Acta Physiologica

Brown adipose tissue (BAT) is a unique tissue with mitochondria specialized for thermogenesis via the BAT-specific uncoupling protein 1 (UCP1). Ucp1-/- mice cannot tolerate acute exposure to cold, illustrating the necessity of UCP1 for efficient mitochondrial thermogenesis. However, these mice adapt to low temperatures through a gradual acclimation process, suggesting a high degree of mitochondrial plasticity in brown and beige fat cells. This phenomenon, which remains to be fully elucidated, indicates the potential for these mitochondria to implement effective thermogenic mechanisms in the absence of uncoupling protein 1 (UCP1). Here, we investigated mitochondrial remodeling in beige and brown fat of Ucp1-/- mice to determine how they fulfill their thermogenic role. Upon gradual acclimation to a cold environment, Ucp1-/- mice exhibited body metabolic parameters and temperatures in the interscapular region similar to those of wild-type mice of BAT, highlighting effective thermogenesis. Interestingly, mitochondrial patch-clamp analysis and a mitochondrial Ca2+ swelling assay revealed a dramatic increase in Ca2+ uptake depending on the mitochondrial calcium uniporter (MCU) in BAT mitochondria from Ucp1-/- mice when robust thermogenesis was required. Mitochondrial remodeling was accompanied by markedly increased tethering between mitochondria and the endoplasmic reticulum (ER) in Ucp1-/- mice, confirming a significant restructuring of the contact sites between the ER and mitochondria, likely to adapt to a new Ca2+ homeostasis. Respiratory complexes also underwent significant reorganization, which partly led to a reduction in their assembly. Levels of ATP synthase and its F1 subcomplex increased, suggesting a major source of ATP consumption and energy expenditure. We propose a new role for MCU as a key regulator of mitochondrial plasticity, enabling efficient thermogenesis in beige and brown adipose tissues in the absence of UCP1.

BackgroundCarnitine plays an obligatory role in energetics owing to its role in the translocation of long-chain fatty acids into the mitochondrion for oxidation. Here, we determined the metabolic and behavioral consequences of systemic carnitine deficiency (SCD) in mice. MethodsFemale C57BL/6J mice were randomized to receive normal drinking water (control, n = 8) or drinking water supplemented with mildronate 4g.L-1 (mildronate, n = 8) for 21 days. Body composition was assessed at baseline and post treatment. Metabolic and behavioral phenotyping was performed continuously over 72 hours following 14 days of control or mildronate treatment. Stable isotope were used to assess whole-body substrate oxidation. Carnitine subfractions were quantified in skeletal muscle and liver, as was mitochondrial respiratory function. Liver and muscle samples also underwent proteomic analysis. ResultsMildronate treatment depleted total carnitine in muscle and liver by [~]97% (P < 0.001) and [~]90% (P < 0.001), respectively. Carnitine depletion was accompanied by lower total energy expenditure (P = 0.01), attributable to lower voluntary wheel running (P = 0.01). Oxidation rates of palmitate (P < 0.01) but not octanoate were lower whereas rates of glucose oxidation were greater in carnitine depleted mice (P < 0.01). Mitochondrial respiratory capacity was unaltered by carnitine deficiency. Carnitine deficiency remodeled muscle and liver proteomes to support lipid oxidation and energy production. SummaryIn mice, carnitine deficiency is characterized by decreased long-chain fatty acid oxidation despite preserved mitochondrial respiratory capacity. Carnitine deficiency resulted in lower voluntary exercise and a concomitant reduction in energy expenditure.

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

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.

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.

