Acta Physiologica

IntroductionVariants in PRKAG2 cause hypertrophic cardiomyopathy (HCM) and conduction disturbances. While prior studies associated PRKAG2-related hypertrophy with increased glycogen storage, many HCM phenotypes remain unexplained. We aimed to uncover how PRKAG2 variants induce myocyte hypertrophy and electrical changes during early cardiac development. MethodsWe generated transgenic zebrafish expressing wild-type (TgWT) or pathogenic variant (TgR299Q) Prkag2 cDNA under a myocardium-specific promoter, and examined cardiac electrophysiology, contractile function, and cytoarchitecture during cardiogenesis and in adult hearts. ResultsTgR299Q fish showed hypertrophic cardiomyocytes and progressive contractile abnormalities, recapitulating human HCM phenotypes. Cardiomyocyte glycogen was elevated in adult but not embryonic hearts. Despite the absence of glycogen accumulation at 6-day post-fertilization, TgR299Q hearts showed electrical abnormalities, including reduced conduction velocity and prolonged action potential and Ca2+ transient durations. We observed decreased AMPK phosphorylation in the TgR299Q hearts. However, AMPK activation did not rescue the electrophysiological abnormalities in TgR299Q. Proximity ligation assays and co-immunoprecipitation identified a physical interaction between AMPK{gamma}2 and myosin, enhanced by the R299Q variant and accompanied by increased AMPK{gamma}2 localization to the myofilament. Na/Ca{superscript 2} exchanger (NCX) inhibition increased Ca2+ duration and diastolic Ca2+ in TgWT but not TgR299Q hearts, indicating reduced free cytosolic Ca2+ for NCX-mediated extrusion in TgR299Q. These findings suggest that enhanced AMPK{gamma}2-myosin interaction may promote myofilament Ca{superscript 2} retention, thereby prolonging Ca{superscript 2} transient duration and APD in the mutant. Notably, the myosin inhibitor mavacamten reduced AMPK{gamma}2-myosin interaction in TgR299Q hearts, and both mavacamten and vmhcl knockdown rescued the early electrophysiological abnormalities. ConclusionsThe PRKAG2 variant altered cardiac excitability, contractility, and Ca2+ handling during cardiogenesis, independent of glycogen accumulation. Enhanced interactions between AMPK{gamma}2 and myosin contributed to these early changes. Our study revealed a novel link between cellular energy sensing and contractile machinery, with therapeutic potential for modulating contractile function in cardiomyopathies.

Elastin haploinsufficiency is a primary determinant of arteriopathy and hypertension that hallmark Williams syndrome (WS), a rare genetic disorder resulting from microdeletion of genes on human chromosome 7, including the elastin gene (ELN). Accumulating evidence suggests renal dysfunction, including enhanced sodium and water retention as an underlying cause of blood pressure elevation resulting from heterozygous deletion of Eln (Eln+/-) in mice that recapitulates the cardiovascular phenotype of WS. However, the underlying pathophysiological mechanisms are poorly understood. Here, we determined whether the activity of neuraminidase-1 (NEU1) of the elastin receptor complex (ERC) contributes to abnormal handling of water and electrolytes by the kidney in Eln haploinsufficiency. Adult male and female Eln+/+ and Eln+/- mice were subjected to acute extracellular fluid volume expansion with normal saline, combined with pharmacological intervention targeting vasopressin V2 receptor (V2R), NEU1, ENaC, and NKCC2. In male Eln+/+ mice, V2R blockade induced a dose-dependent increase in urine flow rate without affecting sodium excretion. Conversely, V2R stimulation with desmopressin markedly increased urinary sodium excretion in male Eln+/+ but not Eln+/- mice, while both sexes of Eln+/- mice exhibited marked suppression of urine flow rate. Abrogation of ERC signaling through NEU1 inhibition produced a modest increase in urinary sodium excretion in male mice of both genotypes but augmented urine flow rate only in male Eln+/+mice. NEU1 blockade strikingly enhanced the natriuretic effect of furosemide and amiloride in male Eln+/+ and modestly in Eln+/-mice. Taken together, we conclude that Eln haploinsufficiency disrupts vasopressin-dependent modulation of sodium and water reabsorption by sex-dependently altering ERC-mediated modulation of NKCC2 and ENaC. These findings reveal a novel mechanism by which abnormal ERC activity due to Eln haploinsufficiency potentially contributes to renal dysfunction and hypertension. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/731713v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1870e8org.highwire.dtl.DTLVardef@9cbd8dorg.highwire.dtl.DTLVardef@60b2deorg.highwire.dtl.DTLVardef@7f31f1_HPS_FORMAT_FIGEXP M_FIG C_FIG AC, adenylyl cyclase; AQP2, aquaporin 2; CD, collecting duct; CNT, connecting tubule; DCT, distal convoluted tubule; EBP, elastin binding protein; Eln, elastin allele; ENaC, epithelial sodium channel; ERC, elastin receptor complex; Gs, stimulatory G subunit; NEU1, neuroaminidase1; NKCC2, sodium-potassium-chloride cotransporter; PPCA, protective protein/ cathepsin A; TAL, loop of Henle thick ascending limb; V2R, vasopressin receptor type 2

