, pulmonary inflammation and hypoxia contribute to the COPD-associated muscle defects. Nevertheless, the precise molecular mechanisms and their effects across muscles with distinct metabolic profiles remain elusive. This study investigated the independent and combined effects of chronic pulmonary inflammation and chronic hypoxia on mitochondrial function, metabolic enzyme activity and fibre type composition in oxidative (soleus) and glycolytic (plantaris) muscles. MethodsAdult male Wistar rats were subjected to chronic hypoxia (FiO2=10%) and/or chronic pulmonary inflammation (induced by bi-weekly intratracheal instillations of lipopolysaccharides). After 4 weeks of exposure, mitochondria from both soleus and plantaris were isolated to measure oxygen consumption rates, reactive oxygen species (ROS) emission, calcium retention capacity (CRC), respiratory complex activities and fatty acid oxidation capacity. Cross-sections from both muscles were analysed for fibre typology, fibre cross-sectional area (fCSA) as well as succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (GPDH) activities. ResultsChronic hypoxia led to a decline in adenosine diphosphate-stimulated complex I (CI) respiration (p<0.01), Hydroxyacyl-Coenzyme A dehydrogenase activity (p<0.01), weight (p<0.05) and fCSA of type IIb fibres (p < 0.05) in plantaris. In contrast, chronic hypoxia increased CI-derived ROS emission (p<0.01) without changes in mitochondrial respiration or mass of soleus. No alterations in fibre typology were noticed in either muscle following the hypoxic exposure. Chronic pulmonary inflammation caused a reduction in mitochondrial CRC and an increase in GPDH activity in type IIa fibres of soleus (p<0.001) without any changes in fibre type distribution. Conversely, chronic pulmonary inflammation induced a downregulation of GPDH activity in plantaris type I and type IIa fibres, in parallel with an elevation in the SDH/GPDH ratio across all fibre types and a rise in the proportion of type IIx fibres. ConclusionsOur results demonstrate fundamental differences in the responses to chronic hypoxia and chronic pulmonary inflammation between soleus and plantaris. While hypoxia affects predominantly the mitochondrial function and mass of plantaris, pulmonary inflammation drives metabolic reprogramming in both muscles that opposes their intrinsic functional specialisation. Additionally, soleus appears more vulnerable to permeability transition pore opening following pulmonary inflammation. Notably, these mitochondrial alterations seem to occur independently of fibre type shifts highlighting the central role of intrinsic mitochondrial maladaptations in the COPD-associated muscle dysfunction.

Anterior cruciate ligament (ACL) rupture leads to muscle deconditioning and downregulation of Na+,K+-ATPase (NKA). Low-load blood flow restriction (LL-BFR) training was shown to improve muscle function after ACL injury, but its effects on NKA are unknown. We analysed expression of NKA and its FXYD regulators in knee muscles from ACL-injured subjects undergoing LL-BFR, low-load training with sham blood flow restriction (LL-Sham), or no training (Control). Additionally, we dissected effects of ischemia components by subjecting cultured human myotubes to glucose deprivation and/or hypoxia. The LL-BFR group had higher vastus lateralis mRNA levels of NKA1, NKA{beta}3, and FXYD5 than the LL-Sham group. In vitro, NKA1, NKA{beta}1, and NKA{beta}3 mRNA and NKA1 and NKA{beta}1 protein levels were downregulated by sustained ischemia and glucose deprivation, but not hypoxia, while FXYD5 protein was upregulated by glucose deprivation. Conversely, intermittent ischemia had no effect on NKA or FXYD expression. In conclusion, our study shows that LL-BFR training induces specific transcriptional adaptations in vastus lateralis after ACL injury, potentially contributing to functional improvements. Moreover, it shows that glucose availability plays a major role in modulating NKA and FXYD expression in muscle cells under ischemic conditions. NEW & NOTEWORTHYO_LIThis is the first study exploring effects of low-load BFR training on Na+,K+-ATPase (NKA) and FXYD expression in knee muscles after anterior cruciate ligament injury. C_LIO_LIEffects of ischemia components were analysed by subjecting cultured human myotubes to glucose deprivation and/or hypoxia. C_LIO_LILL-BFR training induces specific transcriptional adaptations in vastus lateralis after ACL injury, potentially contributing to functional improvements. C_LIO_LIGlucose plays a major role in modulating NKA and FXYD expression in cultured myotubes under ischemic conditions. C_LI