Metabolic dysfunction-associated steatotic liver disease (MASLD) afflicts more than one-third of adults globally, contributing significantly to an increased cardiovascular disease risk. Further, patients with severe liver disease experience muscle weakness (sarcopenic obesity) and fatigue. Hypoxia-inducible factor 2 (HIF2) accumulates in the livers of MASLD patients and has been implicated in disease progression. Here we sought to understand the role of hepatic HIF2 in mediating hepatic and extra-hepatic features of MASLD. Using a well-validated obese mouse model of MASLD, we investigated the impact of hepatocyte-specific HIF2 deletion (hHIF2-/-) on hepatic, cardiac and skeletal muscle metabolism, and cardiac function. Over 28 weeks, mice were exposed to a high-fat, high-fructose, high-cholesterol (GAN) diet, which induced obesity alongside hepatic steatosis, fibrosis and inflammation. In contrast to observations in lean mouse models of liver disease, hHIF2-/- did not protect against MASLD, despite greater hepatic NADH-supported mitochondrial respiration and higher intracellular sphingomyelin levels. Instead, in the hearts of GAN-fed mice, hHIF2-/- caused diacylglycerol accumulation independent of diet, accumulation of long-chain acyl-carnitines and exacerbation of ceramide accumulation. Langendorff-perfused hearts from hHIF2-/- mice showed systolic and diastolic dysfunction, including 24% lower left ventricular developed pressure and 34% lower maximal rate of relaxation (dP/dtmin). However, isolated hearts from hHIF2-/- mice were protected against MASLD-associated sympathetic dominance, determined using autonomic receptor agonist stimulation. Both GAN-feeding and hHIF2-/- were associated with lower lean mass (14% and 5.4% lower than respective controls), whilst hHIF2-/- enhanced OXPHOS-associated protein levels in gastrocnemius muscle. Overall, hHIF2-/- resulted in detrimental extra-hepatic effects, including myocardial lipid accumulation, impaired cardiac function, and loss of whole-body lean mass, with no apparent protection against MASLD disease progression.

Metabolic syndrome (MetS), characterized by abdominal obesity, insulin resistance, dyslipidemia, and hypertension, affects a substantial proportion of the global population and increases the risk for cardiovascular disease, diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD). Despite its prevalence, there are currently no effective pharmacological therapies targeting MetS, highlighting the need to identify novel etiological mechanisms, particularly within the gastrointestinal (GI) tract. Using a mouse model of MetS and healthy lean controls, we assessed the colonic microenvironment through metabolomic, transcriptomic, and microbiome analyses. Colonic organoids were cultured to further explore epithelial alterations. Additionally, human MetS fecal metabolomics data were cross-compared with the mouse model to validate translational relevance. MetS mice exhibited upregulation of colonic anabolic pathways, including glycolysis, the pentose phosphate pathway, and the tryptophan/kynurenine pathway, without evidence of intestinal inflammation. Microbiome analysis revealed an increased abundance of the genus Lactobacillus in MS NASH mice. Colonic organoids from MetS mice showed altered goblet cell differentiation. Comparative analysis with human MetS fecal metabolomics demonstrated similar dysregulated pathways, underscoring the translational relevance of these findings. Our study reveals significant metabolic and microbial alterations in the colon of MS NASH mice, implicating a dysfunctional GI tract as a potential etiological factor in MetS. These findings highlight specific metabolic pathways and microbial signatures that could serve as future therapeutic targets for MetS. NEW & NOTEWORTHYThis study identifies the colon as a metabolically active tissue affected in metabolic syndrome. Despite the absence of intestinal inflammation, MS NASH mice displayed altered colonic metabolism and microbiota composition, with conserved metabolite changes matching those seen in humans with metabolic syndrome. These findings highlight colonic metabolic dysfunction as a potential driver of gut dysbiosis and disease progression in metabolic syndrome and MASLD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/716131v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@1b7c685org.highwire.dtl.DTLVardef@4a832aorg.highwire.dtl.DTLVardef@1e95c66org.highwire.dtl.DTLVardef@1b14209_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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.