Obesity is associated with an elevated risk of pathological cardiac hypertrophy, whereas exercise confers cardioprotective effects; however, the cellular mechanisms underlying these opposing influences remain incompletely defined, particularly in females. We investigated how obesity and exercise affect cardiomyocyte ultrastructure, Ca{superscript 2} release, and contractility in female Zucker Diabetic Fatty rats and their lean littermates. Animals were assigned at 12 weeks to sedentary or aerobic exercise-trained groups and maintained on a standard diet. By 18 weeks, obese rats exhibited increased body mass and myocardial hypertrophy in the absence of diabetes. Sedentary obese animals showed a reduced fraction of compact dyads and diminished stimulated and caffeine-induced Ca{superscript 2} release, while contractility remained preserved. In lean rats, exercise increased dyad density but reduced Ca{superscript 2} release, whereas in obese rats, exercise enhanced both dyad compactness and Ca{superscript 2} release. Across all groups, global cardiomyocyte ultrastructure and contractile function were similar. Type III ANOVA revealed a significant obesity x exercise interaction for dyadic structure and Ca{superscript 2} release. These findings demonstrate that obesity itself, independent of diabetes, triggers early dyadic remodeling and altered Ca{superscript 2} handling in female myocardium before detectable impairment of global cardiomyocyte structure or contractile function. Furthermore, exercise exerts beneficial effects on dyadic ultrastructure and Ca{superscript 2} signaling in obese animals. New & NoteworthyUsing a female rat model of obesity without diabetes, we demonstrate that obesity induces early remodeling of the dyadic system and impairs Ca{superscript 2} release in cardiac myocytes. We further show that the effects of aerobic exercise on dyadic structure and function are obesity-dependent, improving both dyad organization and Ca{superscript 2} signaling. These findings identify the dyadic microdomain as a vulnerable cellular site in obesity and a potential target for exercise-induced recovery.

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.