BackgroundSkeletal muscle in wasting conditions often exhibits a common set of phenotypes that include atrophy, mitochondrial respiratory dysfunction, and fragmentation of the acetylcholine receptor (AChR) cluster at the endplate. Mitochondria are frequently implicated in driving muscle pathology in these conditions, although which aspects of mitochondrial function are most relevant is poorly understood. MethodsTo address this gap, we focused on mitochondrial permeability transition (mPT), a well-established pathological mechanism in ischemia-reperfusion injury and neurodegeneration but poorly studied in skeletal muscle. We performed a broad assessment of the consequences of mPT in skeletal muscle, focusing on features that are common in wasting conditions. We then tested whether tumor-host factors could promote mPT and compared differentially expressed genes (DEGs) with mPT and a mouse model of pancreatic cancer cachexia. ResultsInducing mPT in mouse skeletal muscle bundles in a Ca2+ retention capacity assay progressively altered mitochondrial morphology, beginning with cristae swirling and condensation, progressing to mitochondrial cristae displacement, and culminating in breach of the outer mitochondrial membrane; features that are common in wasting conditions. Inducing mPT with Bz423 in single mouse muscle fibers increased mROS and Caspase 3 (Casp3) activity and was prevented by inhibitors of mPT, mROS or Casp3. Incubating single muscle fibers with Bz423 for 24 h reduced fiber diameter by [~]20% which was prevented by inhibiting mPT, mROS, or Casp3. Inducing mPT caused a complex I-specific mitochondrial respiratory impairment and increased co-localization of lysosomes with mitochondria. Inducing mPT also fragmented the AChR cluster at the muscle endplate and was prevented by inhibiting mPT or Casp3. The Ca2+ threshold for mPT and mitochondrial calcein colocalization were reduced by pancreatic tumor-conditioned media in skeletal muscle or C2C12 myoblasts, respectively, and these effects were counteracted by mPT inhibition or cyclophilin D knockout. Finally, there was significant overlap between the transcriptome of mPT and that seen in diaphragm muscle in a mouse model of pancreatic cancer cachexia, particularly during the muscle wasting phase. ConclusionsWe conclude that inducing mPT in skeletal muscle recapitulates muscle phenotypes common with muscle wasting conditions like cachexia. Furthermore, mPT is engaged by tumor-host factors and had significant overlap with DEGs seen during the muscle wasting phase in a mouse model of pancreatic cancer cachexia, warranting further investigation of mPT as a therapeutic target.

S-nitrosylation of Cx43 gap junction channels critically regulates communication between smooth muscle cells and endothelial cells. This posttranslational modification also induces the opening of undocked Cx43 hemichannels. However, its specific impact on vasomotor regulation remains unclear. Considering the role of endothelial TRPV4 channel activation in promoting vasodilation through nitric oxide (NO) production, we investigated the direct modulation of endothelial Cx43 hemichannels by TRPV4 channel activation. Using the proximity ligation assay, we identify that Cx43 and TRPV4 are found in close proximity in the endothelium of resistance arteries. In primary endothelial cell cultures from resistance arteries (ECs), GSK-induced TRPV4 activation enhances eNOS activity, increases NO production, and opens Cx43 hemichannels via direct S-nitrosylation. Notably, the elevated intracellular Ca2+ levels caused by TRPV4 activation were reduced by blocking Cx43 hemichannels. In ex vivo mesenteric arteries, inhibiting Cx43 hemichannels reduced endothelial hyperpolarization without affecting NO production in ECs, underscoring a critical role of TRPV4/Cx43 signaling in endothelial electrical behavior. We perturbed the proximity of Cx43/TRPV4 by disrupting lipid rafts in ECs using {beta}-cyclodextrin. Under these conditions, hemichannel activity, Ca2+ influx, and endothelial hyperpolarization were blunted upon GSK stimulation. Intravital microscopy of mesenteric arterioles in vivo further demonstrated that inhibiting Cx43 hemichannels activity, NO production and disrupting endothelial integrity reduce TRPV4-induced relaxation. These findings underscore a new pivotal role of Cx43 hemichannel associated with TRPV4 signaling pathway in modulating endothelial electrical behavior and vasomotor tone regulation.