Colorectal cancer (CRC) cachexia induces skeletal muscle dysfunction, impeding quality of life and worsening cancer prognosis. Multiple preclinical models, including the widely used mouse model of subcutaneous inoculation with the C26 colorectal carcinoma cell line, have been developed to study the biological mechanisms of CRC cachexia and elucidate potential new treatments. It has been proposed that a distinct cell line of the same origin, namely CT26, is relatively non-cachexic. However, studies evaluating the relative potential of C26 and CT26 cells to induce cancer cachexia in parallel have been limited. The differences in the biological mechanisms by which C26 and CT26 impact skeletal muscle mass and function have also not been fully elucidated. In the current study, we investigated the differential capacity of C26 and CT26 to induce cancer cachexia using both an in vitro cancer-muscle cell co-culture and an in vivo syngeneic mouse model. Our results show that both C26 and CT26 cells induced significant atrophy of murine C2C12 skeletal myotubes. In the mouse model, while C26 and CT26 both reduced skeletal muscle mass and fat mass, only C26 tumors led to loss of body weight and impaired skeletal muscle force output. We further show that C26 tumor-bearing mice exhibit greater muscle inflammation than CT26 tumor-bearing mice. In addition, mice bearing C26 and CT26 tumors showed differential regulation of the innate immune responses and muscle protein turnover. Overall, our data suggests that although both C26 and CT26 cells do exhibit cachexic effects, C26 cells induce greater loss in body weight, fat mass, skeletal muscle mass, and physical function via promoting chronic inflammation and deregulating protein balance of skeletal muscle.

{beta}2-Adrenergic receptor (Adr{beta}2) is the most abundant form of adrenergic receptors in skeletal muscle. Our previous studies have shown that the ventromedial hypothalamic nucleus (VMH) regulates metabolic benefits of exercise, potentially by skeletal muscle Adr{beta}2. Although a large body of literature has shown the importance of Adr{beta}2 on skeletal muscle physiology, it remains unexplored whether skeletal muscle Adr{beta}2 contributes to metabolic benefits of exercise, such as prevention of diet-induced obesity (DIO). Here, we generated mice lacking Adr{beta}2 in skeletal muscle cells (SKMAdr{beta}2) and tested whether SKMAdr{beta}2 is required for metabolic benefits of exercise on DIO. Deletion of SKMAdr{beta}2 completely abolished the induction of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1) in skeletal muscle by {beta}2-agonist, which is a potent activator of Pgc-1. Exercise upregulates Pgc-1, which regulates a broad range of skeletal muscle physiology, including hypertrophy and mitochondrial function. Deletion of SKMAdr{beta}2 hampers augmented Pgc-1 in skeletal muscle by a single bout of exercise. Intriguingly, we found that deletion of SKMAdr{beta}2 increased endurance capacity. Further, our data showed that body weight in DIO mice lacking SKMAdr{beta}2 is comparable to that of control DIO mice during exercise training, suggesting that deletion of SKMAdr{beta}2 did not affect the metabolic benefits of exercise in DIO. Collectively, our data indicate that SKMAdr{beta}2 contributes to exercise-induced transcriptional changes and endurance capacity, however, it is not required for exercise benefits on bodyweight in DIO mice.

Exercise is a cornerstone therapy for diabetes because working skeletal muscles take up glucose at dramatically greater rates than postprandial insulin-stimulated glucose uptake and, notably, do so without a requirement for insulin. This remarkable ability of working muscles is preserved in diabetes, when muscles become resistant to insulin. However, the mechanism of insulin-independent glucose uptake by working muscles is not fully understood. Here we describe a previously unrecognized glucose uptake pathway in muscle, which we refer to as "mSGLT" based on shared properties with the Sodium Glucose Linked Transporter family. In contrast to the abundant GLUT4 transporter, mSGLT is not regulated by insulin, requires Na,K-ATPase-2 activity, and transports the hexose -methyl-D-glucoside (MDG), a glucose derivative that is handled by SGLTs but not GLUT4. The mSGLT pathway and GLUT transport pathways are independent and additive. In addition to exercise, mSGLT imports glucose under other conditions of adrenergic stimulation, which inhibits pancreatic insulin release and reduces the insulin sensitivity of muscle. SGLT2-specific antibodies recognize a protein in muscle of similar size to the kidney SGLT2; this protein localizes to the muscle t-tubules, together with Na,K-ATPase-2 and MAP17, the regulatory subunit of SGLT2. However, skeletal muscles do not express a full-length transcript of Slc5a2 (SGLT2), and SGLT2-specific inhibitors do not inhibit mSGLT with high affinity. The novel transporter may be a muscle variant of Slc5a2 that results from post-transcriptional or post-translational mechanisms. mSGLT and its regulation offer potential muscle-specific therapeutic targets for treating hyperglycemia and other conditions when insulin-stimulated glucose disposal into muscle is impaired.