BackgroundNeprilysin (NEP) is a zinc-dependent metalloprotease targeted in heart failure therapy to prevent it degrading circulating cardioprotective vasoactive peptides. NEP can also cleave sarcolipin (SLN), the skeletal- and atrial muscle-specific micropeptide regulator of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). A direct pathophysiological role of NEP in ventricular muscle has not been established. MethodsProteomics and immunoblot analysis of human myocardial specimens were used to quantify NEP abundance in failing and non-failing hearts. Heterologous protein expression and biochemical binding assays assessed NEP-mediated cleavage of phospholamban (PLB) and its impact on PLB-SERCA interactions. Functional consequences of NEP expression or inhibition were evaluated in neonatal rat ventricular myocytes and in a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of heart failure. ResultsWe observed increased NEP abundance in failing human myocardium relative to non-failing controls. We demonstrated that NEP cleaves phospholamban (PLB), disrupting PLB-SERCA interactions. Mutation of PLB (V49A), prevented NEP cleavage and preserved PLB-SERCA binding, indicating V49 is critical for NEP substrate recognition. In neonatal rat ventricular myocytes, NEP expression was associated with faster Ca2+ transient decay kinetics and increased SR Ca2+ load, consistent with reduced SERCA inhibition. Inhibition of NEP in a hiPSC-CM heart failure model attenuated the hypertrophic transcriptional responses and reversed Ca2+-transport dysregulation. ConclusionsThese findings implicate increased NEP expression in the sarcoplasmic reticulum of cardiomyocytes as previously unrecognized maladaptive consequence of heart failure contributing to cardiac dysfunction. In this novel pathophysiological mechanism, increased NEP results in PLB cleavage and loss of regulation of SERCA. While this may relieve SERCA inhibition and augment cellular Ca2+ handling, loss of PLB chronically disrupts hearts dynamic response to adrenergic stress, changing heart rate, or other physiological challenges. The data provide new insight into the cardioprotective effects of pharmacological NEP inhibition in clinical practice, reveal a novel mechanism of action of neprilysin inhibition in cardiomyocytes and may help inform future therapeutic strategies for patients with heart failure. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/732949v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@4daf93org.highwire.dtl.DTLVardef@41ef8aorg.highwire.dtl.DTLVardef@d568daorg.highwire.dtl.DTLVardef@d6d213_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cold-induced thermogenesis in brown adipose tissue (BAT) is essential for maintaining energy homeostasis, yet the Ca2+-dependent mechanisms underlying this process remain incompletely understood. Here, we identify Orai1, a component of the store-operated Ca2+ entry pathway, as a regulator of thermogenic activation in BAT. Using a brown adipocyte-specific Orai1 knockout mouse model, we demonstrate that cold exposure is associated with Orai1-dependent Ca2+ influx through a non-canonical mechanism. Orai1 deficiency leads to impaired cAMP-PKA signaling, reduces the expression of lipolytic enzymes and thermogenic genes, and diminished mitochondrial Ca2+ uptake and uncoupling. These defects culminate in cold intolerance, lipid accumulation, and decreased energy expenditure. Mechanistically, Orai1 facilitates Ca2+-dependent activation of adenylyl cyclase 3, linking membrane Ca2+ entry to cAMP production, and promotes mitochondrial remodeling and oxidative metabolism. These findings support a key role for Orai1 in coordinating Ca2+ entry to lipolytic and mitochondrial pathways in brown adipocytes and highlight its potential therapeutic target in metabolic diseases characterized by impaired energy metabolism. HIGHLIGHTSO_LIOrai1 mediates Ca2+ influx in brown adipocytes through a non-canonical, partially STIM1-independent mechanism. C_LIO_LIOrai1-mediated Ca2+ influx promotes both cAMP-PKA-driven lipolysis and mitochondrial oxidative activation. C_LIO_LIOrai1-dependent Ca2+ entry promotes cAMP-PKA signaling and lipolytic activation I nbrown adipocytes. C_LIO_LIOrai1 coordinates mitochondrial Ca2+ uptake to support thermogenic function in brown adipocytes. C_LI

The carotid body (CB) is the key peripheral oxygen sensor. CB mitochondria are hypothesised to be uniquely adapted with unusually low intrinsic oxygen affinity which, in association with nitric oxide (NO) and reactive oxygen species signalling, enables acute responsiveness to hypoxia. However, CB mitochondrial physiology or intrinsic oxygen affinity have never been measured directly. We sought to address this key gap by isolating sheep CB mitochondria and comprehensively characterising their phenotype and contrasting them to a non-oxygen sensing tissue, left ventricular myocardium (LV). High resolution respirometry, liquid chromatography mass spectrometry, enzymatic assays and in silico modelling were used to characterise mitochondrial content, aerobic capacity, oxygen affinity, complex subunit abundance and activity, H2O2 production and NO sensitivity in ovine CB and LV. Mitochondrial oxygen affinity (P50 = 0.089 mmHg) was lower in the CB than the LV (P50 = 0.058 mmHg; p = 0.005). Whilst mitochondrial content was lower in the CB, CB mitochondria had higher respiratory rates and enzymatic activity than LV. H2O2 production and NO sensitivity were similar in the two tissues. While intrinsic mitochondrial oxygen affinity is slightly lower in the oxygen sensing CB than in the non-oxygen sensing LV, this difference is small. Hence, any role of mitochondria in CB oxygen sensing is not due to an intrinsic difference in the O2 affinity of cytochrome oxidase due to differential expression of its subunits. Instead, this work suggests that differences in O2 affinity in vivo are secondary to other factors, perhaps including NO, that alter mitochondrial O2 affinity.

