Acta Physiologica

Objective: Mitochondrial oxidative capacity in striated muscle is governed by the PGC-1/ERR transcriptional axis, which controls not only steady-state respiration but the capacity to expand energy production when demand rises. Whether this reserve function requires dedicated downstream effectors, and whether its loss is detectable at rest, remains unclear. We asked whether Perm1, a striated muscle-specific PGC-1/ERR-induced regulator, is required cell-autonomously for cardiac bioenergetic reserve. Methods: We generated cardiomyocyte-specific Perm1 knockout mice using alphaMHC-nCre, complemented by an adult tamoxifen-inducible alphaMHC-MerCreMer model, and assessed cardiac development, structure, and function longitudinally through 12 months of age in both sexes. We evaluated mitochondrial function by high-resolution respirometry, transmission electron microscopy, and molecular profiling. We tested energetic capacity under load by treadmill exercise with tissue ATP quantification and by beta-adrenergic stimulation of cultured neonatal cardiomyocytes. Results: Perm1 deletion did not perturb embryonic cardiac development, and neither constitutive nor adult-onset deletion impaired cardiac structure, systolic function, or diastolic function through 12 months. Despite this preserved resting phenotype, mitochondrial respiration was reduced by 20 - 25% across complex I- and complex I+II-supported conditions, without changes in citrate synthase activity, mtDNA copy number, or OXPHOS subunit abundance. Instead, the MICOS components IMMT (MIC60) and CHCHD3 (MIC19) were selectively reduced, and mitochondrial subpopulations shifted toward fewer subsarcolemmal and more interfibrillar mitochondria, with unchanged total mitochondrial density. Functionally, Perm1-deficient mice of both sexes showed reduced treadmill capacity at 6 and 12 months and failed to maintain cardiac ATP during exercise at a submaximal workload, while skeletal muscle ATP and plasma lactate were unaffected. This deficit was cardiomyocyte-autonomous: beta-adrenergic stimulation increased ATP content in control cardiomyocytes, but Perm1 knockdown reduced this response. Conclusions. Perm1 is a cardiomyocyte-intrinsic determinant of bioenergetic reserve, dispensable for resting cardiac function but required to expand ATP production under increased demand. These findings identify a mode of metabolic regulation that is invisible to resting functional phenotyping.

Store-operated calcium entry (SOCE) is a ubiquitous calcium (Ca2+) signalling mechanism that replenishes endoplasmic and sarcoplasmic reticulum Ca2+ stores. We previously identified STIM1L, a skeletal muscle-enriched STIM1 splice variant that enables rapid SOCE activation in human myotubes. To determine its physiological role in vivo, we generated a constitutive knockout mouse model that selectively abolishes STIM1L expression while preserving the canonical STIM1 isoform. Stim1l-KO mice developed normally and exhibited no overt abnormalities, but both sexes showed reduced voluntary wheel-running activity, suggesting neuromuscular perturbation. Skeletal muscle deprived of STIM1L did not show any histological alterations or changes in fibre- type and size. Likewise, force-frequency relationships and fatigue resistance measured ex vivo in isolated extensor digitorum longus and soleus muscles were indistinguishable from control muscles. This lack of difference may be explained by the increased expression of STIM1 in several skeletal muscles and suggests that changes in the voluntary running activity of Stim1l-KO mice may arise from STIM1L loss in non-muscular tissues, including the central nervous system. Notwithstanding, primary myotubes derived from Stim1l-KO mice displayed a marked delay in SOCE activation kinetics, whereas SOCE amplitude remained comparable to controls. This was consistent with a compensatory STIM1 upregulation maintaining overall Ca2+ entry capacity. Interestingly, electrically evoked Ca2+ transients in intact Stim1l-KO flexor digitorum brevis fibres exhibited significantly faster decay kinetics than controls. This revealed another role of STIM1L in Ca2+ handling in skeletal muscle that appears independent of its contribution to SOCE activation. Together, these findings establish STIM1L as a regulator of SOCE activation kinetics and indicate an additional role in myofibre Ca2+ clearance, while suggesting that compensatory upregulation of canonical STIM1 may preserve muscle structure and contractile function in its absence in mice.

