Mitochondrial dysfunction is a hallmark of obesity-associated metabolic disease, with the mitochondrial permeability transition pore serving as a key regulator of cellular energy homeostasis. Cyclophilin D, encoded by Ppif, is the principal regulatory component of this pore and has been implicated in insulin secretion, adipocyte survival, and whole-body energy balance. However, whether Ppif loss produces a consistent metabolic phenotype when challenged with high fat diet (HFD) exposure, and whether systemic metabolic alterations reflect tissue-specific changes in mitochondrial oxidative capacity, remains unresolved. Here, we characterized male, 21-weeks-old mice with germline Ppif deletion longitudinally over 34 weeks under standard (SD) and HFD using body composition analysis, indirect calorimetry, glucose and insulin tolerance testing and tissue-resolved, Seahorse-based mitochondrial respirometry. Under SD, knockout mice displayed elevated fat mass without differences in food intake or energy expenditure. High-fat feeding progressively amplified this adiposity, with knockout mice developing significantly greater body weight and fat mass than wild-type littermates by weeks 28 and 36. Glucose tolerance and insulin sensitivity remained comparable between genotypes under either diet. ANCOVA revealed significant effects of lean mass (p < 0.001) and genotype (p = 0.017) on energy expenditure, as well as a significant lean mass × genotype interaction (p = 0.025). Consistent with a distinct contribution of lean mass to EE in KO vs. WT mice, we found increased ADP-stimulated mitochondrial respiration in quadriceps of knockout mice under both diets, without corresponding differences in brown adipose tissue or liver, or changes in mitochondrial content across tissues. These findings establish cyclophilin D as a diet- and tissue-specific regulator of energy homeostasis, informing therapeutic strategies targeting the permeability transition pore or cyclophilin D interactome in metabolic disease.

Skeletal muscle disuse during injury decreases muscle mass and function. Previous studies have determined differential gene expression associated with atrophy, but changes in alternative splicing (AS) in response to unloading is poorly defined. This study aimed to identify global changes in differential alternative splicing (DAS) in skeletal muscle following unloading. Twenty-one healthy males (26.0 ± 5.7 years; 81.2 ± 11.6 kg) underwent 14 days unilateral knee-brace immobilization. Participants underwent strength and muscle mass assessment before and after the immobilization and vastus lateralis muscle biopsies were taken pre-immobilization, and on days 3 and 14. RNA-sequencing was performed, and DAS events were identified using the rMATS package. Leg strength decreased - 21.5 kg (P < 0.0001) while mid-thigh circumference was reduced ~ 2% (P = 0.016). A total of 157,464 and 159,395 splicing events affecting 15,219 and 15,374 genes identified on day 3 and 14, respectively. Most DAS events occurred in coding sequence regions and were primarily due to exon skipping. A higher number of DAS events were observed by day 14 than day 3, indicating time-dependent regulation. Gene Ontology analysis of DAS at day 14 revealed 37 enriched GO biological processes related to MAPK signalling, mitochondrial function, and organelle organization. Unloading-induced atrophy was associated with widespread changes in AS, particularly in genes linked to muscle maintenance and protein synthesis. AS appears to promote down-regulation of target genes and subsequent differentially enriched biological pathways associated with the functional outcomes of decreased muscle mass and strength. These findings indicate changes in alternative splicing is a regulatory mechanism in muscle wasting during immobilization.

The transcapillary filtration of fluid is governed by the Starling forces. The purpose of the present study was to simultaneously estimate all Starling parameters in living humans by using likelihood mathematics. First, the capillary filtration was estimated by volume kinetic analysis at 3,015 time points during 193 intravenous infusion experiments using crystalloid and colloid fluid in adult volunteers and surgical patients placed in the supine position. Second, the obtained capillary filtration and the measured plasma colloid osmotic pressure were entered into a new kinetic analysis to estimate the five remaining parameters in the Starling equation. The result shows that the capillary hydrostatic pressure was 16 mmHg (Pc), the interstitial hydrostatic pressure - 2.2 mmHg (Pi), the interstitial colloid osmotic pressure 1.6 mmHg (πi), the reflection coefficient 0.79, and the capillary filtration coefficient (Kf) 306 mL/min/mmHg for the whole body when crystalloid volume loading was performed. Kf was significantly lower when colloid fluid (albumin 5% and 20%) was infused, and πi was lower in patients with subacute inflammation. Association plots showed that Pc was low in hypovolemia and inflammation but markedly higher when albumin was infused. Plots over time suggested that Kf was high early during an infusion, which was probably due to release of nitric oxide due to shear stress, but then decreased gradually. In conclusion, the parameters in the Starling equation could be estimated with acceptable confidence during fluid infusion experiments in humans. Changes of Kf appeared to be key parameter that modulated the capillary filtration rate during volume loading.

