Function

RationaleRegular aerobic exercise protects against vascular aging and reshapes the circulating molecular milieu, but the relation between vascular function, circulating molecules, and exercise dose at extreme volumes remains poorly defined. The vascular and molecular consequences of chronic, multi-stage ultra-endurance running are particularly unclear. ObjectiveTo define circulating molecular signatures associated with vascular dysfunction following the 64-stage, 4,486-km Trans Europe Foot Race (TEFR). Methods and ResultsIntegrated multiomics analysis (proteomics, lipidomics, metabolomics) of plasma from 27 finishers revealed a coordinated systemic shift driving an oxidative phenotype. Specifically, we identified altered arginine metabolism and a universal upregulation of lipotoxic ceramides consistent with incomplete fatty acid oxidation. In conjunction, we identified upregulation of innate immune system pathways including the acute phase response and the complement system. Central pulse wave velocity (cPWV) increased significantly after the race, consistent with arterial stiffening. To test whether the post-race circulating milieu could directly influence vascular mechanics, naive murine aortic rings were incubated with participant plasma. Post-race plasma acutely increased aortic elastic modulus, and this effect was attenuated by the superoxide dismutase mimetic TEMPOL, supporting a ROS-dependent component. In human aortic endothelial cells (HAECs), post-race plasma increased reactive oxygen species generation without detectable changes in eNOS phosphorylation, total eNOS abundance, or stimulated nitric oxide production. Endothelial ROS responses were associated with components of the terminal complement pathway. ConclusionsExtreme multi-stage ultra-endurance exercise induces a distinct systemic milieu associated with arterial stiffening through ROS-sensitive mechanisms. This response is characterized by remodeling of arginine-related metabolism, ceramide accumulation, innate immune activation, and oxidative stress, without evidence of reduced measured eNOS abundance or stimulated NO production. These findings identify candidate molecular pathways linking prolonged metabolic stress to vascular dysfunction.

BackgroundEndothelial response to flow is key to vascular function in health and disease. Our earlier studies demonstrated that endothelial Kir2.1 is essential for flow-induced Akt1/eNOS signaling and for flow-induced vasodilation (FIV) but the mechanistic integration between Kir and other flow signaling pathways remained poorly understood. MethodsWe use a combination of electrophysiological recordings in real time of flow exposure, Ca2+ imaging, pressure myography of resistance arteries, and echocardiography. ResultsWe demonstrate that Kir2.1 is essential for flow-induced PI3K phosphorylation, whereas expression of myristoylated Akt1, which bypasses PI3K-dependent membrane recruitment, restores flow-induced Akt1/eNOS phosphorylation in Kir2.1-deficient endothelium. It also restores FIV in Kir2.1-deficient mesenteric arteries. We further demonstrate that Kir2.1 is essential for flow-induced Ca{superscript 2} influx mediated by Piezo1 and TRPV4 channels, whereas Ca{superscript 2} influx induced by pharmacological activation of these channels is Kir2.1 independent. Deficiency of Piezo1 does not affect endothelial Kir2.1 channels. We also discover that flow activation of endothelial Kir2.1 requires Syndecan1, thus creating a link between glycocalyx and downstream effects. Physiologically, we find that endothelial Kir2.1 is suppressed by infusion of Angiotensin-II and by advanced aging, resulting in significant impairment of FIV. In both cases, FIV is fully restored by endothelium-specific over-expression of Kir2.1. ConclusionsOur study reveals that Kir2.1 serves as a mechanistic linker between endothelial glycocalyx to Piezo1-mediated Ca2+ influx and downstream signaling suggesting a new integrated model of endothelial mechanotransduction. A functional loss of endothelial Kir2.1 is shown to play a significant role in FIV impairment in Angiotensin-induced hypertension and aging.