Adrenal chromaffin cells are key effectors of the sympathoadrenal response and play a central role in the organisms adaptation to environmental and physiological challenges. While cholinergic and pituitary adenylate cyclase-activating polypeptide (PACAP)-dependent mechanisms have long been recognized as major regulators of catecholamine secretion, increasing evidence indicates that connexin-mediated gap junctional communication provides an additional and highly dynamic level of control. Whether early-life experience modifies the adult capacity of chromaffin-cell networks to undergo stress-induced connexin remodeling remains unclear. Here, we examined adrenal medullary connexin expression in adult rats exposed to neonatal maternal separation (MS; 3 h daily, postnatal days 2-15) and later challenged with an 8-day unpredictable mild stress (UMS) protocol. Under basal adult conditions, MS did not produce an overt change in adrenal medullary Cx36 or Cx43 immunoreactivity relative to animal-facility-reared controls. In contrast, UMS increased connexin immunoreactivity in the adrenal medulla, and this response was amplified in animals with a history of MS. MS+UMS animals also displayed enhanced corticosterone responses to acute restraint stress. These findings suggest that neonatal MS does not impose a constitutively altered adult chromaffin-cell phenotype, but instead primes the future stress responsiveness of adrenal medullary connexin remodeling. We propose that chromaffin-cell gap junctions represent a substrate sensitive to stress history, through which developmental experience may influence sympathoadrenal and endocrine adaptation in adulthood.

The delta-9 desaturase stearoyl-CoA desaturase-1 (SCD1) catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids (MUFA) and is highly expressed in liver and adipocytes. Previous studies have demonstrated that treating mice with dextran sulfate sodium (DSS), a chemical inducer of ulcerative colitis, results in severe downregulation of SCD1 in the liver. However, the specific role of hepatic SCD1 in modulating colitis severity, as well as the impact of DSS on SCD1 and other lipogenic factors in other tissues has not been investigated. Here we show that downregulation of hepatic SCD1 following DSS treatment is not accompanied by changes to other lipogenic genes in the liver. In contrast, adipose tissue demonstrates coordinated reductions in lipogenic genes, including SCD1 and SCD2, while the colon does not display any perturbation of these targets. Furthermore, we demonstrate that the downregulation of hepatic SCD1 occurs independently of sterol regulatory element binding protein-1c (SREBP-1c) and does not require an intact gut microbiome. Interestingly, a distinct model of colitis induced by IL-10 deficiency does not result in downregulation of hepatic SCD1. Concomitant with transcriptional changes, DSS treatment is associated with significant remodeling of the hepatic lipidome, including reductions in total phospholipids (PLs) and reduced MUFA-containing PLs and triacyglycerols (TAGs), consistent with the observed reduction in SCD1. Interestingly, hepatic cholesterol esters and plasma lipids including free cholesterol and glycerophospholipids were significantly elevated following DSS treatment. Given the significant reduction in hepatic SCD1 following DSS treatment, we tested a role for liver SCD1 in modulating colitis sensitivity. Mice with a targeted deletion of hepatic SCD1 were not more prone to colitis, indicating that the loss of hepatic SCD1, while a consequence of DSS-induced colitis, does not mediate colitis sensitivity in vivo. SynopsisHepatic SCD1 does not modulate colitis severity upon DSS exposure. However, DSS-induced colitis elicits significant lipid metabolism dysfunction, demonstrated by elevated plasma and liver lipids, particularly plasma cholesterol and hepatic cholesterol esters, highlighting a role for gutliver crosstalk following colonic inflammation.