Brain activity is costly, and aerobic metabolism supplies [~]90% of the ATP for cellular and synaptic function. Accordingly, oxygen consumption varies to match activity demands of neural circuits, and mitochondrial defects that reduce aerobic metabolism cause various neurological disorders. Brain activity in frogs has energy demands typical of an average vertebrate, but surprisingly, improves function upon stopping oxidative metabolism from a few minutes to hours following hibernation. While this represents a large capacity to shift to glycolysis as a lone ATP source in an adult brain circuit, we hypothesized activitys reliance on oxygen may be globally reduced. We tested this by simultaneously assessing tissue oxygen consumption and neural activity from a brainstem motor network. We show that hibernation triggers a reduction in the oxygen consumed by neural activity and activity-independent mitochondrial respiration. In accordance with lower aerobic requirements, network output remained stable over a wide range of tissue oxygen levels, from baseline to anoxia, whereas controls were disrupted by moderate hypoxia. Despite operating with reduced aerobic metabolism, network activity was similar to controls, and activity increases did not accelerate oxygen consumption until seizure-like bursts ensued. Therefore, circuits in the vertebrate brain have the surprising capacity to uphold normal network functions with reduced aerobic metabolism. These findings introduce low-cost states can lie dormant within otherwise energetically expensive circuits and raise the question why costly designs are the default when they, in some cases, may be unnecessary and predispose organisms to metabolic pathologies.

Given its well-documented effects on human physiology, hypoxia has garnered increasing interest for its potential to enhance specific adaptations to exercise. However, the molecular response of skeletal muscle to exercise under normobaric hypoxia remains poorly understood. To address this gap in knowledge, ten healthy young males completed a crossover study in which exercise in hypoxia was compared to exercise in normoxia matched by either absolute or relative intensity. This design allowed us to identify shared transcriptomic responses across all three conditions, as well as changes that were specific to exercise intensity or hypoxic exposure. Skeletal muscle biopsies were collected before, immediately after, and at 3 and 24 hours following each exercise session, with RNA sequencing performed to assess changes in gene expression. Following exercise, a greater number of differentially expressed genes were observed in hypoxia compared to normoxia at 24 h post-exercise. This hypoxia-specific response involved the downregulation of multiple mitochondrial pathways and appears to be regulated by a transcriptional network comprising both positive and negative regulators of HIF-1 activity. These findings highlight the ability of normobaric hypoxia to influence exercise-induced gene expression and suggests that it may promote distinct molecular adaptations in skeletal muscle following longer-term training.

Signaling molecules released from the urothelium by mechanical stretch are known to play a sensory role in bladder contractions via neuronal signaling. It is also theorized that these molecules released from the urothelium could act locally to induce urothelial cell-mediated local bladder contractions. In this study, we specifically stimulated the urothelial cells using optogenetics to investigate how the urothelial released signaling molecules can influence bladder contractions locally. Using an ex-vivo whole bladder preparation, we stimulated the urothelial cells by activating channelrhodopsin-2 (ChR2) with blue light and initiated the urothelial cell-mediated local bladder contractions. The P2X receptor antagonist, PPADS, nearly abolished the contractions. The muscarinic receptor antagonist, atropine, significantly inhibited the contractions. Nifedipine, which blocks extracellular Ca2+ entry, abolished the contractions. G protein-coupled receptor inhibitor YM-254890 significantly inhibited the contractions. Hemichannel inhibitor carbenoxolone disodium and the exocytotic pathway of transmitter release inhibitor brefeldin A also showed significant inhibition of the contractions. In conclusion, we validated the previous hypothesis that urothelial release factors can influence bladder contractions locally without the need for signaling from the central nervous system. Further studies are needed to determine the relevance of this signaling pathway in normal bladder physiology and pathophysiologic conditions.

BackgroundIn vascular smooth muscle cells (VSMCs), LRRC8A volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNF), angiotensin II and stretch. LRRC8A physically associates with NADPH oxidase 1 (Nox1) and supports its production of extracellular superoxide (O2-*). Methods and ResultsMice lacking LRRC8A exclusively in VSMCs (Sm22-Cre, KO) were used to assess the role of VRACs in TNF signaling and vasomotor function. KO mesenteric vessels contracted normally to KCl and phenylephrine, but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). 48 hours of ex vivo exposure to TNF (10ng/ml) markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 M, 20 min) enhanced dilation of control rings and restored impaired dilation following TNF exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 35 proteins that interacted with LRRC8A. Pathway analysis revealed actin cytoskeletal regulation as the most closely associated function of these proteins. Among these proteins, the Myosin Phosphatase Rho-Interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots which revealed LRRC8A binding at the second Pleckstrin Homology domain of MPRIP. siLRRC8A or CBX treatment decreased RhoA activity in cultured VSMCs, and MYPT1 phosphorylation at T853 was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNF exposure. ConclusionsInteraction of Nox1/LRRC8A with MPRIP/RhoA/MYPT1/actin may allow redox regulation of the cytoskeleton and link Nox1 activation to both inflammation and vascular contractility.