BackgroundPeripheral artery disease (PAD) and its severe form, chronic limb-threatening ischemia (CLTI), significantly impair blood flow to the lower extremities, affecting millions of adults globally. Intramuscular adipose tissue (IMAT) and fibrosis accumulation distinguish patients with CLTI from those with mild PAD, suggesting a role in CLTI pathobiology. However, the functional consequences of IMAT in CLTI remain unclear. MethodsWe compared gastrocnemius muscle samples from patients with PAD/CLTI, intermittent claudication, and non-PAD individuals. We analyzed bulk RNA sequencing, proteomic, lipidomic, and single-cell/nucleus RNA sequencing datasets. Additionally, we used murine models of hindlimb ischemia (HLI) with genetic manipulation of Ppar{gamma}, a key adipogenic transcription factor, specifically in fibro-adipogenic progenitor cells (FAPs), the cellular source of IMAT, to modulate IMAT formation and assessed the impact on limb function and pathology. ResultsPatients with CLTI exhibited significantly elevated expression of adipogenic genes and proteins in muscle specimens when compared to non-PAD controls. Murine models showed that increasing IMAT formation significantly worsened ischemic limb muscle strength and work output. In contrast, preventing IMAT formation significantly improved ischemic limb muscle strength and work output. These findings were consistent across both male and female mice, although females had greater tendency to form IMAT compared with male mice. ConclusionsIMAT accumulation is a key determinant of limb function in PAD/CLTI. Our studies demonstrate that targeting IMAT formation could improve limb function in mice with experimental PAD. Together, these findings suggest that developing strategies to limit or reduce IMAT may improve limb function and walking performance in patients with PAD/CLTI, providing a novel therapeutic avenue to address a critical unmet need. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABSO_LIIntramuscular adipose tissue accumulation (IMAT) distinguishes patients with chronic limb-threatening ischemia from those with milder peripheral artery disease or those without PAD and directly impairs ischemic limb muscle function. C_LIO_LIGenetic gain- and loss-of-function mouse models demonstrate that increasing IMAT worsens, while preventing IMAT formation improves, ischemic limb strength and performance independent of perfusion. C_LIO_LIAdipogenic signatures in human calf muscle negatively correlates with muscle strength and disease severity, identifying IMAT as a functional biomarker and modifiable target in PAD/CLTI. C_LI What are the clinical implications?O_LIIMAT accumulation represents an underappreciated, non-vascular mechanism contributing to leg dysfunction in PAD/CLTI. C_LIO_LITherapies aimed at limiting or reversing IMAT formation may improve leg strength and walking performance in patients with PAD/CLTI, addressing a critical unmet clinical need. C_LIO_LIIdentifying and targeting cellular pathways regulating IMAT formation from fibro-adipogenic progenitors may complement vascular interventions to enhance functional recovery after revascularization. C_LI

Leucine Rich Repeat Containing 8C (LRRC8C) anion channels modulate NADPH oxidase 1 activity and allow extracellular superoxide influx promoting inflammatory signaling. Here we studied chimeric 8C/8D channels and identified oxidant-dependent current modulation within the N-terminus (NT) and first transmembrane domain (TM1). Chloramine-T (ChlT) elicited inhibitory and activating current responses, whereas other redox agents had comparatively little impact. ChlT moderately inhibited wild-type (WT) 8C current and abrogated block by DCPIB. Substitution of the 8D NT (8D1-22) conferred ChlT-dependent current activation, as did 8D2-4, 8D5-11, I2F, and I2Y substitution. M48T (distal TM1) substitution enhanced WT 8C current inhibition and impaired activation in NT mutants. An M48D mutation diminished 8C current block by DCPIB by [~]50%. WT 8D currents were potently inhibited by ChlT. Substitution of the 8C first extracellular loop (EL1) weakened inhibition, while 8C EL1 + TM145-49 substitution produced ChlT-mediated current activation. 8C45-49 or T48M substitutions in 8D resulted in rapid disruption and loss of initial current inhibition, and a progressive increase of non-rectifying current. These results provide evidence that NT2-4, particularly I2/F2, in combination with M48 are primary determinants of activating vs. inhibitory current modulation by ChlT. M48 oxidation limits 8C inhibition and is required for activating responses, while T48 and 8D EL1 promote 8D signature current inhibition. ChlT exposure disrupts subsequent or preexisting channel block by DCPIB, consistent with a common site of interaction. Thus, factors that alter NT pore stability and mobility may regulate inhibition vs. activation of LRRC8C by redox stress.

Cerebral blood flow (CBF) control is essential for normal brain function and is disrupted in pathological conditions. Arterial diameters are tightly regulated to provide on demand increases in blood flow in regions of neuronal activity. Pericytes (PCs) exhibit robust myogenic tone and may also respond to neuronal activity to fine-tune local resistance and blood flow. Thus, mural control of microcirculatory resistance may extend beyond arteries and arterioles. Yet, PCs electrophysiology and contractility have not been thoroughly characterized, and this prohibits an integrated view of brain blood flow control. In this study, we develop a detailed mathematical model of mural cell electrophysiology, Ca2+ dynamics and biomechanics. The model is informed by electrophysiological data in smooth muscle cells (SMCs) or PCs and predictions are compared against pressure-induced responses in isolated arterioles and capillaries, respectively. Simulations recapitulate myogenic constrictions and examine differences in contractile dynamics as we move from arterioles to proximal and distal capillaries. In arteriole-to-capillary transitional (ACT) zone PCs, increased mechanosensitivity, more Ca2+ influx through non-selective cation (NSC) channels and/or a higher sensitivity of the contractile apparatus to Ca2+ can compensate for reduced L-type voltage-operated (VOCC) Ca2+ influx and allow for robust constrictions at the lower operating pressures of capillaries relative to the arterioles. A significant Ca2+ influx through NSC relative to VOCC, however, can decouple the PCs contractile apparatus from electrical signaling. Vasoactivity to chemomechanical stimuli along the arteriole to capillary axis is progressively driven by VOCC-independent Ca2+ influx and Ca2+ sensitization with slow kinetics. The proposed cell model can form the basis for detailed multiscale and multicellular models that will examine physiological function at a single vessel or vascular network levels and investigate CBF control in health and in disease. Key pointsO_LIA mural cell model of electrophysiology, calcium (Ca2+) dynamics and biomechanics is informed by data and adapted for modeling cerebral arteriole smooth muscle cells and capillary pericytes. C_LIO_LIIon channel activities are characterized by patch-clamp electrophysiology in isolated cerebral smooth muscle cell and pericytes, and capillary and arteriole electromechanical responses to transmural pressure changes are assessed using novel ex vivo preparations. C_LIO_LIMyogenic constrictions in arterioles can be reproduced by pressure-induced non-selective cation channel (NSC) activation that depolarizes the cell, opens L-type Ca2+ channels (VOCCs) and increases Ca2+ influx. C_LIO_LIRobust myogenic constrictions in arteriole-to-capillary transition (ACT) zone pericytes may reflect significant Ca2+ influx through NSC, increased mechanosensitivity, or higher sensitivity of the contractile apparatus to Ca2+, potentially compensating for reduced VOCC density relative to arteriolar smooth muscle. C_LIO_LIA significant contribution of NSC relative to VOCC in Ca2+ influx, can decouple the contractile apparatus from electrical signaling. C_LIO_LIThe model shows how gradients in ionic activities, mechanosensitivity and/or Ca2+ sensitivity can alter contractile phenotype and electromechanical coupling along the arteriole to capillary continuum. C_LIO_LIThe proposed model can form the basis for detailed multiscale and multicellular models that will investigate cerebral blood flow control in health and in disease. C_LI

