Physiological Reports

Myocyte Enhancer Factor 2D (Mef2D) is a member of the Mef2 family. As a transcription factor, Mef2D regulates the expression of genes that impinge on cellular viability, tissue development, and fuel metabolism in a tissue dependent manner. Mef2D is expressed in the beta-cell, and overexpression and knockdown have been shown to modulate glucose stimulated insulin secretion. We sought to understand the role of Mef2D on beta-cell function and survival. To determine the function of Mef2D in the beta-cell, we built overexpression and knockdown INS-1 832/13 cell lines. We determined the effect of Mef2D overexpression or knockdown on mitochondrial respiration, insulin secretion, cell survival, and gene expression. Our data demonstrates that Mef2D knockdown enhances mitochondrial respiration, insulin secretion, and cell survival. Conversely, Mef2D overexpression inhibits mitochondrial respiration, insulin secretion, and cell survival. We demonstrate that some of this effect is due to modulated expression of the mitochondrial gene mtND6. These findings demonstrate that Mef2D overexpression is detrimental to beta-cell function and that Mef2D knockdown is beneficial. These data suggest that Mef2D may be a viable target to enhance functional beta-cell mass as a treatment for Type 1 and Type 2 Diabetes.

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

Right ventricular failure (RVF) is a serious disease with a high mortality but no effective pharmacologic treatments. We reported RVF was reversed by chronic treatment with an 1A-adrenergic receptor (1A-AR) agonist. Recent studies suggest mitochondrial dysfunction contributes to RVF. Therefore, we investigated if reversal of RVF by chronic 1A-AR agonist treatment involved improved mitochondrial function. A mouse model of RVF caused by pulmonary artery constriction (PAC) for 2 wk was chronically treated for a further 2 wk. with a low dose of the 1A-AR agonist A61603 (10 ng/kg/day) or vehicle (no drug control). RV dysfunction was assessed from the fractional shortening of the RV outflow tract (RVOT FS). RVOT FS for sham controls (46.5 {+/-} 1.3 %, n = 9) was reduced 4 wk after PAC (27.6 {+/-} 1.5 %, n = 13, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (34.5 {+/-} 0.6 %, n = 14, P < 0.001). RV myocardial respiration rate (O2 consumption) for sham controls (776 {+/-} 51 pM/s/mg, n = 9) was reduced 4 wk after PAC (493 {+/-} 28 pM/s/mg, n = 15, P <0.0001), but was higher after PAC plus 2 wk A61603 treatment (634 {+/-} 30 pM/s/mg, n = 11, P <0.05). RV myocardial ATP level for sham controls (3.3 {+/-} 0.1 mM, n = 10) was reduced 4 wk after PAC (1.9 {+/-} 0.1 mM, n = 6, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (2.6 {+/-} 0.13 mM, n = 7, P < 0.01). In conclusion, reversal of RVF after chronic A61603 treatment involved reversal of mitochondrial dysfunction. Consistent with our previous studies, this study suggests that the 1A-AR is a therapeutic target to treat RVF. HighlightsRV failure is reported to involve mitochondrial dysfunction which might impair RV contraction by decreasing cardiomyocyte ATP level. Using the pulmonary artery constriction model of RV failure, we found that chronic treatment with an 1A-adrenergic receptor agonist increased RV myocardial respiration rate, increased RV myocardial ATP level, and increased RV function. These findings suggest that the 1A-adrenergic receptor is a therapeutic target for treating RV failure, and that the mechanism involves improved RV cardiomyocyte bioenergetic status.

PurposeDetrended fluctuation analysis alpha-1 (DFAa1) has emerged as a promising non-invasive biomarker for exercise intensity assessment. However, the standard 2-min analysis window lacks temporal resolution necessary for real-time training applications. This study systematically investigated the validity of shortened DFAa1 windows (30s and 1min) versus the 2-min reference across different intensities. MethodsPhysically active males completed three continuous cycling protocols: low-intensity training at the first lactate threshold (LOW, n=19), moderate-intensity training at the second lactate threshold (MOD, n=19), and a 30-min self-paced time trial (TT30, n=18). DFAa1 was calculated using 30-s, 1-min, and 2-min moving windows, advancing in 1s increments. Validity was assessed using intraclass correlation coefficients (ICC), Bland-Altman analysis, and standard error of measurement (SEM). ResultsDuring LOW, both shortened windows showed poor agreement with the 2-min reference (30s: ICC=0.02, mean bias of -0.05; 1min: ICC=0.37, -0.02). During MOD, the 30-s window remained unreliable (ICC=0.32, -0.01), while the 1-min window achieved moderate reliability (ICC=0.63, 0.00). During TT30, both shortened windows substantially improved performance (30s: ICC=0.78, -0.02; 1min: ICC=0.95, -0.01), with the 1-min window achieving excellent reliability. ConclusionDFAa1 analysis window validity is intensity-dependent, with shortened windows showing progressively improved agreement as exercise intensity and heart rate increases. While the 2-min window remains essential for low-intensity monitoring, 1-min or 30-s windows provide appropriate validity during high-intensity exercise, enabling more-responsive real-time feedback. These results support adaptive windowing strategies that dynamically adjust window length based on exercise intensity and the number of included data points, to optimize the analytical validity-temporal responsiveness trade-off.