White, beige and brown adipocytes store energy as lipids, secrete hormones and produce heat, playing an important role in the regulation of energy balance through not completely defined mechanisms. We investigate herein the impact of the almost complete absence of mature adipocytes (severe lipoatrophy) in the determination of energy balance (energy intake and expenditure) and homeothermy in mice. For this, mice with severe lipoatrophy induced by adipocyte deletion of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) (PPAR{gamma} flox adiponectin-Cre) and littermate controls (PPAR{gamma} flox) were evaluated for energy balance, thermoneutral zone, core body temperature, locomotor activity, and gene expression profiles at different ambient temperatures. Severely lipoatrophic mice are heavier, hypermetabolic and hyperphagic and feature a widened thermoneutral zone, lower ambulatory activity, and metabolic inflexibility at both 23 and 17{degrees}C, along with unstable thermal behavior characterized by hyperthermia at 30{degrees}C, normothermia at 23{degrees}C, and bouts of hypothermia at 17{degrees}C. Noteworthy, lipoatrophic mice hypermetabolism at 30{degrees}C is not due to thyroid hormones, impaired insulation or increased body and lean masses and is not altered by pharmacological blockade of either {beta}-adrenergic receptor signaling with propranolol or skeletal muscle sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) and sarcolipin (SLN)-mediated calcium cycling with dantrolene, but is partially attenuated by pharmacological inhibition of acetyl-CoA carboxylase (ACC) and de novo lipogenesis with ND-630. In conclusion, severe lipoatrophy causes hypermetabolism and hyperthermia at 30{degrees}C partly through the activation of liver de novo fatty acid synthesis.

A hallmark of dilated cardiomyopathy (DCM) is calcium mishandling, including reduced transport activity of the SERCA calcium pump in cardiac muscle cells. This has focused attention on SERCA as mechanism of disease and potential therapeutic target. Previously, diminished SERCA activity has been attributed to decreased protein expression, but recent studies suggest SERCA levels are unchanged in DCM. Thus, another mechanism must be responsible for the deficit. Since proteolysis is increased and proteosome function is impaired in DCM, we reasoned that accumulation of toxic protein fragments may contribute to SERCA dysfunction. In particular, previous studies showed diverse species of hydrophobic -helices can inhibit SERCA, so we hypothesized that SERCA may become congested with transmembrane peptides that mimic endogenous regulatory partners. We purified cell membranes from non-failing and DCM human ventricles and subjected them to mass spectrometry to identify protein species upregulated in DCM. Select candidates were screened for binding and inhibition of SERCA. Several small membrane proteins and membrane protein fragments bound avidly to SERCA and significantly reduced cellular calcium stores. The data suggest a novel pathophysiological mechanism in which transmembrane protein debris obstructs SERCA function and regulation, contributing to cardiac muscle dysfunction in heart failure.

BackgroundDietary sodium restriction is a common nutritional and physiological challenge that activates electrolyte-conserving endocrine pathways, but its impact on systemic lipid metabolism remains incompletely defined. We examined whether short-term dietary sodium deprivation alters the circulating lipidome and identifies lipid signatures of metabolic adaptation. MethodsMale Sprague-Dawley rats were maintained on sodium-sufficient (NaS) or sodium-deprived (NaD) diets for 7 days (n=3 per group). Serum lipids were profiled by untargeted LC-MS/MS in positive and negative ion modes. Lipidomic differences were evaluated using class-level and species-level analyses, principal component analysis, volcano plots, heatmaps, and pathway-oriented interpretation. ResultsNaD rats exhibited a distinct serum lipidomic profile compared with NaS controls, indicating global remodeling of circulating lipid composition. Sodium deprivation produced class-specific and species-resolved changes, including selective depletion of subsets of neutral lipid species, prominent wax ester remodeling, increased phosphatidylcholine and lysophosphatidylcholine abundance, and altered acylcarnitine profiles. These signatures are consistent with coordinated changes in lipid storage, membrane phospholipid turnover, and mitochondrial fatty-acid handling. ConclusionsDietary sodium deprivation induces coordinated serum lipidome remodeling in rats, supporting the concept that nutritional electrolyte status can influence systemic lipid metabolism. These exploratory findings identify sodium deprivation as a metabolic stressor linked to neutral lipid mobilization, phospholipid remodeling, and altered mitochondrial substrate handling, and provide a foundation for future mechanistic studies.