Sickle cell disease (SCD) causes heterogeneous mineral imbalances including variable degrees of hypocalcemia. The kidney controls systemic calcium by reabsorbing calcium from the glomerular filtrate via paracellular transport and transcellular transport in the nephron tubules, yet it is unknown whether these processes are modulated by genetic or environmental factors or disrupted in SCD. Using SCD mouse models and single-cell multiomics, we identify the distal convoluted tubule (DCT) as the nephron segment most susceptible to calcium reabsorption dysfunction in SCD, mainly via reduction of calcium buffer protein calbindin 1 (CALB1). We show that CALB1 and its encoding mRNA are decreased in DCT cells in SCD, alongside decreased Klotho (KL)-dependent fibroblast growth factor (FGF) 23 signaling and intracellular calcium signaling. Dietary iron restriction reduces CALB1, KL, and calcium exporter SLC8A1 levels in SCD kidneys. Loss of CALB1 shifts DCT cells toward energy-inefficient glycolysis with the metabolite 2,3-diphosphoglycerate impairing KL-dependent FGF23 signaling to create a feed-forward loop suppressing calcium reabsorption. Analysis of gene expression and protein quantitative trait loci data from kidneys of genetically diverse mice revealed that Calb1 expression levels are highly heritable and co-regulated with Slc8a1, identifying a genetic axis that dictates differential capacities for calcium buffering and trafficking toward blood in the kidney. Together, these findings support a model in which genetic variation, dietary iron status, and FGF23 signaling converge on DCT calcium buffering to reduce renal calcium reabsorption in the SCD kidney. This points to personalized, genotype- and iron-dependent strategies for managing mineral metabolism in SCD patients.

Transverse (t)-tubules enable rapid, synchronous Ca release required for efficient cardiac contraction by bringing L-type Ca channels into close apposition with ryanodine receptors. In heart failure with reduced ejection fraction (HFrEF), t-tubule disorganisation and loss occur alongside cardiac microtubule remodelling, contributing to impaired Ca handling and contractile dysfunction. Despite their canonical function in contraction, how t-tubules develop is unknown. Microtubules support delivery of L-type Ca channels to t-tubules via Amphiphysin-II/BIN1, yet whether microtubules directly regulate t-tubule formation and maintenance is unclear. Here, we investigated a role for microtubules in t-tubule development and homeostasis. Neonatal rat ventricular myocytes (NRVMs), which lack endogenous t-tubules, were used as a reductionist model in which BIN1 overexpression induces nascent membrane tubules. Microtubule depolymerisation with nocodazole before BIN1 overexpression impaired BIN1-driven tubule formation, reducing tubule density and length. Dynein inhibition with EHNA produced similar effects, indicating a requirement for microtubule-based motor activity during tubule elongation. Knockdown of the microtubule +TIP tracking protein CLIP-170 also reduced BIN1-driven tubule density, implicating BIN1-CLIP-170-dependent microtubule capture in tubule initiation. Microtubules were also required to maintain existing tubules. In NRVMs with established BIN1-driven tubules, microtubule depolymerisation, microtubule stabilisation or dynein inhibition each reduced tubule density and length. Consistent with this, acute microtubule depolymerisation or stabilisation disrupted native t-tubule networks in isolated adult sheep left atrial myocytes. Together, these findings identify cardiac microtubules as active regulators of t-tubule architecture. We propose that BIN1-dependent tubule formation requires CLIP-170-mediated microtubule plus-end capture and dynein-dependent elongation, while ongoing microtubule dynamics are necessary to preserve mature t-tubule structure.

Maternal hyperglycemia can affect offspring development and metabolism, but how its effects on skeletal muscle change during postnatal life remains unclear. We investigated skeletal muscle phenotypes in male offspring of streptozotocin-induced hyperglycemic mice at 4 and 8 weeks of age, representing early post-weaning and later postnatal stages, respectively. Maternal hyperglycemia resulted in lower offspring body and skeletal muscle weights at both time points. Myofiber cross-sectional area was reduced without a reduction in total myofiber number, indicating impaired myofiber growth rather than myofiber loss. At 4 weeks, skeletal muscle showed type I/IIA hybrid myofibers and increased expression of fetal, myogenic, and proliferation-associated genes. This immature profile was not maintained at 8 weeks; instead, the transcriptional profile shifted toward reduced mitochondrial and oxidative metabolism. DNA methylation changes at 4 weeks showed limited correspondence with transcriptional alterations. Taken together, maternal hyperglycemia impaired postnatal myofiber growth at both time points, with distinct molecular profiles at 4 and 8 weeks.