Obesity is the most common cause of hypertension. We have previously shown that high levels of circulating leptin in diet-induced obese (DIO) mice induced hypertension by increasing expression of Transient Receptor Potential Melastatin-subfamily member 7 (TRPM7) in the carotid bodies (CB). In addition, we demonstrated in rat PC12 cells that leptin increases Trpm7 gene expression by inducing CpG site-specific demethylation within the 5 regulatory region containing a signal transducer and activator of transcription 3 (STAT3) binding site. This leptin-induced Trpm7 upregulation was prevented by inhibition of JAK-STAT3 signaling. Based on these findings, we hypothesized that reversing region-specific methylation at the Trpm7 promoter in the CB could attenuate obesity-associated hypertension. Compared with lean controls, DIO mice exhibited increased Trpm7 expression and the STAT3- binding site-specific promoter demethylation in the CB. Administration of methylated DNA oligonucleotides targeting the STAT3 binding site attenuated CpG site-specific DNA demethylation and reduced Trpm7 transcription in the CB of DIO mice. This intervention resulted in decreased carotid sinus nerve activity and reduced arterial blood pressure, especially during the light phase. Our results suggest that targeted modulation of CpG site-specific DNA methylation at the Trpm7 promoter using DNA oligonucleotide may represent a novel therapeutic strategy for obesity-induced hypertension.

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.

Hypoxemia occurs in intermittent forms, such as obstructive sleep apnea, and in continuous forms, such as at high altitude, and is increasingly recognized as a modulator of cardiometabolic risk. Although hypoxemia alters postprandial glucose and lipid metabolism, its effects on ketone bodies remain unclear. Using a randomized crossover design, we examined whether six hours of normoxemia or intermittent hypoxemia (15 hypoxemic cycles/hour targeting [~]85% peripheral oxyhemoglobin saturation with 100% medical-grade nitrogen) alters plasma {beta}-hydroxybutyrate (BHB) concentrations in 12 young adult females (mean [SD]: 21 [3] years) following a high-fat meal (33% of estimated daily energy requirements; 59% of calories from fat). In a follow-up session, a subset (n = 8) completed six hours of continuous hypoxemia (fraction of inspired oxygen [~]12.0% in a normobaric chamber). Postprandial data were analyzed using baseline-adjusted linear mixed-effects models, with Bonferroni post hoc tests. A time x condition interaction (P = 0.010) indicated that BHB concentrations at 360 minutes were higher during continuous hypoxemia (0.247 mmol/L; 95% CI: 0.218-0.275) than normoxemia (0.176 mmol/L; 95% CI: 0.153-0.200; PBonferroni = 0.029) and intermittent hypoxemia (0.163 mmol/L; 95% CI: 0.139-0.186; PBonferroni = 0.002), representing increases of 13.0% and 14.2% in estimated marginal means, respectively. This response was accompanied by higher postprandial plasma glucose and triglyceride concentrations during continuous hypoxemia than during normoxemia and intermittent hypoxemia (PBonferroni [&le;] 0.002), despite similar plasma insulin and non-esterified fatty acid responses across conditions (P [&ge;] 0.081). These findings indicate that continuous hypoxemia increases late postprandial plasma BHB concentrations in young adult females. New FindingsO_ST_ABSWhat is the central question of this study?C_ST_ABSWhat are the effects of normoxemia, intermittent hypoxemia, and continuous hypoxemia on plasma {beta}-hydroxybutyrate (BHB) concentrations in young adult females after a high-fat meal? What is the main finding and its importance?Compared to normoxemia, young adult females showed higher postprandial plasma BHB concentrations during continuous hypoxemia, but not during intermittent hypoxemia, despite similar changes in plasma concentrations of two main regulators of BHB production (non-esterified fatty acids and insulin) across experimental conditions. These findings suggest that continuous hypoxemia modifies postprandial BHB concentrations through mechanisms not fully explained by circulating non-esterified fatty acids or insulin concentrations alone.