Impaired contractility and reduced myocardial energetic reserve underlie heart failure with reduced ejection fraction. Catecholaminergic inotropes such as dobutamine are used to augment cardiac output. However, dobutamine increases Ca{superscript 2} cycling, raising ATP demand and worsening energetic stress. The myotrope CK-138 increases contractility by directly activating myosin, sparing the added energetic cost of Ca{superscript 2} handling. This study compares CK-138 and dobutamine with respect to the relationship between contractile performance and myocardial energetic state, including high-energy phosphate balance, energetic efficiency, and substrate-specific metabolic fluxes. Isolated rat hearts were perfused with escalating concentrations of CK-138 or dobutamine. Contractility was assessed by measuring left ventricular pressure and rate-pressure product. Myocardial energetics were analyzed using 31P-NMR, and metabolic fluxes by 13C NMR and mass spectrometry. Unlike dobutamine, CK-138 increased LV contractility without increasing heart rate or LV end-diastolic pressure. CK-138 preserved ATP and phosphocreatine levels, maintaining a stable phosphocreatine-to-ATP ratio and free energy of ATP hydrolysis, whereas dobutamine progressively depleted both. At comparable workload, dobutamine exhibited higher glycolytic flux and lactate production, indicating greater reliance on glycolysis relative to mitochondrial oxidative metabolism, whereas CK-138 exhibited a 13% higher rate of ATP synthesis and [~]50% lower anaplerotic flux, consistent with preserved mitochondrial efficiency. In conclusion, CK-138 enhances cardiac contractility while preserving myocardial energetic state and substrate utilization. Unlike dobutamine, which depletes ATP reserves and shifts metabolism toward glycolysis, CK-138 maintains ATP homeostasis and supports oxidative metabolism. These findings support cardiac myosin activators, including CK-138 and omecamtiv mecarbil, as a mechanistically distinct class of energy-efficient inotropes.

Skeletal muscle homeostasis depends on the coordinated regulation of protein turnover and mitochondrial quality control; however, the molecular mechanisms linking these processes remain unclear. In this study, we examined the physiological role of leucine aminopeptidase 3 (LAP3), a post-proteolytic aminopeptidase, using constitutive LAP3-deficient mice. LAP3 deficiency preferentially affected skeletal muscle, causing reduced muscle mass and mitochondrial enlargement in both sexes. Female LAP3-deficient mice also showed reduced myofiber size, impaired endurance capacity, increased energy expenditure, elevated lipid oxidation, and lipid droplet accumulation adjacent to the mitochondria. Proteomic analyses revealed remodeling of pathways related to lipid metabolism and protein homeostasis. Consistent with these findings, LAP3 deficiency increased the expression of Pink1 and Tax1bp1 and promoted the accumulation of ubiquitinated proteins, suggesting alterations in mitochondrial quality control and proteostatic regulation. In cultured myogenic cells, LAP3 localized to mitochondrial fractions, and both LAP3 knockdown and overexpression altered mitochondrial morphology. Taken together, these results identify LAP3 as a regulator of skeletal muscle homeostasis and support a role for LAP3 in linking intracellular peptide turnover to mitochondrial homeostasis, with female skeletal muscle showing greater susceptibility to LAP3 deficiency.