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

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

BackgroundDyspnea and exercise intolerance are the primary clinical symptoms of heart failure. Heart failure patients experience frequent hypoxemic episodes, yet underlying mechanisms and relevance remain poorly understood. In a cohort of heart failure patients and multiple animal models, we identify pulmonary capillary rarefaction driven by excessive autophagy in endothelial cells as a novel mechanism of hypoxemia and cardiac disease progression. MethodsA cohort of heart failure with preserved ejection fraction (HFpEF) patients was analyzed for parameters of left ventricular (LV) dysfunction and pulmonary gas exchange. Morphological and cellular mechanisms of impaired pulmonary oxygenation were assessed in three animal models of heart failure, namely two HFpEF models, SU5416-treated ZSF1 obese rats and high fat diet/L-NAME treated mice, and in rats subjected to aortic banding. Lung microvascular rarefaction was quantified by micro-computed tomography, stereology, flow cytometry and dye efflux. Cellular mechanisms of capillary loss were analyzed by single-cell transcriptomics, electron microscopy and immunofluorescence, and in mice with endothelial-specific deletion of the autophagy gene Atg7 (Atg7EN-KO). ResultsIn 234 HFpEF patients, advancing NYHA class was associated with progressive worsening of arterial oxygen saturation at rest and during exercise and a reduced lung diffusing capacity. Impaired gas diffusion correlated with indices of LV diastolic dysfunction. Impaired oxygenation and reduced exercise capacity were similarly evident in animal models of left heart disease, which showed a distinct loss of pulmonary microvessels and capillaries. Lung microvascular endothelial cells in HFpEF showed characteristics of increased autophagic flux and apoptosis. Relative to their wild type HFpEF controls, Atg7EN-KO mice had less capillary loss, restored normoxemia, improved exercise tolerance, and mitigated LV diastolic dysfunction. Additional studies in HFpEF mice corroborated the functional relevance of impaired gas exchange for the progression of left heart disease by demonstrating that additional hypoxia aggravated, whereas moderate hyperoxia improved LV function. ConclusionOur findings identify pulmonary microvascular rarefaction as a novel pathomechanism in heart failure that i) contributes to dyspnea and exercise intolerance, ii) impairs pulmonary gas exchange and iii) accelerates LV disease progression. Strategies targeting this axis such as moderate oxygen therapy may mitigate cardiopulmonary morbidity in heart failure. Clinical Trial RegistrationRegistered in the DRKS (Deutsches Register fur klinische Studien) as trial# DRKS00032974 at https://drks.de/search/en/trial/DRKS00032974.