RationaleAutonomic dysfunction is a hallmark of sepsis pathophysiology, yet its quantification remains challenging. Multiscale entropy (MSE) derived from heart rate variability (HRV) offers a dynamic measure of physiological complexity and may serve as a biomarker of early deterioration associated with subsequent organ failure, vasopressor escalation, or mortality. ObjectiveTo determine whether MSE computed across multiple temporal scales during the first 24 hours of Intensive Care Unit (ICU) admission is associated with short-term mortality and longer-term organ dysfunction in patients with sepsis, and whether these relationships vary across vasopressor exposure. Unlike prior studies that focused on short-term HRV metrics, we applied MSE across multiple temporal scales and incorporated these features into machine learning models to evaluate their prognostic utility in septic shock. MethodsThis retrospective cohort study included adult ICU sepsis patients at Emory University Hospital from January 2016 to December 2019. Of 2,076 eligible patients, 958 were propensity matched into two cohorts: fluids-only and fluids-plus-vasopressor, with norepinephrine as the primary vasopressor. High-resolution electrocardiogram (ECG) waveforms were analyzed to compute MSE across 20 temporal scales. Machine learning models using (1) MSE features alone and (2) MSE combined with demographic and vital sign data (MSE-DV) were compared against traditional HRV measures based model and severity of illness scores for predicting outcomes. Model performance was assessed using the area under the receiver operating characteristic curve (AUROC), with a primary outcome of mortality at day 7 and secondary outcome of persistent organ dysfunction at day 28. ResultsIn the fluids-plus-vasopressor cohort, MSE-based models demonstrated superior predictive performance for 7-day mortality (AUROC 0.84) compared to severity of illness scores (AUROC 0.64). MSE-DV models also predicted organ dysfunction including 28-day renal (AUROC 0.75), neurological (AUROC 0.79), and respiratory (AUROC 0.71) dysfunction. Patients receiving second-line and third-line vasopressors and corticosteroids exhibited progressively lower MSE values, particularly at mid-range and long-range scales. ConclusionMSE features in the first 24 hours of ICU stay predict mortality and organ dysfunction with higher discrimination than traditional severity of illness scores. Future work should validate these findings, assess longitudinal MSE trends, and race-specific autonomic patterns to refine predictive models.

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

Exercise affects the immune function and induces pro- and anti-inflammatory effects. The alterations concerning the immune system linked to physical activity have been documented across various studies with complex exercise tests. However, the characterization of the non-pathological, exercise-induced immunological stress regulation is highly relevant in numerous clinical and non-clinical areas for a better understanding of normal physiological adaptations and differentiation from non-healthy adaptations. Thus, it is valuable and necessary to establish simple immune-metabolic response triggering exercise tests for use in clinical and non-clinical settings. The aim of this study was to examine the effects of the 1-minute sit-to-stand test (STST) on immune-metabolic stress indices and to determine whether it elicits a sufficiently high intensity to qualify as an anaerobic exercise test, thereby supporting its application in investigating exercise-induced immunological stress regulation. 28 study participants performed the 1-minute STST. Capillary blood was taken 20 and 10 minutes before the test, immediately after, and 5, 10, 15, 30, and 45 minutes after exercise. Lactate, glucose and blood counts were determined. Lactate concentration increased significantly immediately after the STST (p < 0.001) and remained significantly elevated until 45 minutes post-exercise. Glucose concentration was significantly decreased after 10 minutes post-exercise (p < 0.05) and again 30 and 45 minutes post-exercise (p < 0.01 for both). Leucocyte count increased significantly post-exercise (p < 0.001) and returned to baseline levels 30 minutes afterwards. Lymphocyte and granulocyte count increased significantly after the test (p < 0.001 for both) and lymphocyte count slightly decreased below baseline values 30 minutes post-exercise (p = 0.07) while granulocyte count remained significantly elevated (p < 0.05). Furthermore, decreased NLR (p < 0.001) and SII (p < 0.01) immediately after the test and increased levels of NLR, SII and SIRI post-exercise could be observed. The 1-minute STST caused an increase in lactate level above the anaerobic threshold, therefore the test can be evaluated as an anaerobic exercise test. Furthermore, it was demonstrated that the STST induced shifts in leucocyte, lymphocyte, and granulocyte counts, which means that even a short intense anaerobic exercise, such as the STST causes changes in immune cell counts and therefore, the test is suitable for analyzing the exercise-induced immunological stress response.