BackgroundDiabetes-associated neurodegeneration is amplified by methylglyoxal (MGO)-driven dicarbonyl stress linking hyperglycemia to neuronal insulin resistance and maladaptive neuroinflammation. We tested the neuroprotective activity of MNP-021, a non-electrophilic TRPA1 modulator, in neurons and glial cells in vitro. MethodsSH-SY5Y neurons were pretreated with MNP-021 and challenged with MGO, then profiled by high-content imaging, RNA-seq, Seahorse OCR/ECAR, glycolytic stress assays and AKT/ERK/CREB immunoblotting {+/-} insulin. In parallel, HMC3 glial cells were treated with MNP-021, exposed to LPS/TNF- or A{beta}(25-35) and tested for viability and inflammatory markers by ELISA and qRT-PCR. ResultsMGO increased nucleus-to-cytoplasm area ratio by 49% and dysregulated glucose handling, increasing 2-NBDG uptake by [~]25%, with GLUT1/GLUT4 membrane redistribution; MNP-021 normalized morphology, uptake, and transporter localization without cytotoxicity up to 10 {micro}M. RNA-seq identified 754 MGO-deregulated genes, including ISR/metabolic nodes (GCK, SESN2, PHGDH/PSAT1, PCK2); MNP-021 buffered stress-induced transcription with limited baseline effects, remodeled mitochondrial redox readouts consistent with controlled ROS signaling, while improving mitochondrial content/architecture and blunting stress-evoked compensatory glycolysis. MNP-021 restored pro-survival signaling (pAKT/pERK and nuclear pCREB), including insulin responsiveness during MGO exposure. MNP-021 reduced IL-6/TNF- release while increasing IL-10 and ARG1 ([~]1.9-fold vs LPS/TNF-) in HMC3 glial cells, shifting them toward a pro-resolving IL-10/ARG1 program with reduced A{beta}(25-35)-evoked cytokine release with GLP-1 remaining very low ([≤]10 pg/mL) and not significantly increased in this system. ConclusionsMNP-021 coordinates transcriptomic restraint, transporter-level glucose handling, mitochondrial resilience, and pro-survival/pro-resolving signaling across neuron-microglia compartments, supporting TRPA1-tuned small-molecule modulation as a candidate strategy against dicarbonyl-linked neuro-metabolic stress.

BackgroundIn the context of increased salt intake in the world population, the understanding of the mechanisms that contribute to its correct renal excretion and therefore, avoid variation of blood volume and blood pressure is of major importance. MethodsMolecular, ex vivo microperfusion on isolated tubules, and integrative analysis, was used to identify, characterize and investigate a Na+ secretion pathway in the collecting duct. ResultsIn collecting duct of mice, salt load induced an increase of the type A intercalated cells (AIC) number, an overexpression of the H(Na),K-ATPase type 2 (HKA2) catalytic subunit Atp12a and a stimulation of the bumetanide-sensitive Na+ secretion in isolated and microperfused tubules. Surprisingly, HKA2KO mice fed a high-salt diet exhibit a strong dysregulation of their Na+ and water balance with a pronounced loss of Na+ and fluid, alkalosis, hypokalemia and low blood pressure. This Bartter-like phenotype is due to an over-inhibition of the thick ascending limb (TAL) related to an elevated PGE2 production. ConclusionOur findings establish that activation of Na+ secretion in AIC act as the fine-tuning knob in the regulation of renal Na+ excretion in response to high salt intake. Its absence is overcompensated by an inhibition of the Na+ transport system of the TAL.