Repetitive cytosolic Ca2+ spikes in pancreatic acinar cells, elicited by low (physiological) concentrations of acetylcholine (ACh), cholecystokinin (CCK) and gastrin releasing peptide (GRP), control secretion of digestive enzymes, whereas high-intensity stimulation induces sustained Ca2+ elevation initiating acute pancreatitis. Since inositol trisphosphate (IP3) was discovered as an intracellular Ca2+ releasing messenger, it has been assumed that a major class of G-protein coupled receptors relies on this pathway. We have now compared the mechanisms of action of the three physiological stimulants, all acting on different receptors, but each coupled to the IP3 pathway. Low concentrations of CCK and GRP cannot elicit Ca2+ signals without co-operation of an additional intracellular mechanism. CCK-elicited Ca2+ signalling requires activation of intracellular receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), whereas this is not the case for the action of GRP that nevertheless relies on the operation of CD38, the enzyme involved in the synthesis of both cyclic ADP ribose and NAADP. Even Ca2+ signals elicited by ACh are partially dependent on CD38. It is engagement of these additional non-IP3 pathways that allows low concentrations of secretagogues to elicit safe Ca2+ spiking and therefore secretion, obviating the need for potentially toxic high levels of secretagogues.

Background: Loss- and gain-of-function variants in SCN5A, encoding the cardiac sodium channel Nav1.5, are major causes of inherited cardiac channelopathies. Their pathophysiology has almost exclusively been attributed to altered cardiomyocyte excitability and cardiac conduction. However, growing evidence indicates that autonomic dysfunction contributes to these disorders, raising the possibility that Nav1.5 dysfunction may also affect the intrinsic cardiac nervous system. Objectives: We investigated whether Nav1.5 is functionally expressed in murine intrinsic cardiac neurons and evaluated its contribution to neuronal excitability. Methods: Scn5a expression was investigated by digital PCR and immunocytochemistry in isolated murine intracardiac neurons. Whole-cell patch-clamp recordings were used to characterize sodium currents and action potential properties in wild-type and Scn5a+/- mice. Results: Scn5a transcripts and Nav1.5 protein were detected in intrinsic cardiac neurons. Voltage-clamp recordings identified a tetrodotoxin-resistant sodium current accounting for approximately 8% of the total sodium current and displaying pharmacological and biophysical properties consistent with Nav1.5. Activation and inactivation analyses indicated that Nav1.5 is the predominant sodium conductance available at most polarized membrane potentials. Accordingly, Scn5a haploinsufficiency selectively altered action potential threshold, maximal upstroke velocity, overshoot and duration in neurons resting below -60 mV, whereas more depolarized neurons were essentially unaffected. No structural or neurochemical remodeling of intrinsic cardiac ganglia was detected. Conclusion: Nav1.5 is a functional determinant of intrinsic cardiac neuronal excitability. These findings identify the intrinsic cardiac nervous system as a new cellular target of SCN5A dysfunction and suggest that inherited cardiac sodium channelopathies could involve alterations in both myocardial and intrinsic neuronal electrophysiology.