Chronic hyperglycemia and cerebral insulin resistance are the major contributors to cognitive impairment in the ageing population. Due to the increased number of the worldwide type - 2 diabetic mellitus (T2DM) population, the prevalence of cognitive decline and dementia has also increased. It is well documented that moderate aerobic exercise dramatically improves cognitive function in individuals with diabetes. However, the impact of exercise on T2DM-associated cognitive decline modulating TGF-β/Smad-3 signalling is yet to be explored. In this study, we assessed the impact of treadmill exercise on cognitive health in a preclinical model of T2DM, focusing on neuroinflammation, mitochondrial function, and the TGF-β/Smad-3 signalling pathway. To develop an experimental type - 2 diabetic model, rats were fed a high-fat diet (HFD), followed by an intraperitoneal injection of a single low dose of streptozotocin (STZ, 35 mg/kg). After confirmation of diabetes, rats underwent moderate-intensity treadmill exercise for 14 weeks. We observed a significant alternation in behavioural patterns in diabetic rats compared to controls, which reversed on exercise treatment. Moreover, chronic Type - 2 diabetes compromises mitochondrial function and increases inflammation in the hippocampal region of the brain. An increase in pro-inflammatory cytokines, such as IL-1β and TNF-α, was observed in the hippocampus of T2DM animals and was reduced by exercise. Oxidative stress is markedly increased in the type - 2 diabetic brain, resulting in compromised mitochondrial function with elevated hippocampal MDA and decreased GSH and ATP levels, which are reversed in the exercise group. Furthermore, we evaluated the role of exercise on TGF-β/Smad-3 signalling in the type - 2 diabetic brain. Exercise suppresses the overexpression of this signalling cascade in the diabetic brain. In summary, moderate-intensity treadmill exercise ameliorated type - 2 diabetes-associated cognitive impairment by decreasing oxidative stress, neuroinflammation, mitochondrial dysfunction and downregulating the TGF-β/Smad-3 signalling pathway.

Heart failure is associated with systemic skeletal muscle remodeling, where sex-specific differences suggest differential peripheral involvement in disease progression. Skeletal muscle macrophages regulate angiogenesis and tissue remodeling and may contribute to early alterations in muscle structure and function during cardiac pressure overload. We tested whether chronic pressure-overload induces sex-specific skeletal muscle remodeling and alters macrophage abundance and paracrine signaling in male and female Sprague Dawley rats that underwent transverse aortic constriction (TAC) or sham surgery at 4 weeks of age and were studied after 40 wk. TAC induced 12% greater heart mass (p = 0.049) and 17% greater heart-to-body mass ratio (p = 0.018) in males, while females showed minimal cardiac remodeling. In extensor digitorum longus (EDL) muscle, TAC males exhibited ~ 10% smaller fiber perimeter corresponding with higher capillary-to-fiber perimeter exchange (p < 0.05 for both), while female TAC rats had no difference in fiber size and 17% lower capillary-to-fiber ratio than female sham (p = 0.033). In soleus muscle, TAC rats had higher capillarization indices (p < 0.05) regardless of sex. Muscle macrophage abundance was 19% lower in TAC males than sham males (p = 0.005). TAC macrophages secreted less MCP-1 (- 35%, p = 0.0058), IL-1β (- 16%, p = 0.0167), TNF-α (- 16%, p = 0.0337), and VEGF-A (- 16%, p = 0.0120) and produced less complex endothelial networks (18% larger mesh size, p = 0.001). These findings indicate that TAC is associated with skeletal muscle remodeling and macrophage immunosuppression, with some changes occurring in a sex-specific manner. This suggests the potential for immune-microvascular coupling in peripheral adaptations to cardiac pressure overload.