Spinal cord injury (SCI) disrupts supraspinal autonomic pathways that regulate cardiovascular function, producing marked blood pressure instability and contributing to secondary injury in peripheral organs. The kidney is particularly vulnerable to these disturbances because renal blood flow (RBF) depends on tightly regulated interactions between neural, myogenic, and vascular control mechanisms. However, how SCI level and chronicity alter dynamic renal autoregulation remains poorly defined. Here, we investigated the effects of high- and low-thoracic SCI on renal hemodynamic control using in vivo blood pressure and RBF recordings in female mice. Hemodynamics were assessed at baseline and during acute sympathetic stimulation induced by norepinephrine (NE; 10 g/kg, i.v.) at 24 h and 4 wk following spinal cord transection at thoracic level 3 (T3) or thoracic level 10 (T10). Time-domain analyses quantified systolic blood pressure recovery, while frequency-domain analyses were used to resolve myogenic and sympathetic contributions to RBF regulation. High-thoracic SCI caused marked disruption of renal vascular responses to acute hypertension, producing paradoxical increases in RBF during NE-induced pressure elevations and sustained reductions in baseline and evoked RBF activity within frequency ranges associated with myogenic and sympathetic vasomotion. These impairments were most pronounced during the chronic phase of injury, consistent with loss of dynamic autoregulatory control and vascular remodeling. In contrast, low-thoracic SCI preserved baseline renal vasomotor activity and demonstrated recovery of dynamic autoregulatory responses over time. These findings identify SCI level and chronicity as critical determinants of renal microvascular regulation and demonstrate that high-thoracic SCI produces persistent autonomic-vascular uncoupling. This disruption of dynamic renal autoregulation represents a previously underappreciated mechanism of secondary organ vulnerability following neurotrauma.

BackgroundExercise is an effective way to improve metabolic health, and the modulation of adipose tissue (AT) secretory functions may play a significant role in this process. AT produces various lipokines, including fatty acid esters of hydroxy fatty acids (FAHFA), which increase insulin sensitivity and have anti-inflammatory effects. While factors such as sex, age, obesity, and genetics influence FAHFA levels, their impact on exercise-induced FAHFA regulation remains unclear. MethodsFirst, sex-specific responses to an acute bout of exercise were assessed in wild-type (WT) and ADTRP-deficient (ADTRP KO) mice. Fasted mice underwent acute treadmill exercise until exhaustion, followed by analysis of non-esterified fatty acids in plasma, ex vivo lipolysis in the presence or absence of a hormone-sensitive lipase (HSL) inhibitor, and FAHFA release from AT (measured by LC-MS). Second, obese male WT and ADTRP KO mice fed a high-fat diet underwent 7 weeks of regular treadmill exercise (5 days/week), after which parameters of glucose homeostasis, plasma and AT FAHFA levels, and AT lipid profiles were analyzed. ResultsAcute exercise-induced increases in plasma non-esterified fatty acid levels, AT lipolysis, and FAHFA release from AT explants were more pronounced in male mice of both genotypes. Conversely, pharmacological inhibition of HSL using BAY 59-9435 increased FAHFA release from AT explants only in females. In obese sedentary ADTRP KO mice, insulin sensitivity was improved compared with their WT counterparts. Although regular exercise suppressed weight gain in obese animals of both genotypes, insulin sensitivity improved only in WT mice. Chronic exercise generally had no effect on plasma FAHFA levels in mice fed ad libitum; however, in WT mice, it increased the levels of FAHFA-containing triacylglycerol estolides, which were associated with improved insulin sensitivity. ConclusionsAcute exercise revealed sex-specific differences in AT lipolysis and FAHFA metabolism, with HSL playing an important role in FAHFA hydrolysis. Chronic exercise in obesity increases insulin sensitivity and FAHFA storage in AT; however, this effect is absent in ADTRP KO mice, which exhibit elevated FAHFA levels in AT, a condition associated with improved insulin sensitivity even in non-exercising animals.