Maintenance of whole-body pH is essential for human health. The kidneys play a crucial role in defending pH homeostasis by excreting excess acid in the urine and returning alkali buffers to the blood. Consequently, renal insufficiency causes serious and harmful effects on pH balance. While a serious and common complication of chronic kidney disease (CKD), pH imbalances themselves appear to be catalysts of kidney injury. Renal adaptations to pH imbalances contribute to compensated acid-base disorders and are vital to correcting whole-body pH. However, overstimulation of these adaptive processes can cause renal inflammation and lead to long-term kidney injury. Surprisingly, the acute and chronic effects of pH challenges on the whole kidney are poorly defined. The upregulation of ammoniagenesis in the proximal tubule due to acidosis, and the coordinated secretion of protons from the collecting ducts is a well-documented phenomenon. However, there is a significant gap in knowledge regarding how the other segments of the nephron respond to acidosis or alkalosis. Therefore, to determine the cell-specific impact of overt metabolic acidosis and alkalosis on the kidney, we performed single-nucleus RNA sequencing on male and female WT mice following 48-hours of acid-base challenge (280mM NH4Cl (acid), 280mM NaHCO3 (alkali), 280mM NaCl (isosmotic control)). The results of our studies reveal the sex-specific single-cell transcriptional response by the kidney to pH imbalances, including a proximal straight tubule cell cluster that arises de novo following both acidosis and alkalosis. We label these proximal tubule cells PT S3a and demonstrate that their transcriptional profile is distinct from other injured PT cells that arise from ischemic injury. These studies lay the foundation for future research into the long-term renal adaptations to pH challenges that may lead to renal insufficiency and the development of CKD.

BackgroundFailure of cerebrovascular reserve is a fundamental determinant of ischemic vulnerability, yet the mechanisms by which vascular smooth muscle dysfunction compromises reserve and predisposes the brain to injury remain incompletely defined. We therefore tested whether a pathogenic smooth muscle mutation produces a baseline failure of cerebrovascular reserve sufficient to render the brain vulnerable to hypoperfusion, even in the absence of fixed arterial occlusion. MethodsWe examined cerebrovascular structure, hemodynamics, and reserve in a genetically defined mouse model of ACTA2-associated multisystemic smooth muscle dysfunction syndrome with systemic or brain-restricted expression of the mutant allele. Cerebral artery morphology was assessed using magnetic resonance angiography and black ink angiography. Vascular smooth muscle phenotype was evaluated by immunohistochemistry and proliferation assays. Blood pressure reactivity and cerebral blood flow (CBF) were measured simultaneously using femoral arterial catheterization and laser speckle flowmetry during vasoactive challenges and controlled hypotension. Cerebrovascular stress responses were tested using unilateral common carotid artery occlusion. Downstream brain effects were assessed by histology, resting state functional connectivity imaging, and behavioral testing. ResultsImpaired smooth muscle contractility drove rectification and narrowing of major cerebral arteries, downregulation of contractile markers, and increased vascular cell proliferation. These structural changes produced a distinct physiological phenotype: mutant mice exhibited blunted vasoreactivity, diminished spontaneous vasodynamic activity, and a downward shift in the blood pressure-CBF relationship across a wide range of arterial pressures, consistent with loss of cerebrovascular reserve. As a result, CBF was reduced at baseline and could not be maintained during hypotension or acute vascular stress. During carotid occlusion, mutant mice showed impaired compensatory perfusion, greater physiological instability, and worse behavioral outcomes. Chronic reserve failure coincided with white matter loss, reduced neuronal density, disrupted large-scale functional connectivity, and deficits in locomotion, anxiety-related behavior, and working memory. ConclusionsPathogenic smooth muscle dysfunction caused by ACTA2 mutation produces a baseline failure of cerebrovascular reserve that renders the brain vulnerable to hypoperfusion and stress-induced ischemic injury. These findings establish cerebrovascular reserve failure as a central physiological mechanism linking vascular dysfunction to end-organ brain injury and identify reserve preservation as a critical, potentially actionable determinant of brain health in hypotension-prone vascular disease. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABS- ACTA2 smooth muscle dysfunction produces baseline cerebrovascular reserve impairment, with reduced cerebral blood flow and a downward-shifted pressure-flow relationship in the absence of critical large-vessel occlusion. - Vascular tone dysregulation is coupled to end-organ brain injury, including white matter and neuronal loss, disrupted functional connectivity, and behavioral deficits. - The results support complementary disease mechanisms in ACTA2 vasculopathy: baseline reserve limitation and injury-provoked occlusive remodeling. Clinical Implications- Patients with ACTA2 vasculopathy may be vulnerable to ischemic brain injury during hypotension or systemic stress despite the absence of critical stenosis or occlusion on routine imaging. - Peri-procedural and acute-care management should emphasize preserving perfusion pressure and cerebrovascular reserve (e.g., during anesthesia, dehydration, or systemic illness). - More broadly, cerebrovascular reserve is a clinically relevant, potentially modifiable determinant of brain health in hypotension-prone vasculopathies and conditions characterized by impaired vascular reactivity.