Skeletal muscles secrete a variety of cytokines in response to inflammatory stimuli such as lipopolysaccharide (LPS); however, the contributions of resident macrophages or other non-muscle cells to the secretory responses are not well understood. To determine the potential impact of resident macrophages to inflammatory cytokine production, we tested the LPS responsiveness of isolated mouse soleus muscle when a critical toll receptor adapter protein (Myd88) was knocked down only in myeloid-derived cells within the muscle (e.g. resident macrophages). The phenotype is referred to as LyzMyd88-/- ; the litter mate controls were Myd88fl/fl. In solei from LyzMyd88-/- mice, cytokine secretory rates for interleukin-6 (IL-6) and keratinocyte-derived cytokine (KC, CXCL1) were significantly reduced to 56.3%, and 60.6% of control, respectively, over the first hour of LPS exposure. In the second hour, secretion of granulocyte colony stimulating factor (G-CSF), IL-6, KC(CXCL1) and monocyte chemoattractant protein-1 (MCP-1, CCL2) were greatly elevated by 5-10-fold in both phenotypes compared to the first hour. However, only MCP-1 secretion was decreased to 70.6% of control in the second hour. We also tested the secretory response to buffer containing 1% sterile mouse plasma because dilute plasma is known to amplify the responses of macrophages to LPS. Treatment with 1% plasma alone affected baseline measures of some cytokines but resulted in no further increases in secretion during either hour of exposure. However, small and gradual increases in secretory rates were observed for several cytokines over the study period, with or without plasma, with the largest responses seen in IL-6 and KC. Overall, the results are consistent with a significant early contribution of myeloid-derived, resident immune cells to the cytokine secretory responses of intact oxidative skeletal muscle. In addition, small quantities of plasma in the buffer have no independent stimulatory effects on cytokine secretion

Elephant seals experience extreme hypoxemia during diving bouts. Similar depletions in oxygen availability characterize pathologies including myocardial infarction and ischemic stroke in humans, but seals manage these repeated episodes without injury. However, the real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture system to assess the molecular response to prolonged hypoxia. Seal and human cells exposed to 1% O2 for up to 6 h demonstrated differential responses to both acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1 from angiogenic signaling at both the transcriptional and cellular level. Rapid upregulation of genes involved in the glutathione (GSH) metabolism pathway supported maintenance of GSH pools and increases in intracellular succinate in seal but not human cells during hypoxia exposure. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurred in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. In sum, our studies show that in contrast to human cells, seal cells adapt to hypoxia exposure by dampening angiogenic signaling, increasing antioxidant protection, and maintaining mitochondrial morphological integrity and function.

Neuropathic pain, a debilitating condition with unmet medical needs, can be charactarised as hyperexcitability of nociceptive neurons caused by dysfunction of ion channels. Voltage-gated potassium channel type 7 (Kv7), responsible for maintaining neuronal resting membrane potential and thus neuronal exitability, resides under tight control of G protein-coupled receptors (GPCR). Calcium-sensing receptor (CaSR) is a GPCR that is known to regulate activity of numerous ion channels, but whether CaSR could control Kv7 channel function has been unexplored until now. Our results demonstrate that CaSR is expressed in recombinant cell models, human induced pluripotent stem cell (hiPSC)-derived nociceptive-like neurons and mouse dorsal root ganglia neurons, and its activation induced depolarisation via Kv7.2/7.3 channel inhibition. The CaSR-Kv7.2/7.3 channel crosslink was mediated via the Gi/o protein/adenylate cyclase/cyclic adenosine monophosphate/protein kinase A signalling cascade. Suppression of CaSR function rescued hiPSC-derived nociceptive-like neurons from algogenic cocktail-induced hyperexcitability. To conclude, this study demonstrates that CaSR-Kv7.2/7.3 channel crosslink via the Gi/o protein signalling pathway effectively regulates neuronal excitability, providing a feasible pharmacological target for neuronal hyperexcitability management in neuropathic pain.

Skeletal muscle regulation is responsible for voluntary muscular movement in vertebrates. The genes of two essential proteins, teneurins and latrophilins (LPHN), evolving in ancestors of multicellular animals, form a ligand-receptor pair, and are now shown to be required for skeletal muscle function. Teneurins possess a bioactive peptide, termed the teneurin C-terminal associated peptide (TCAP) that interacts with the LPHNs to regulate skeletal muscle contractility strength and fatigue by an insulin-independent glucose importation mechanism. CRISPR-based knockouts and siRNA-associated knockdowns of LPHN-1 and-3 shows that TCAP stimulates an LPHN-mediated cytosolic Ca2+ signal transduction cascade to increase energy metabolism and enhance skeletal muscle function via increases in type-1 oxidative fiber formation and reduce the fatigue response. Thus, the teneurin/TCAP-LPHN system is presented as a novel mechanism likely to regulate the energy requirements and performance of skeletal muscle.