Background: The neuromuscular junction (NMJ) is the site of communication between myofibers and a-motor neurons. Cellular and molecular mechanisms that determine, maintain, and remodel the neuromuscular synapse are poorly understood. Coactivator-associated arginine methyltransferase 1 (CARM1) post-translationally modifies target proteins by methylating arginine residues and has emerged as a key determinant of skeletal muscle biology. Methylarginine signalling is required for the maintenance and repair of the NMJ, but the direct role of CARM1 on the NMJ in health and disease remains unexplored, particularly in humans. Methods: We generated Carm1 skeletal muscle-specific knockout-out (mKO) mice to gain a basic understanding for the role of the enzyme in NMJ biology under homeostatic and denervated conditions. Additionally, we investigated CARM1 activity in severe mouse models of neuromuscular disorders (NMDs) including D2.mdx and Smn2B/- mice, which replicate Duchenne's muscular dystrophy (DMD) and spinal muscular atrophy (SMA), respectively, and exhibit chronic remodelling of the NMJ. Lastly, to evaluate if methylarginine signalling is implicated during NMJ instability in human skeletal muscle, we obtained samples from healthy volunteers before and after 14 days of single leg immobilization as well as from patients with myotonic dystrophy type 1. Results: Our results demonstrated that Carm1 mRNA expression and activity are elevated (P<0.05) in NMJ-enriched regions of healthy murine skeletal muscle. Carm1 muscle-specific deletion reduced NMJ compactness (-9.3%; P<0.05), increased fragmentation (+33%; P<0.05), and disrupted the expression of synapse-specific transcripts basally and following sciatic nerve transection. In skeletal muscle from pre-clinical models of NMDs, we observed a compensatory upregulation in CARM1-dependent arginine methylation as evident by +54% and +71 increases (P<0.05) in asymmetric dimethylarginine (ADMA)-marked CARM1 substrates in DMD and SMA mice, respectively. Similarly, muscle CARM1 was hyperactive with increased NMJ instability during neuromuscular disuse (+22%; P<0.05), and disease (+30%; P<0.05), in humans. In a cohort of muscular dystrophy patients and healthy volunteers, elevated CARM1 signalling was negatively correlated with clinical metrics of skeletal muscle health including grip strength (r = -0.583; P<0.05) as well as positively correlated with mRNA expression of NMJ machinery such as CHRNA1 (r = 0.578; P<0.05). Conclusion: In summary, we highlight that muscle-specific CARM1 is required for maintaining NMJ morphology and transcriptional regulation. Insults to NMJ stability during muscle disuse or in myopathic conditions were associated with enhanced CARM1-mediated methylarginine signalling in mice and humans. Collectively, our findings demonstrate CARM1 as a key mediator of NMJ biology and plasticity in health and disease.

Arginine vasopressin (AVP) increases excitability of myometrial smooth muscle cells (MSMCs) through Gq-coupled AVP receptors. Although excitability requires membrane depolarization, the mechanisms linking AVP receptor activation to membrane depolarization and Ca{superscript 2} signaling are incompletely understood. Here, we show that AVPR1 is the predominant AVP receptor in primary MSMCs. In Xenopus oocytes, AVP signals through AVPR1 to inhibit SLO2.1-mediated potassium currents, reducing current amplitude to approximately 60% of control currents. Consistent with suppression of a hyperpolarizing conductance, AVP depolarized a myometrial cell line (hTERT-HM) and increased intracellular Ca{superscript 2} signaling. Analysis of Ca{superscript 2} dynamics revealed that the initial Ca{superscript 2} peak was largely preserved under conditions limiting extracellular Ca{superscript 2} entry, consistent with intracellular store release. Conversely, the oscillatory phase depended on extracellular Ca{superscript 2} influx and was reduced by SLO2.1 knockdown. Together, these findings support a model in which AVP preferentially signals through AVPR1A to inhibit SLO2.1, depolarize myometrial cells, enhance VDCC-dependent Ca{superscript 2} entry, and promote excitability, enhancing conditions for uterine contraction.

Nitrogenous waste excretion is essential for all developmental stages of fish. Embryonic fish excrete urea, transitioning to cutaneous and later branchial ammonia excretion. In zebrafish, ammonia excretion involves rhesus glycoproteins Rhbg and Rhcgb in keratinocytes and ionocytes, but the developmental moment they appear in the gill remains unclear. Potential redundancy between Rhbg and Rhcgb in ammonia excretion is also not fully investigated, nor is the difference in response to low pH. We hypothesized that rhesus glycoproteins are partially redundant, and that they differ in their response to low pH as ammonia excretion enables ionocytes to exchange Na+ and H+ (Rh-NHE-metabolon). We predicted that a loss of rhbg or rhcgb induces compensatory responses. We characterized the transition from urea to branchial ammonia excretion from 0 to 8 days-post fertilization (dpf) and the response to pH 5.0 on the expression and localization of rhesus glycoproteins in control zebrafish and rhbg or rhcgb-crispants. Effects of high external ammonia (HEA, 500 M NH4Cl) and 10 mM HEPES-buffering were further characterized in rhcgb-crispants. Rhag and Rhbg appeared in the gill at 5 dpf, while Rhcgb appeared at 6 dpf. A loss of rhbg or rhcgb did not impact baseline N-excretion, illustrating that zebrafish can maintain ammonia excretion without the full complement of rhesus glycoproteins. We observed no compensatory increase in rhesus glycoproteins, but expression of the transporter hippocampus-abundant transcript 1b increased. HEA-exposed rhcgb-crispants switched to urea as primary nitrogen waste. Together, these findings underline the plasticity of the larval in dealing with nitrogenous waste.