BackgroundCoronary autoregulation is the ability of the normal heart to maintain constant coronary blood flow (CBF) over a wide range of coronary driving pressures (CDP). Despite being vital for survival, the mechanism of coronary autoregulation is unknown. We hypothesized that GPR39, present in vascular smooth muscle cells, together with its endogenous agonist 15-hydroxyeicosatetraenoic acid (15-HETE) orchestrate coronary autoregulation. MethodsWe created coronary stenoses of varying degrees in open-chest, anesthetized dogs where we measured CBF and CDP. In a subset of animals, coronary venous blood was sampled for eicosanoid, adenosine, endothelin-1, polyunsaturated fatty acids, and prostaglandins levels. Stenoses were recreated during intravenous administration of VC108, a specific GPR39 antagonist and systemic, pulmonary, and coronary hemodynamics measured. ResultsGPR39 was identified in coronary arterioles by immunohistochemistry and in heart tissue by western blot. In-vivo, 15-HETE correlated linearly with CDP over the autoregulatory range (r2=0.47, p=0.0024). Apart from 6-keto PGF1 no other metabolite had any relation with CDP. Prior to administration of VC108, CBF did not change within the autoregulatory range. VC108 had no effect of systemic and pulmonary hemodynamics but increased CBF (p=0.02 versus vehicle) by decreasing coronary microvascular resistance (p=0.01 versus vehicle), indicating that GPR39 participates in control of normal coronary vascular tone. With VC108, coronary autoregulation was abolished and CBF became CDP dependent (r2=0.96, p=0.004). ConclusionGPR39 and its endogenous agonist 15-HETE together orchestrate coronary autoregulation when CDP is reduced. These novel findings provide a mechanism for coronary autoregulation and could direct pharmacological treatment of various coronary syndromes in humans.

Short-term heat acclimation (HA) induces cardiovascular and fluid-regulatory adaptations, but its impact on markers of renal tubular injury and acute kidney injury risk (AKI) during exercise-heat stress remains unclear. Fourteen healthy endurance athletes were randomised to five days of isothermic HA (HOT; n = 7; 32 {degrees}C, 70% relative humidity; target core temperature [&ge;]38.5 {degrees}C), or matched exercise in thermoneutral conditions (TEMP, n = 7). Heat stress tests (HST; 45 min cycling at 32 {degrees}C, 70% RH) were performed pre- and post-intervention. Blood biomarkers of kidney tubular stress (NGAL, KIM-1), fluid-regulation (copeptin, serum osmolality) and sympathetic activity (plasma normetanephrine) were measured at rest and immediately post-HST. HA reduced resting heart rate (-8 {+/-} 5 bpm, p = 0.007, d = 1.0), increased plasma volume (+7.3 {+/-} 5.1%, p = 0.022) and sweat loss (+500 {+/-} 539 mL, p = 0.018, d = 1.1). Copeptin rose during the pre-intervention HST in both groups (HOT: +11 {+/-} 6; TEMP: +12 {+/-} 13 pmol{middle dot}L-1, p < 0.05), but not post-intervention. NGAL increased only in TEMP during HST1 (+45 {+/-} 29 g{middle dot}L-1, p = 0.030), while KIM-1 remained unchanged. No group x time interactions were observed for any biomarkers (p > 0.05). Five days of HA improved cardiovascular and thermoregulatory responses but did not alter renal stress markers or fluid-regulatory responses during exercise in the heat. These findings suggest short-term HA enhances heat tolerance without reducing acute renal biomarker responses under hot, humid conditions. New & NoteworthyFive days of isothermic heat acclimation improved cardiovascular and thermoregulatory responses, related to a lower resting heart rate, plasma volume expansion, and greater sweat loss. However, these benefits did not reduce renal tubular stress markers (NGAL, KIM-1), fluid-regulatory strain (copeptin), or sympathetic activity (normetanephrine) during exercise in the heat. Short-term heat acclimation lowers cardiovascular strain but does not mitigate renal biomarker responses, suggesting kidney stress risk remains unchanged in hot, humid conditions.