Background The relative contributions of molecular size, electrostatic charge, and filtration rate to glomerular transport remain controversial. We hypothesized that glomerular sieving data contain a limited number of underlying transport modes that can be identified directly from experimental measurements. Methods Glomerular sieving coefficients were measured in anesthetized rats using neutral and anionic polysucrose during baseline conditions and glucagon-induced hyperfiltration. Data were analyzed using aligned-rank two-factor ANOVA, nonlinear mixed-effects regression of an electrostatic distributed two-pore model, pairwise correlation analysis, and principal component analysis. Results Hyperfiltration reduced the sieving of small and intermediate polysucrose molecules, whereas anionic polysucrose exhibited lower sieving coefficients than neutral polysucrose over a broad range of molecular sizes. An electrostatic distributed two-pore model accurately reproduced the observed effects of filtration rate and molecular charge and yielded an effective pore-wall charge density of 5.4 mC/m2 (95% confidence interval, 4.5 to 6.6). Pairwise correlation analysis revealed strong coupling between neighboring molecular sizes throughout the entire measured size range. Principal component analysis of the 2.5-8.0 nm size-selective region showed that the first principal component explained 96.3% of the variance and the first two principal components explained 99.9% of the variance. Separate analyses of the 2.5-5.0 nm and 5.0-8.0 nm transport regions showed that the first principal component explained 99.4% and 89.5% of the variance, respectively. Conclusions Glomerular sieving curves exhibited a highly constrained low-dimensional structure despite differences in molecular charge, filtration rate, and individual animals. The observed transport structure was consistent with distinct small-pore and large-pore transport domains and enabled highly effective principal component-based denoising of experimental sieving data.

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

Nutritional strategies to mitigate obesity and type 2 diabetes (T2D) have largely focused on dietary fat and carbohydrate composition, with less attention given to protein sources. While total dietary protein intake is recognized as an important modulator of energy balance and glucose metabolism, it remains unclear how the composition of dietary proteins can influence energy metabolism and body weight gain. Here, we investigated the metabolic effects of three distinct protein sources from meat (pork), dairy (casein) and plant (soy) on either a low-fat low sucrose (LFLS) or a high-fat high sucrose (HFHS) diet. While protein sources failed to influence metabolic homeostasis on LFLS, mice kept on the HFHS diet were distinctly impacted by the dietary protein sources. Pork and to a lesser extent soy protein feeding exacerbated obesity, glucose intolerance, and hepatic insulin resistance. Remarkably, livers of mice fed pork or soy protein on the HFHS diet were characterized by extensive microvesicular steatosis compared to the predominant macrovesicular steatosis in HFHS fed mice fed casein protein. Liver transcriptomic and metabolomic signatures in pork and soy protein fed mice were consistent with increased mitochondrial beta-oxidation. Intake of pork and soy proteins in HFHS fed mice lead to a striking reduction in hepatic acetyl CoA carboxylase 2 (ACC2) protein levels relative to casein fed HFHS mice. Pork and soy feeding raised carnitine exposure in the post-prandial period and we determined that exposure of hepatocytes to carnitine provokes downregulation of ACC2 and hepatic insulin resistance in the presence of palmitate:oleate and fructose. Collectively, these findings identify a novel mechanism by which dietary proteins modulate obesity and associated metabolic disturbances through a carnitine-mediated regulation of ACC2 protein and mitochondrial lipid handling in liver.

BackgroundVertical sleeve gastrectomy (VSG) improves glycaemic control in type 2 diabetes (T2D) through mechanisms that extend beyond weight loss. The interaction between glucocorticoid metabolism and inflammation in this context remains unclear. MethodsWe investigated the role of 11{beta}-hydroxysteroid dehydrogenase type 1 (11{beta}HSD1) in mediating the metabolic effects of VSG in humans and mice. Subcutaneous adipose tissue biopsies were collected before and 6 months after VSG. Parallel studies were conducted in lean and high-fat diet-fed mice undergoing VSG or sham surgery, alongside 11{beta}HSD1 knockout models. Glucose tolerance and expression of 11{beta}HSD1 and interleukin-6 (IL6) were assessed. Mechanistic interactions were examined in IL6-treated human hepatocytes. ResultsVSG reduced 11{beta}HSD1 and IL6 expression in human adipose tissue and improved insulin resistance. In lean mice, VSG improved glucose tolerance and downregulated both markers independently of weight loss. 11{beta}HSD1 knockout mice exhibited improved glucose tolerance despite increased adiposity, partially recapitulating the VSG phenotype. Both interventions reduced circulating and tissue IL6 levels. IL6 stimulation increased HSD11B1 expression in hepatocytes. Conclusions11{beta}HSD1 links glucocorticoid metabolism, inflammation, and glucose homeostasis following VSG. Targeting this pathway may offer a strategy to replicate key metabolic benefits of metabolic bariatric surgery.