The past decade has seen remarkable progress in the molecular resolution of the influx and efflux components of mitochondrial Ca2+ handling, but little progress in matrix Ca2+ chelation that is central to mitochondrial calcium signaling. Among possible chelators, inorganic phosphate (Pi) is dynamic, and its uptake can lessen during Ca2+ uptake the rundown of the membrane potential, the primary driving force for Ca2+ uptake. Thus, PiC, the major mitochondrial Pi transporter, is well-positioned to regulate mitochondrial Ca2+ handling. To test this, we depleted (KD) PiC in murine skeletal muscle. We show that PiC depletion enhances the matrix free Ca2+ rise across a range of Ca2+ uptake activities, and surprisingly, causes elevated mitochondrial Ca2+ uptake, which seems to arise from an increased abundance of the mitochondrial Ca2+ uniporter. With protein levels of non-mitochondrial Ca2+ handling proteins unaltered, the greater mitochondrial Ca2+ uptake may drive a suppressed cytoplasmic Ca2+ response to tetanic stimulation, via lesser Ca2+-mediated positive feedback on Ca2+ release channels, contributing to an exercise deficit in KD mice. Alternatively, less buffering of matrix Ca2+ might be beneficial. We test these possibilities by lowering MCU, the uniporters pore forming subunit, in skeletal muscle of adult PiC KD mice, and find a worsened exercise deficit. This study establishes the requirement for PiC to maintain a bound fraction of Ca2+ in the matrix, and reveals a fitness benefit for elevated [Ca2+]m in striated muscle depleted of PiC.

Background: Succinate dehydrogenase (SDH) is a heterotetrameric enzyme in both the tricarboxylic acid (TCA) cycle and the electron transport chain. SDH-deficiency has been shown to drive the oncogenesis of cancer types including gastrointestinal stromal tumors (GIST), paraganglioma/pheochromocytoma, and clear cell renal cell carcinoma. Previous work on SDH loss has shown increases in succinate, decreases in fumarate, as well as increase in the succinate to -ketoglutarate ratio, and correlated these changes to metabolite signaling and epigenetic dysregulation. Alterations to other TCA intermediates, including succinyl-CoA, the acyl-donor for succinylation, and the resulting cellular effects has not been fully explored. Methods: We used liquid chromatography high resolution mass spectrometry to quantify TCA cycle metabolites across the NCI-60 panel of cell lines, three SDH-competent and SDH-deficient model systems, and human tumor samples. We measured histone post-translational modifications in SHD-deficient and SDH-competent models and examined the correlation and potential pharmacological targeting of described desuccinylase enzymes by protein and cell assays. Results: We found an increase in succinyl-CoA in multiple SDH-deficient models, but no correlation between succinate to fumarate ratio and succinyl-CoA concentrations in the NCI-60. Interestingly, we found that while there was an increase in succinyl-CoA in the SDH-deficient cells, there was no corresponding increase in histone succinylation. We also observed a decrease in lysine malonylation and glutarylation, two other anionic lysine modifications. Cell viability and clonogenic assays revealed very weak or no selective effect of desuccinylase targeting in SDH-deficient models. Similarly, using SDH-deficient versus SDH-competent GIST patient samples, we examined both protein expression and metabolite abundance at the bulk tumor level and found no significant increase in succinyl-CoA or desuccinylase expression, despite the increase in succinate to fumarate ratio. Conclusions: We found that SDH-deficiency led to altered central carbon metabolism including increases to succinate and succinyl-CoA. Despite data that suggested the upregulation of desuccinylases may mediate a disconnect between succinyl-CoA concentration and histone succinylation, we found weak or no reductions in cell viability in response to pharmacological inhibition of desuccinylases. Additionally, no correlation of SDH loss with desuccinylase expression and succinyl-CoA in bulk tumor samples was observed.