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

General anesthetics enable invasive experimentation but can affect cardiovascular and respiratory physiology, biasing preclinical outcomes. We compared five anesthetic regimens in adult male Wistar rats, tribromoethanol (TBE, 250 mg/kg i.p.), chloral hydrate (CH, 400 mg/kg i.p.), ketamine-xylazine (KX, 80/10 mg/kg i.p.), thiopental (TP, 80 mg/kg i.p.), and isoflurane (ISO, 4% induction, 2% maintenance), to investigate integrated cardiorespiratory and biochemical markers. Femoral arterial catheterization allowed continuous blood pressure (BP) and derived heart rate (HR) recordings, while ventilation was assessed through pletysmography at baseline (awake), during induction, and recovery phases of anesthesia. Variability was evaluated in the time and frequency domains, including HR, systolic blood pressure (SBP), and spontaneous baroreflex sensitivity. In an independent cohort of rats, butyrylcholinesterase (BChE), CK-MB, cTnI, and LDH were measured. Baseline BP was unchanged by TBE and TP, whereas all anesthetics affected HR. Minute ventilation and breathing frequency were reduced with all agents, while tidal volume decreased with KX and TBE only. LDH and cTnI were unaffected, BChE was reduced by KX, TBE, and ISO, and CK-MB increased with CH and KX. Variability analysis showed that all anesthetics depressed pulse-interval and SBP variability and shifted spectral power toward higher frequencies, while baroreflex sensitivity and effectiveness were consistently reduced. During recovery, KX and TP restored most variability indices, whereas CH, TBE, and ISO showed persistent suppression. These findings highlight distinct profiles of cardiovascular depression and biomarker responses across anesthetics and underscore the importance of accounting for autonomic variability when selecting different anesthetics in experimental protocols. HighlightsO_LIFive anesthetic regimens were tested in rats. C_LIO_LIAll anesthetics reduced ventilation, and KX and TBE also reduced tidal volume. C_LIO_LICH and KX increased CKMB, while KX, TBE and ISO reduced BChE. C_LIO_LIAll anesthetics reduced blood pressure variability and baroreflex sensitivity. C_LIO_LIVariability recovered with TP and KX, whereas CH, TBE and ISO showed persistent suppression. C_LI

AimsCaveolae are plasmalemmal microdomains regulating stretch-dependent, nitric oxide (NO), and other signalling pathways governing myocardial structure, function and resilience. We have reported that global deletion of the scaffold protein cavin-1 disrupts caveolar biogenesis and impairs ventricular compliance and tolerance to ischaemic injury. However, cardiomyocyte-specific and sex-dependent roles of cavin-1 and caveolar complexes remain unresolved. Methods and ResultsWe generated a floxed Cavin-1 transgenic mouse, enabling cardiomyocyte-specific knockdown via adeno-associated virus (AAV) mediated expression of iCre recombinase driven by a cardiac-specific troponin T promoter. Knockdown was confirmed by RNA, protein, and immunofluorescence analyses, and cardiac function was assessed via echocardiography, left ventricular pressure-volume (PV) catheterisation, and ex vivo PV analysis of perfused hearts. Conditionally deleted hearts and myocytes exhibited up to 50% knockdown of Cavin-1 mRNA together with 15% deficiency in muscle-specific Caveolin-3, 70% depletion of caveolae, and mislocalisation of NO synthase (NOS) within cardiomyocytes. This was associated with elevated heart rate and shortened PR interval; reduced intraventricular and systolic blood pressures and peripheral resistance; and sex-dependent impairment of ventricular filling (females only). Diastolic dysfunction was detectable ex vivo, to a greater extent in male vs. female hearts. Mechanisms were sex-dependent, linked to interstitial fibrosis in females and NOS overactivity (inhibited by 100 {micro}M L-NAME) in males. Female hearts also exhibited increased susceptibility to ischaemia-reperfusion injury. Coronary function appeared preserved in both sexes, with intact reactive hyperaemic responses. ConclusionThis model identifies cardiomyocyte caveolae and cavin-1 as key determinants of myocardial function and compliance, involving sex-dependent remodelling and NOS signalling. By linking cardiomyocyte disruption to whole-organ and -body dysfunction, this model provides mechanistic insight into impaired function in heart failure and ageing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/717104v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@51bfe4org.highwire.dtl.DTLVardef@10d4323org.highwire.dtl.DTLVardef@1b2baa7org.highwire.dtl.DTLVardef@fc5f21_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.