BackgroundChronobiology research has historically focused on circadian rhythms; however, longer infradian rhythms are prevalent in human physiology and may have important implications for health and wellbeing. Previous studies have identified widespread infradian rhythms across human physiology, often in the context of hormonal regulation and disease. Despite growing evidence of their ubiquity, the mechanisms, significance, and clinical relevance of these rhythms remain poorly understood, largely due to lack of longitudinal datasets and robust detection methods. The emergence of new wearable technologies enables rich, continuous data capture within individuals, allowing physiological rhythms to be studied at scale. MethodsThis study analyzed a cohort of healthy, young adults (N=623), with up to four years of wearable and questionnaire data collected through the University of Notre Dames (USA) NetHealth project. Participants who recorded at least three months of continuous (>80% adherence) heart rate data were included and significant infradian rhythms were identified using wavelet analysis. Unsupervised non-negative matrix factorization was performed to cluster similar wavelet power spectrum distributions. Individuals heart rate rhythms were compared to known environmental cycles (day-of-week, lunar, seasonal) and considering demographics and social networks. A second, smaller cohort (N=70) with heart rate and menstrual timing were included to analyze the interplay of hormonal regulation on monthly cycles. Multinomial logistic regression, and statistical tests (i.e., one-way ANOVA) were applied to quantify the effects of environmental, behavioral and demographic factors on heart rate rhythms. FindingsSignificant infradian rhythms of heart rate were detected in 69.7% (365/523) of the cohort and 35.9% (188/523) had two or more rhythms. Annual, biannual and 10-week rhythms were the most common. Within the 4-45-day band, individuals clustered into four multiday chronotypes based on dominant periodicities in their wavelet power spectra: weekly ([~]7 days), shorter-monthly ([~]25 days), longer-monthly ([~]35 days), and multi-month (>35 days). Heart rate rhythms were influenced by environmental cycles (day-of-week and seasonality) but were not tightly correlated to external cues. Additionally, heart rate rhythms were synchronized to the menstrual cycle in most menstruating females, although monthly rhythms were also observed in males and menopausal women. InterpretationThe prevalence of infradian, or multiday heart rate rhythms in healthy young people motivates further scientific investigation to understand the mechanisms of these rhythms and their potential association with autonomic function, and risk of disease or disease-specific symptoms. Characterizing physiological rhythms can drive new insights into how multiscale fluctuations modulate disease symptoms across neurological, psychiatric, and broader health conditions.

The population of kisspeptin neurons located in the rostral periventricular area of the third ventricle (RP3V) is thought to have a key role in generating the GnRH surge that triggers ovulation. Using a modified GCaMP fibre photometry procedure, we have been able to record the in vivo population activity of RP3VKISS neurons across the estrous cycle of female mice. A marked increase in GCaMP activity was detected beginning on the afternoon of proestrus that lasted in total for 13{+/-}1 hours. This was comprised of slow baseline oscillations with a period of 91{+/-}4 min and associated with high frequency rapid transients. Very little oscillating baseline or transient activity was detected at other stages of the estrous cycle. Concurrent blood sampling showed that the peak of the LH surge occurred 3.5{+/-}1.1 h after the first baseline RP3VKISS neuron baseline oscillation on the afternoon of proestrus. The time of onset of RP3VKISS neuron oscillations varied between mice and across subsequent proestrous stages in the same mice. To assess the impact of estradiol on RP3VKISS neuron activity, mice were ovariectomized and given an incremental estradiol replacement regimen. Minimal patterned GCaMP activity was found in OVX mice, and this was not changed acutely by any of the estradiol treatments. However, on the afternoon of the expected LH surge, the same oscillating baseline activity with associated transients occurred for 7.1{+/-}0.5 h. These observations reveal an unexpected prolonged oscillatory pattern of RP3VKISS neuron activity that is dependent on estrogen and underlies the preovulatory LH surge as well as potentially other facets of reproductive behavior.