The urinary bladder functions to store and release urine, yet how the sensation of bladder fullness is conveyed and perceived to the central nervous system is not understood. During bladder filling, the detrusor smooth muscle (DSM) generates phasic contractions, resulting in pressure fluctuations within the bladder. These transient pressure events drive bursts of afferent nerve activity, yet the underlying mechanism leading to rhythmic contractions remains unclear. Here, we examined the role of Gq protein-coupled receptor (GqPCR) activity on DSM excitability and contractility. Using ex vivo pressurized urinary bladder preparations and sharp microelectrode experiments on bladder strips from mice, we evaluated whole bladder transient pressure events, whole bladder DSM Ca2+ activity, and membrane potential in bladder strips. We found that global inhibition of urinary bladder GqPCR activity with YM-254890 abates phasic contractility and transient pressure events through a reduction in DSM Ca2+ activity and propagation of Ca2+ waves. Further, we found inhibition of GqPCR significantly hyperpolarizes DSM, reducing action potentials and decreasing excitability, and activation of protein kinase C restores membrane potential to baseline levels. These findings highlight that GqPCR activity mediates DSM excitability and contractility in such a way as to result in phasic detrusor contractions and transient pressure events.

Cerebrospinal fluid (CSF) provides a specialized extracellular environment for the central nervous system, which is predominantly produced by the choroid plexus, a highly vascularized epithelial structure whose ion transport processes are fundamental to CSF secretion, composition, and homeostasis. The mechanisms of Na+ entry into choroid plexus epithelial cells (CPECs) from the interstitial side remain disputed. The slc4a10 gene product encoding the Na+-dependent Cl-/HCO3- exchanger, Ncbe, was suggested as a key transport mechanism based on its impact on the cell's Na+-dependent regulation of intracellular pH and its basolateral membrane expression. The current study was undertaken to directly assess the contribution of Ncbe to the Na+ uptake into CPECs. Intracellular Na+ was recorded by fluorometry using the Na+ probe Sodium Binding Fluorescent Indicator in clusters of CPECs with access to both the luminal and basolateral membranes. Removal of extracellular Na+ reduced the apparent ex vivo intracellular [Na+] to ~5 mM from a baseline of ~43 mM in the absence of CO2/HCO3- and ~54 mM in the presence of CO2/HCO3-. Flame photometry estimated the intracellular [Na+] ex vivo to ~28 mM. The CO2/HCO3--dependent rate of [Na+] recovery amounted to ~53% of the total recovery rate upon re-addition of Na+. Experiments with access to only the luminal membrane show a [Na+] recovery of a similar rate as observed in the absence of CO2/HCO3- in the clusters. The CO2/HCO3--independent [Na+] recovery was inhibited to ~50% by the NKCC1 inhibitor bumetanide and to ~30% by the TRPv4 inhibitor RN1734. NHE contributed to a minor extent to the CO2/HCO3--independent transport. The HCO3- transport inhibitor DIDS, however, inhibited the total [Na+] recovery rate to ~50%, indicating a role for Ncbe rather than NBCn1 in the cellular [Na+] recovery. Indeed, docking of DIDS into Ncbe and NBCn1 indicated that both proteins can accommodate the binding of DIDS. However, the orientation of the DIDS poses in Ncbe suggests a binding mode more similar to that found in the Anion Exchangers (SLC4A1-3), which seems to accommodate the covalent-type docking more than NBCn1. The Ncbe inhibition by DIDS was supported by the rate of [Na+] recovery that was significantly higher in CPECs from Ncbe-wt than Ncbe-ko mice in the presence of CO2/HCO3-. As both NKCC1 and TRPv4 are localized to the luminal membrane, the findings collectively suggest that Ncbe is the most prominent mechanism for Na+ entry into CPECs expressed at the basolateral side. We suggest Ncbe as the rate-limiting mechanism in the vectorial Na+ transport driving CSF secretion.