AimSecretin was recently found to play a pivotal role in the renal adaptation to acute base excess. Here, secretin increases pendrin-dependent HCO3- secretion from the beta-intercalated cells in the cortical collecting ducts. Whether secretin and its receptor play a role during prolonged base-loading remains unknown. MethodsUrine and blood acid-base analyses were carried out in secretin receptor (SCTR) KO and WT mice at baseline and after 1 and up to 8 days of base-loading with NaHCO3-enriched drinking water. Changes in pendrin protein abundance and function were assessed by immunoblotting and isolated tubule perfusion experiments. Plasma secretin levels and renal SCTR expression were assessed after 24 hours of acid/base-loading by radioimmunoassay and qPCR, respectively. ResultsSCTR KO mice responded with diminished urine alkalization and a lesser reduction of urinary acid excretion when base-loaded for 48 hours. Concordantly, SCTR KO mice presented with increased blood base retention compared with WTs. Base-loaded SCTR WT and KO mice showed comparable total pendrin protein abundance. Despite this, pendrin function was markedly lower in SCTR KO mice. Base-loaded mice had higher plasma secretin and renal SCTR levels compared with acid-loaded mice. Higher arterial HCO3- associated with higher renal SCTR mRNA expression. ConclusionPlasma secretin and renal SCTR levels are modulated by systemic acid-base status. Loss of the SCTR diminishes renal base excretion capacity and exacerbates systemic base accumulation during prolonged base-loading. These findings further support a central role of secretin and its receptor in the regulation of both acute and prolonged base excess.

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder characterized by skeletal muscle degeneration and cardiomyopathy without a cure. This study examined the therapeutic potential of the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (EMPA) on cardiac function in the dystrophin-deficient mdx mouse model of DMD. Male mice were fed control chow or EMPA-containing chow ([~]25 mg/kg/day), and cardiac function was evaluated longitudinally by four-dimensional ultrasound imaging. EMPA did not alter left ventricular mass or chamber volume but preserved ejection fraction (EF) for 12 weeks, maintained significantly higher EF through 24 weeks, and attenuated global impairment of systolic and diastolic myocardial deformation. These functional improvements were accompanied by reduced cardiomyocyte hypertrophy and decreased expression of cardiac stress genes. EMPA reduced mitochondrial DNA damage, increased mitochondrial DNA copy number, and induced transcriptional signatures consistent with enhanced fatty acid and ketone metabolism, contributing to increased myocardial ATP content. Systemically, EMPA improved body mass trajectory, preserved relative lean mass, enhanced skeletal muscle torque, and did not adversely affect renal function. Together, these findings demonstrate that EMPA improves cardiac performance and mitochondrial integrity while enhancing myocardial energy availability in mdx mice, supporting SGLT2 inhibitors as a promising therapeutic strategy for individuals with DMD.

BackgroundWingless/Int-1 (Wnts) proteins are canonical Frizzled receptor ligands. Recent evidence indicates that some Wnts, including Wnt9b and Wnt5a, bind to polycystin 1 (PKD1), a transmembrane protein which can couple to polycystin 2 (PKD2) to form a non-selective cation channel. The functional significance of Wnts binding to PKD1 is unclear. Here, we tested the hypothesis that Wnts act through PKD1/PKD2 channels on endothelial cells (ECs) to regulate arterial contractility and blood pressure and investigated the cellular source and secretory regulation of vasoactive Wnt proteins. MethodsA wide variety of approaches, including inducible EC-specific PKD1 and PKD2 knockout mice, reverse-transcription polymerase chain reaction, Western blotting, immunofluorescence, pressurized artery myography, blood pressure measurements, patch-clamp electrophysiology, in vivo and in vitro Wnt and nitric oxide assays, and Wnt secretion assays. ResultsIntravascular Wnt9b or Wnt5a administration stimulates an EC PKD1/PKD2-dependent dilation in pressurized resistance-size arteries. Wnt9b and Wnt5a are present in serum and plasma and intravenous infusion rapidly stimulates a blood pressure reduction which requires EC PKD1. Wnts stimulate a PKD1-dependent non-selective cation current in ECs which through Ca2+ signaling activates endothelial nitric oxide synthase (eNOS) and small conductance Ca2+-activated K+ channels to induce vasodilation. Wnt9b acts solely via PKD1/PKD2 channels, whereas Wnt5a stimulates signaling through PKD1/PKD2, Frizzled-7 (Fzd-7), Dishevelled and c-Jun N-terminal kinase (JNK). Intravascular flow stimulates angiotensin II type 1 (AT1) receptors, which through Gq/11 and Porcupine activate Wnt9b and Wnt5a secretion in ECs. Wnts secreted in response to flow activate PKD1/PKD2 signaling in ECs and contribute to flow-mediated vasodilation. ConclusionsIntravascular flow activates AT1 receptors, which through Gq/11 and Porcupine stimulate Wnt9b and Wnt5a secretion in ECs. Wnt9b activates PKD1/PKD2 channels whereas Wnt5a stimulates both PKD1/PKD2 and Fzd-7 in ECs to induce vasodilation. Wnts contribute to flow-mediated autocrine/paracrine dilation and reduce blood pressure. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/712518v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@158bad1org.highwire.dtl.DTLVardef@5113eforg.highwire.dtl.DTLVardef@f3b94eorg.highwire.dtl.DTLVardef@10ab479_HPS_FORMAT_FIGEXP M_FIG C_FIG