For decades, ischemic neuronal death was understood largely as a story about glutamate. Acid-sensing ion channels (ASICs) have revised that account. When cerebral blood flow stops, the penumbra can develop prolonged extracellular acidosis, and experimental studies implicate ASIC1a in mediating sufficient Ca[2+] entry to injure neurons even when glutamate receptors are blocked, a contribution shaped by channel expression, subunit composition, and the intracellular signaling context. However, sustained acidosis does not necessarily imply sustained channel opening, given ASIC1a desensitization. Besides acidosis, glutamate, polyamines, a paradoxical intracellular alkalization, and oxidative stress can enhance ASIC1a activity or expression, and the channel also signals through a non-conducting route, recruiting RIPK1 to trigger necroptosis independently of ion flux. The relative contributions of these conducting and non-conducting mechanisms in vivo remain incompletely defined. Endogenous mechanisms may constrain injury by internalizing the channel, altering subunit expression, including conditioning-associated ASIC2a upregulation, and degrading ASIC1a through estrogen signaling. We argue that these responses are better understood as constraints on injury than as a genuine survival program, a distinction with mechanistic and therapeutic consequences. Critically, the contribution of these mechanisms may vary over time. The same channel acts as a target of homeostatic regulation before stroke, a driver of death during ischemia, a potential target at reperfusion when acidosis persists but restored flow finally permits drug delivery, and an impediment to repair for weeks afterward. The question in ASIC-targeted neuroprotection is not only whether to inhibit the channel, but also when and for how long.

Type 2 long QT syndrome (LQT2) increases the risk of cardiac arrest and sudden death due to reduced rapid delayed rectifier potassium currents (IKr) in the heart. Caused by loss-of-function genetic variants in the hERG K[+] channel, LQT2 might benefit from variant-targeted therapy approaches. T1019PfsX38 and Q1070X are LQT2-causing variants reported in Oman and Saudi Arabia. Although nonsense-mediated mRNA decay (NMD) has been proposed to contribute to reduced expression of these truncating variants, it remains unclear whether residual mutant hERG channel function can be pharmacologically enhanced. We used the patch-clamp technique and examined the effects of the hERG potentiator NS1643 on wild-type-, T1019PfsX38- and Q1070X-hERG channels expressed in HEK293 cells. We started by assessing the response of these variants under the influence of a ventricular action potential clamp protocol, then used additional protocols to elucidate the drug's mechanism of action. NS1643 [10 µM] enhanced the repolarization currents in wild-type-, T1019PfsX38- and Q1070X-expressing cells. Its effect was associated with accelerated activation rates and a negative shift in the voltage dependence of activation. In addition, NS1643 suppressed fast deactivation transitions. We conclude that the functions of T1019PfsX38- and Q1070X-hERG channels can be augmented by hERG potentiators if NMD is incomplete, which may facilitate the development of a mechanism-based rescue approach.

Circadian rhythms regulate physiological and behavioural processes, with the light-dark cycle acting as the principal environmental cue synchronising the biological clock; disruption of this cue can affect brain function and behaviour. Using zebrafish (Danio rerio) as a translational model, we examined how chronic photoperiod alteration, applied from the larval stage to adulthood, affects exploratory activity, anxiety-like behaviour, aggression, and social preference, and whether these changes are paralleled by shifts in gut microbiota composition and brain transcriptomic profile. Zebrafish were reared under three photoperiod regimes: 14L/10D (control), 20L/4D (extended light), and 4L/20D (extended dark), followed by behavioural, gut metagenomic, and brain transcriptomic analyses. Extended darkness (4L/20D) reduced anxiety-like behaviour, whereas extended light (20L/4D) increased aggression both extended photoperiods altered social behaviour, but with distinct behavioural profiles. Gut microbiota in the 20L/4D group showed phylum-level co-dominance of Actinomycetota and Pseudomonadota, including a greater presence of potentially pathogenic taxa, while the 4L/20D group was dominated by Bacillota. Brain transcriptome profiling, based on a single pooled sample per condition, identified transcripts showing the largest expression differences in each group: in 20L/4D, transcripts linked to phototransduction (gnat1, saga, pde6ga, exorh), ribosomal function (rpl13a, rpl9, knop1), melatonin synthesis (asmt), calcium signalling (plcb4a), and glycolysis (eno1a) were elevated; in 4L/20D, transcripts linked to neuroprotection (nr4a1), DNA repair and chromatin remodelling (fance, uimc1, histh1l), synaptic plasticity (serpina10a), neurodevelopment (six6a), and cell-cycle regulation (btg2) were elevated. These findings indicate that photoperiod shapes zebrafish behaviour, gut microbial composition, and brain gene expression, providing a hypothesis-generating basis for future studies incorporating biological replication.