BackgroundAldosterone promotes endothelial dysfunction and cardiovascular injury through mineralocorticoid receptor (MR) activation. Autophagy is essential for endothelial homeostasis, yet its role in aldosterone-mediated vascular dysfunction remains unclear. We tested whether aldosterone impairs autophagic flux and whether restoring autophagy via Beclin1 (BCN1) activation protects vascular and cardiac function. MethodsEndothelial and vascular responses to aldosterone were assessed in wild-type mice, BCN1 gain-of-function mice (Becn1), and mice treated with spermidine or a BCN1- activating TB-peptide. Vascular function, nitric oxide (NO)/reactive oxygen species (ROS) production, autophagy markers, endothelial migration, and cardiac fibrosis were evaluated using wire myography, fluorescence assays, Western blotting, confocal microscopy, migration assays, and histology. ResultsAldosterone impaired endothelium-dependent relaxation, decreased NO, increased ROS, and disrupted autophagic flux in an MR-dependent manner, indicated by LC3 accumulation and reduced p62 and BCN1 expression. Spermidine restored endothelial function and normalized NO and ROS levels. BCN1 gain-of-function mice were protected from aldosterone-induced endothelial dysfunction and exhibited reduced coronary and myocardial fibrosis. TB-peptide activation of BCN1 enhanced autophagic flux, improved vascular function, decreased cardiac fibrosis, and rescued endothelial migration impaired by aldosterone. ConclusionsAldosterone induces endothelial dysfunction by suppressing autophagic flux through MR activation. Genetic or pharmacologic enhancement of BCN1-dependent autophagy restores endothelial homeostasis and prevents vascular and cardiac injury, identifying autophagy activation as a promising therapeutic approach for cardiovascular diseases associated with mineralocorticoid excess.

Elephant seals are remarkable breath-hold divers, capable of remaining submerged for up to two hours during diving bouts. These dives entail repeated, extreme hypoxia/reoxygenation events that would induce severe lipid peroxidation and tissue dysfunction in most mammals. Here, we show that primary vascular endothelial cells derived from elephant seals possess an intrinsic resistance to lipid peroxidation. Comparative transcriptomic and lipidomic profiling across seal, human, and sheep cells identified ferroptosis - an iron-dependent, lipid peroxidation-driven cell death pathway - as uniquely regulated in seal cells following hydroperoxide exposure. Mechanistically, seal cells exhibit robust baseline expression of acyl-CoA synthetase long-chain family member 3 (ACSL3), alongside rapid, seal-specific induction of the sole mammalian iron exporter, ferroportin (SLC40A1). Functional validation using genetic and pharmacological approaches revealed that seal cells are naturally enriched in monounsaturated fatty acids and triglycerides and utilize lipid droplet biogenesis and active iron export as dual protective axes to evade lipid peroxidation. Together, these findings show that elephant seal cells employ a coordinated cytoprotective network of lipid remodeling and iron handling to withstand the severe challenges of deep diving. SIGNIFICANCE STATEMENTDeep-diving marine mammals repeatedly experience extreme hypoxia-reoxygenation events that would induce severe oxidative damage in most terrestrial mammals. However, vascular cells derived from seals naturally resist lipid peroxidation, a major driver of ischemia-reperfusion injury. Here, we show that elephant seal endothelial cells evade lipid peroxidation through two complementary mechanisms: lipid droplets that sequester peroxidation-prone phospholipids, and rapid iron export that limits lipid peroxide formation. These findings reveal naturally evolved cellular strategies that protect against vascular oxidative stress, offering new insights into physiological resilience against ischemia-reperfusion injury.

Introduction: Adipose tissue fibrosis has been associated with metabolic dysfunction-associated steatotic liver disease (MASLD) and suboptimal weight loss after bariatric surgery. The objective was to characterize changes in adipose tissue fibrosis and extracellular matrix (ECM) components in adipose tissues from two abdominal fat compartments, subcutaneous and omental, in response to bariatric surgery; and determine the effect of weight loss on the association between adipose tissue fibrosis and MASLD feature changes with weight loss. Methods: Liver and adipose tissue biopsy samples were obtained before and after weight loss from patients who underwent two-stage bariatric surgery. Adipose tissue pericellular fibrosis was evaluated histologically with picrosirius red staining for collagen quantification and normalized to adipocyte count per microscope field. Transcript levels of ECM remodeling and inflammatory genes were measured by RNA sequencing in subcutaneous adipose tissue pre- and post-surgery. Results: At baseline, adipose tissue pericellular fibrosis did not associate with MASLD features, metabolic dysfunction-associated steatohepatitis or hepatic fibrosis in patients with severe obesity. Changes in liver histology after surgery were unrelated to changes in adipose tissue fibrosis. Transcripts for most genes involved in ECM remodeling and inflammation were downregulated in subcutaneous fat one year after bariatric surgery. Omental, but not subcutaneous, adipose tissue fibrosis decreased after surgery when adjusted for cell number (mean {+/-} SD: 0.41 {+/-} 0.12 vs 0.32 {+/-} 0.08 {micro}m2/cell, p<0.005). Higher subcutaneous adipose tissue fibrosis correlated with reduced weight loss (estimate {+/-} SE: -2.04 {+/-} 0.99, p<0.05) and BMI loss (-0.73 {+/-} 0.35, p<0.05), while increased omental adipose tissue fibrosis after weight loss was associated with worsening hepatic fibrosis (0.31 {+/-} 0.14, p<0.05). Conclusion: In individuals with severe obesity undergoing bariatric surgery, adipose tissue fibrosis is poorly correlated with MASLD features. Notably, pericellular fibrosis in subcutaneous abdominal, but not omental, adipose tissue was associated with a reduced weight-loss response to surgery.