Maintenance of skeletal muscle function is essential for functional independence, quality of life and healthspan. Muscle RING-finger protein-1 (MuRF1) negatively regulates muscle function and mass through ubiquitination and degradation of muscle proteins. Accordingly, genetic and pharmacological inhibition of MuRF1 attenuates muscle wasting and weakness under catabolic stress. To explore the potential of MuRF1 inhibitors (e.g., MyoMed-205) to improve muscle health, we investigated here the long-term effects of MyoMed-205 on functional capacity and muscle physiology in rats under basal conditions. Wistar rats were randomized to control or MyoMed-205 groups and were followed for 4 or 8 weeks. Body weight, food and water intake, and exercise capacity were monitored weekly. At each endpoint, the soleus muscle was collected for histological analyses. MyoMed-205-treated rats showed normal basic survival-related behaviors and body growth. After 8 weeks, MyoMed-205-treated animals exhibited enhanced exercise capacity (speed (m/min): +45%, p = 0.01; endurance (min): +47%, p = 0.03; and distance covered (m): +87%, p = 0.04) compared with baseline performance. Conversely, no differences were found in soleus fiber type distribution, cross-sectional area, or lipid and collagen content. Our findings indicate that MyoMed-205 enhances functional exercise capacity independently of changes in soleus muscle structure in rats under basal conditions.

Skeletal muscle hypertrophy results from the integrated regulation of anabolic and proteolytic processes in response to mechanical loading. Although increases in resistance training (RT) volume are used to increase mechanical stress, it remains uncertain whether large and abrupt volume progressions could exceed muscle adaptive capacity by disrupting the balance between anabolic and catabolic signaling. The present study investigated whether a large increase in weekly RT volume (+120%) leads to impaired hypertrophic outcomes and intracellular regulatory responses compared with a modest increase (+20%). Twenty-five resistance-trained men and women (18-35 years old) completed an 8-week randomized, single-blind, within-subject unilateral intervention. Each participant trained both legs twice weekly, with one leg assigned to the large (VOL120) and the contralateral leg to the modest (VOL20) weekly volume progressions relative to habitual training volume. Vastus lateralis muscle cross-sectional area (mCSA) was assessed by ultrasonography before and after training. Muscle biopsies were obtained at baseline, post-intervention, and 24 h after the last session to quantify muscle fiber cross-sectional area (fCSA), satellite cell myonuclear content, and anabolic/catabolic signaling markers. Both protocols induced increases in mCSA over time (p<0.001), with no protocol vs. time interaction. No significant effects were observed for fCSA nor satellite cell number or myonuclear content. Additionally, molecular responses related to translational regulation and protein degradation were largely similar between protocols. Collectively, these data indicate that a large, abrupt increase in weekly set volume does not impair hypertrophic adaptations or meaningfully alter the anabolic-catabolic signaling profile in resistance-trained individuals.

Due to aging, the efficiency of kidney function begins to decrease. Dysfunction in mitochondria and their cristae is a hallmark of aging. Therefore, age-related decline in kidney function could be attributed to changes in mitochondrial ultrastructure, increased reactive oxygen species, and alterations in metabolism and lipid composition. We sought to understand how mitochondrial ultrastructure is altered over time in tubular kidney cells. A serial block facing-scanning electron microscope and manual segmentation using the Amira software were employed to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we demonstrate that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney during aging. Disruption of the MICOS complex resulted in altered mitochondrial metabolic function and increased ROS levels. We found significant, detrimental structural changes in the mitochondria of aged kidney tubules, suggesting a potential mechanism underlying the increased frequency of kidney disease with aging. We hypothesize that disruption of the MICOS complex exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, which impacts kidney health. Impact and ImplicationsDue to aging, the efficiency of kidney function begins to decrease, and the risk of kidney diseases may increase; however, the specific regulators of mitochondrial age-related changes are poorly understood. This study demonstrates that the MICOS complex may be a target for mitigating age-related mitochondrial changes. The MICOS complex is associated with oxidative stress and calcium dysregulation, which also arise in many kidney pathologies. HighlightsO_LIAging alters the MICOS mRNA levels and disease markers. C_LIO_LIAging reduces cristae architecture, mitochondrial volume and complexity in murine kidney ultrastructure C_LIO_LIReducing MIC60 and CHCHD6 lowers Ca2+ uptake and retention and induces oxidative stress in HEK cells. C_LIO_LIMetabolomic Profiling revealed that NAD+ and amino acid metabolism were altered in aged kidneys. C_LIO_LIMICOS deficiency alters the reduced basal, ATP-linked, maximal capacity and spare capacity. C_LIO_LIDecreased modeled expression of CHCHD6 in individuals of European genetic ancestry is linked to chronic kidney disease, whereas decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic kidney disease. C_LI Graphical AbstractKidney aging causes a decline in the MICOS complex, concomitant with metabolic, lipidomic, and mitochondrial structural alterations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/598108v3_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@80123aorg.highwire.dtl.DTLVardef@2c9f1eorg.highwire.dtl.DTLVardef@1827e26org.highwire.dtl.DTLVardef@280f4f_HPS_FORMAT_FIGEXP M_FIG C_FIG