The epithelial sodium channel (ENaC) is a key regulator of electrolyte homeostasis and blood pressure. A characteristic ENaC feature is sodium self-inhibition (SSI), a rapid autoregulatory mechanism in which extracellular Na+ ions allosterically bind ENaC and facilitate transition into a low open probability state. Although SSI is an important functional parameter for assessing ENaC activity and gating, its quantification has traditionally relied on manual two-electrode voltage-clamp (TEVC) recordings with limited scalability and throughput. Here, we improved and evaluated experimental strategies and analytical methods for robust quantification of ENaC SSI in manual and automated TEVC systems: (1) The determination of SSI from extrapolation of transient current recordings in response to a jump in the extracellular Na+ concentration; (2) the estimation of SSI by comparison of steady-state currents before and after chemical modification of a βS520C-ENaC mutant with MTSET; and (3) quantification of SSI from the analysis of steady-state current-voltage relationships recorded at different extracellular Na+ concentrations. The latter procedure is the most reliable for SSI quantification in perfusion rate-limited automated TEVC systems. These methods can expand the capabilities of automated electrophysiology and support medium- to high-throughput investigation of autoregulatory mechanisms in ENaC variants and the discovery of novel ENaC modulators.

Excessive dietary salt intake is a global health concern, affecting cardiovascular, renal, and bone health. While the renin-angiotensin-aldosterone system (RAAS) is a known regulator of dietary salt-induced hormonal responses, the impact of adrenal cortisol remains unclear. Here, we performed a retrospective analysis in individuals (n = 321) consuming a random diet. Dietary salt intake positively correlated with urinary cortisol and inversely correlated with plasma fibroblast growth factor 23 (FGF23), a bone-derived hormone regulating phosphate and vitamin D homeostasis. Controlled salt diets in healthy individuals confirmed a dose-dependent increase in urinary cortisol and suppression of plasma FGF23. In mice, oral corticosterone, a cortisol analogue, reduced circulating FGF23 levels. RNA-seq analysis of corticosterone-treated MC3T3 osteoblasts identified suppression of FGF23 via glucocorticoid receptor activation, anti-inflammatory pathways, and reduced osteoblast activity. Our findings provide evidence for an endocrine cascade in which high salt intake elevates cortisol signaling and suppresses FGF23.

Heart rate variability (HRV) is widely used as a non-invasive marker of cardiac autonomic regulation in human and experimental animal research. Its accessibility, repeatability, and applicability across clinical, psychophysiological, wearable, and preclinical settings make HRV a valuable translational tool. However, HRV indices are highly sensitive to physiological and methodological context, including recording duration, signal quality, respiratory pattern, posture, behavioral state, circadian timing, medication use, anesthesia, temperature, species, strain, artefact correction, frequency-band selection, and analytical method. Human HRV standards provide a strong methodological foundation, but they cannot be directly transferred to animal models without considering species-specific differences in basal heart rate, respiratory frequency, autonomic regulation, recording conditions, and experimental constraints. In animal studies, additional sources of heterogeneity include telemetry versus surface ECG recordings, conscious versus anesthetized states, handling and restraint, surgical recovery, body temperature control, and inconsistent frequency-domain definitions. This review synthesizes current methodological knowledge on HRV measurement in human and experimental animal studies and highlights major challenges that limit reproducibility and translational interpretation. Attention is given to common methodological pitfalls, and the risk of overinterpreting single indices such as LF/HF as direct measures of sympathovagal balance. We also propose a minimum reporting framework for human and experimental animal HRV studies, with shared translational items intended to support authors, reviewers, and editors. Transparent reporting of acquisition, preprocessing, analysis, and physiological context may improve comparability across studies and strengthen the role of HRV as a bridge between mechanistic animal research and human autonomic assessment.Clinical trial number: not applicable.

The gut microbiota, a complex community of bacteria, fungi, viruses, and other microorganisms, plays a critical role in regulating host physiology through the gut-brain axis. The gut microbiota interacts with the central nervous system through a range of interconnected pathways. These include humoral signaling, immune-related mechanisms, sensory afferent pathways, and the modulation of enteric neural circuits. Recent research has uncovered specialized epithelial sensory cells, like neuropod cells, that can directly detect microbial molecules and send signals via vagal afferents. This discovery highlights a previously unknown aspect of communication between the gut and brain. The review investigates the structural and functional diversity of the ENS, highlighting components like mechanosensory and chemosensory neurons, enteroendocrine cells, and neuropods that interact with vagal afferents. Specific focus is placed on pattern recognition receptors such as TLR5, which allow gut epithelial neuropod cells to sense bacterial flagellin and dynamically regulate appetite via PYY release and vagal signaling. Further examination addresses the impact of microbial metabolites including short-chain fatty acids, bile acids, tryptophan derivatives on neuronal excitability, neurotransmitter synthesis, and neuroimmune interactions. These insights underscore the gut as a sensory organ equipped with neural circuits specialized for detecting microbial cues, reimagining traditional concepts of host-microbe communication. By redefining microbial detection as a distinct sense comparable to vision or taste, the neurobiotic sense offers a transformative perspective on how gut microbes influence behavior, feeding patterns, and brain function. This emerging framework holds the potential to drive novel therapeutic strategies for treating metabolic, inflammatory, and neuropsychiatric disorders.