Longitudinal muscle growth occurs through the addition of sarcomeres in series, a process termed serial sarcomerogenesis. To investigate contraction mode dependence of sarcomerogenesis in Sprague-Dawley rats (n=36, 14 to18 weeks, ~468g), we compared the effects of isometric, eccentric, and concentric training (4 to 6 weeks; 3 times/week) on morphological and mechanical adaptations of the plantar flexors. Isometric training was performed at a joint angle corresponding to the plateau nearing the descending limb of the force length relationship, eccentric contractions began at a shorter muscle length and lengthened to this same joint position, and concentric contractions started at this lengthened position but then actively shortened. Owing to the rapid regulation of serial sarcomere number the 4-and 6-week timepoints were collapsed. While force output increased across all training modes (+16%; p<0.05), longitudinal muscle growth was regulated by contraction type. Both isometric and eccentric training were highly effective at increasing serial sarcomere number when optimal loading conditions (high forces at longer lengths) are provided (+7-9%; p<0.05). Concentric training, even when initiated at long lengths with high forces was not an effective stimulus for longitudinal remodelling (+2%; p>0.05). Therefore, these findings identify the mechanical loading environment associated with isometric and eccentric training as a key driver of serial sarcomerogenesis.

Gastrointestinal dysfunction is a prominent feature of neurodevelopmental disorders and contributes significantly to visceral discomfort, but the specific etiology is unclear. In particular, it is unknown which cell types within central, autonomic or enteric nervous systems, gut muscle, epithelium or immune system are the principal drivers of gut dysfunction. We report that the Mecp2 mouse model of Rett syndrome, a neurodevelopmental disorder with profound gut dysmotility, exhibits impaired whole gut transit times and constipation in vivo as well as striking alterations to propagating neurogenic contractions in ex vivo colons. Selective elimination of Mecp2 in neurons, but not glia or smooth muscle, recapitulates these dysmotility phenotypes. Bulk RNA sequencing of colonic external muscle, containing the myenteric compartment of the enteric nervous system, showed significant expression changes in specific neurochemical signaling pathways. Pharmacological modulation of one, the M1 acetylcholine receptor, modified the aberrant neurogenic contractile phenotype in mutant mice and reproduced it in wild-type mice. These results identify disruptions to neuronal muscarinic signaling in the enteric nervous system in a mouse model of Rett syndrome and provide an experimental strategy to identify translationally relevant cellular and molecular pathways underlying gastrointestinal dysmotility in neurodevelopmental disorders.

To address the long-standing question of the respective physiological contributions of classical brown versus beige adipocytes to adaptive nonshivering thermogenesis, we generated mice with lineage-specific ablation of UCP1 in thermogenic adipocytes of myogenic origin. This selectively targeted the major classical brown adipocyte lineage while preserving UCP1 expression in the remaining thermogenic adipocytes, reducing total UCP1 content by approximately 80 %. Unexpectedly, despite this profound reduction in UCP1 abundance, cold acclimation-recruited thermogenic capacity, assessed by adrenergic stimulation, remained largely preserved. In contrast, complete UCP1 deficiency abolished the adrenergically induced thermogenic response, demonstrating that UCP1 is indispensable for adaptive nonshivering thermogenesis. These findings indicate that in cold-acclimated mice only a fraction of the UCP1 normally present is required to sustain maximal thermogenic capacity. We further establish that the capacity to support UCP1-dependent oxidative metabolism, rather than UCP1 abundance, is the principal constraint on maximal thermogenic output under these conditions.