This study aimed to determine the impact of inborn metabolic fitness and early life exercise training on whole body and brown adipose tissue (BAT) energetics. We carried out comprehensive metabolic phenotyping on 4-week old rats bred for high (high-capacity runner, HCR) and low (low-capacity runner, LCR) running capacity following randomization to voluntary wheel running (VWR) or control (CRTL) for 6-weeks. High-resolution respirometry and untargeted proteomics were then employed to determine the impact of inborn fitness and early life exercise on BAT function. When accounting for differences in body mass, early life exercise (VWR) resulted in greater basal and total energy expenditure, irrespective of strain (P < 0.0001 for both). Both leak and uncoupling protein 1 (UCP1) dependent respiratory capacities in isolated BAT mitochondria were greater in rats randomized to VWR compared to CTRL in both HCR (P < 0.01) and LCR (P < 0.05) strains. Similarly, mitochondrial sensitivity to the UCP1 inhibitor GDP was greater in both HCR (P < 0.01) and LCR (P < 0.05) rats randomized to VWR versus control. The BAT proteome differed in CTRL HCR and LCR rats, were there was enrichment in proteins related to branched chain oxidation and mitochondrial fatty acid oxidation in HCR rats. VWR remodeled the BAT proteome, where 151 proteins were differentially expressed in LCR BAT and 209 differentially expressed in LCR BAT following VWR. In both stains, there was an enrichment in proteins related to metabolism mitochondrial function in response to VWR. However, when comparing strains, 39 proteins were differentially expressed in BAT in HCR rats compared to LCR rats in response to VWR. These proteins were related to carboxylic acid and amino acid metabolism. Collectively, inborn fitness impacts body mass and composition, exercise behaviors, and the BAT proteome in early life. Early life exercise alters whole body and BAT energetics irrespective of inborn fitness, augmenting basal and total energy expenditure and BAT thermogenic capacity and function.

Skeletal muscle is the most abundant tissue in the human body and is essential for locomotion and the regulation of whole-body metabolism. The maintenance of skeletal muscle mass is essential for health, yet the molecular and signaling mechanisms that control skeletal muscle mass remain poorly understood. Transforming growth factor-{beta}-activated kinase 1 (TAK1) is a key signaling protein that regulates multiple intracellular pathways. Recent studies have demonstrated that TAK1 is a critical regulator of skeletal muscle mass. However, the mechanisms by which TAK1 regulates muscle mass and whether its role is sex dependent remain incompletely understood. In this study, we show that targeted inactivation of TAK1 induces muscle atrophy more rapidly in male than in female mice. Loss of TAK1 activity also abolished mechanical overload-induced phosphorylation of p70S6K and rpS6, and the induction of myofiber hypertrophy in both sexes. RNA-Seq analysis further revealed that TAK1 inactivation in skeletal muscle disrupts the gene expression of various molecules involved in catabolic processes, calcium signaling, muscle structure development, and aerobic respiration. Moreover, TAK1 inactivation impairs fatty acid oxidation and promotes lipid accumulation in skeletal muscle of adult mice in a sex-independent manner. Collectively, our findings demonstrate that TAK1 regulates skeletal muscle mass and growth by coordinating distinct intracellular pathways in both male and female mice.

Duchenne muscular dystrophy (DMD) is a multisystem disorder affecting striated muscle, metabolism, and the central nervous system (CNS). Although glucocorticoids remain the standard therapy, muscle-centric evaluations typically fail to capture how dosing regimen and compound selection affect CNS and metabolic phenotypes. Here, we compared daily and weekly dosing of prednisolone and vamorolone in juvenile mdx mice over six weeks to determine how these variables influence multisystem outcomes. Multiorgan efficacy and adverse effects were quantified across behavioural, endocrine, metabolic, cardiovascular, and muscle domains using behavioural assays, in vivo and functional muscle testing, haemodynamic evaluation and histopathology. Daily glucocorticoid dosing failed to improve muscle function or strength, whereas weekly vamorolone produced the most robust improvements in functional and in vivo muscle strength. Daily prednisolone reduced circulating creatine kinase levels, but this biochemical change did not translate into enhanced muscle function outcomes. Daily regimens also induced severe adrenal cortical atrophy, yet these endocrine alterations were dissociated from CNS stress and anxiety responses, which remained unchanged by treatment. In addition, daily dosing caused pronounced systemic metabolic consequences, whereas weekly regimens substantially attenuated these effects, identifying dosing frequency as a key determinant of safety. Together, these findings demonstrate that glucocorticoid regimen selection fundamentally reshapes the efficacy-adverse effect profile and underscores the value of integrated multiorgan evaluation in DMD. This work highlights the need to expand therapeutic assessments beyond muscle pathology and raises new questions about how glucocorticoid signalling differentially engages peripheral and central physiological systems.