Acid Sensing Ion Channels (ASICs) are widely expressed in the nervous system and involved in an increasing number of functions such as nociception, mechanoreception, and taste transduction, to name a few. In the CNS, ASICs are involved in synaptic plasticity, learning/memory, and in acidosis-mediated neuronal injury. In retina, ASICs are expressed in rodent, rabbit and monkey retina and pharmacological and gene knockdown studies have indicated changes in the earliest phases of electroretinogram that point to effects on phototransduction. This finding led us to investigate the electrophysiological/pharmacological properties of ASICs in cultured primate rod and cone cells. Patch-clamp recordings showed transient ASIC currents with a threshold pH of ~ 7.0 and pH0.5 of 6.15 ± 0.03 and 6.33 ± 0.03 for cultured rod and cone cells, respectively. The currents were almost completely blocked by amiloride and highly sensitive to PcTx-1. Our results for the first time demonstrate the expression of functional ASICs on primate photoreceptors and suggest that ASIC currents in these cells are mediated predominantly by homomeric ASIC1a channels.

Strenuous exercise induces microstructural muscle damage, leading to delayed onset muscle soreness and reduced performance. Although structural changes are well documented, subsequent inflammatory responses are not fully understood. Research has mainly examined leukocyte infiltration and cytokine expression, while low-abundance bioactive lipid mediators remain understudied. Nutritional strategies, such as palmitoylethanolamide (PEA) supplementation, can modulate these mediators and promote anti-inflammatory, pro-resolving conditions, which may improve recovery. Therefore, the present study evaluated the effect of PEA on the abundance of bioactive lipid mediators in skeletal muscle tissue of healthy males following muscle damaging exercise (MDE). 10 participants were included in a double-blind crossover study where they received PEA (2 × 350 mg/d, Levagen+) or placebo (PLA, maltodextrin), in a randomized order. In each experimental condition participants performed an MDE bout (24 × 10 eccentric contractions of the knee extensors on an isokinetic dynamometer). Muscle biopsies were collected at baseline and 48 h following MDE and analysed for lipid mediator profile via LC-MS/MS-based lipidomics. Many lipid mediators decreased 48 h post-exercise in PLA, whereas they remained unchanged or increased in PEA. This pattern was observed for mediators derived from AA (12-HHTrE, 11-HETE, 12-HETE, 15-HETE, 15-epi-LXA4, 14,15-EpETrE, 5,6-DiHETrE), DHA (4-HDoHE), LA (9-HODE, 13-HODE) and ALA (9-HOTrE, 13-HOTrE). MCTR3 levels were consistently higher in PEA compared to PLA, whereas concentrations of LXB4 and AT-RvD3 were consistently lower in PEA compared to PLA, independent of exercise. In conclusion, PEA supplementation attenuated or counteracted the decline in lipid mediators following exercise, indicating that PEA partially enhances bioactive lipid tone in response to strenuous exercise.

While under physiological conditions, extracellular pH in the brain is reasonably constant, several mechanisms induce transient and localized pH fluctuations. Multiple pathologies involve prolonged changes in pH. Therefore, detecting and monitoring pH changes is crucial for the proper functioning of the nervous system. Acid-Sensing Ion Channels (ASICs) are Na+-permeable ion channels, predominantly expressed in the nervous tissue and activated by protons. Thus, ASICs act as pH sensors, leading to neuronal excitation when the pH drops. ASICs belong to the epithelial sodium channel/degenerin (ENaC/DEG) family of amiloride-sensitive transmembrane ion channel proteins. There are at least eight different ASIC subunits encoded by five genes. Functional ASICs assembled in the plasma membrane are homo- or heteromeric trimers. Trimers containing ASIC1a are of particular interest as, in addition to sodium ions, they also conduct calcium ions and thus can regulate multiple cellular processes. ASICs are involved in numerous physiological functions and pathological states. Biophysical properties of ASICs, their evolutionary origins, regulation and pharmacology, their roles in the peripheral nervous system and in the regulation of synaptic transmission have been recently reviewed in many details. Here we shall focus on brain ASICs in the context of the following issues. (i) The role of ASICs in the homeostasis of extracellular рН in the brain; (ii) interactions of ASICs with major neurotransmitters and (iii) ASICs as emerging key factors and drug targets in brain pathology with focus on stroke, neurological consequences of neonatal hyperbilirubinemia, and Alzheimer's disease.