Salt-sensitive hypertension, a condition in which the blood pressure (BP) increases with an increase in salt intake, is influenced by behavioral, genetic, and environmental factors. Salt sensitivity is associated with variants of the G protein-coupled receptor kinase 4{gamma} (GRK4{gamma}) and the renal sodium bicarbonate cotransporter 2 (NBCe2), encoded by the solute carrier family 4 member 5 (SLC4A5). The R>65L variant (rs2960306) of human GRK4 (hGRK4{gamma} 65L) contributes to salt sensitivity through a signaling pathway and gene-gene interaction with SLC4A5. Global expression of GRK4{gamma} 65L in transgenic mice results in salt-sensitive hypertension, due in part to an increase in endogenous GRK4 and angiotensin type 1 receptor (AT1R) expression. Grk4 knockout (Grk4-/-) mice have decreased blood pressure and are salt-resistant. The expression of hGRK4{gamma} 65L only in the kidney of Grk4-/- mice increases BP in response to a high salt diet. The renal expression of SLC4A5 is increased in hGRK4{gamma} 65L transgenic mice, relative to mice expressing wild-type (WT) human GRK4 (hGRK4 65L), without endogenous mGrk4. Human renal proximal tubule cells (hRPTCs) endogenously expressing GRK4 WT and SLC4A5 WT, SLC4A5 variants, GRK4 65L, and both GRK4 65L and SLC4A5 variants were studied. SLC4A5 expression is increased in hRPTCs expressing GRK4 65L and in cells expressing both GRK4 65L and SLC4A5 variants compared with GRK4 WT and SLC4A5 WT. Luminal and basolateral sodium transport in hRPTCs is increased in the presence of both hGRK4 65L and SLC4A5 variants. GRK4 interacts with nuclear histone deacetylases (HDACs). Mice expressing hGRK4 65L only in the kidney have decreased expression but increased phosphorylation of HDAC1. HDAC1 expression is decreased and HDAC1 but not HDAC2 phosphorylation is increased in hRPTCs expressing both hGRK4 65L and SLC4A5 variants. The presence of hGRK4{gamma} 65L decreased HDAC1 expression but increased AT1R expression in the kidneys of mice on high salt diet. Our results show that GRK4{gamma} 65L causes salt-sensitive hypertension by increasing renal SLC4A5 and AT1R expressions by inhibiting the HDAC1 pathway.

Exercise provides broad health benefits, including improved emotional well-being and cognitive function. Emerging evidence suggests that exercising at different times during the day can have differential effects. However, how circadian phase and sex influence behavioral and physiological responses to exercise remains unclear. To address this question, we examined male and female wild-type mice maintained in either regular (REG, lights on/off at 7AM/7PM) or inverted (INV, lights off/on at 10AM/10PM) light cycles. Mice were then subjected to daily 20-min group swimming exercise sessions at ZT2-3 for 3 weeks. Exercised and sedentary controls mice were then subjected to an open field test (OFT) and blood corticosterone (CORT) measurements 24 hours post-exercise. We quantified several behaviors during swimming: escape attempts, floating, climbing and collisions. We also identified a novel swimming behavior: floating with only nostrils-above-water events (NAWEs). We found that expression of these behaviors was differentially modulated by sex, light-cycle and their interaction. Notably, behavioral differences were more pronounced in REG mice (rest phase). REG mice also lost weight after exercise and had elevated CORT levels compared to mice kept in INV conditions (active phase). Interestingly, OFT behaviors showed significant differences primarily in INV mice, particularly females, when comparing exercised vs sedentary groups. Our novel findings reveal that circadian rhythms and sex significantly interact to shape swimming exercise and stereotyped behaviors in mice. This emphasizes the need to consider the animals circadian phase when designing preclinical studies to match intended behavioral and physiological outcomes. HIGHLIGHTSCircadian phase and sex jointly shape swimming behavior patterns. Newly identified swimming behavior is more prevalent during rest-phase Restphase exercise produced stronger behavioral and physiological effects. Rest-phase exercise resulted in weight loss and elevated stress markers. Active-phase exercised females showed the strongest open field behavioral differences.

Insulin-stimulated glucose uptake (ISGU) in adipocytes is central to maintain systemic glucose homeostasis. Understanding how ISGU relates to adipocyte traits, such as cell-size, is critical for elucidating pressing questions related to metabolic dysfunction connected to adipose hypertrophy and hyperplasia. Cell size is considered a central trait reflecting multiple aspects of adipocyte metabolism. However, a robust quantitative approach to estimate ISGU for a specific cell size is currently missing. Here, in an attempt to move towards such a method, we have formulated an approach using a mathematical framework. The framework consists of a linear equation: the product of the known number of cells (calculated using coulter counter-based cell-size distributions) and the unknown ISGU/cell, compared to the absolute ISGU (measured using 14C-glucose tracer assays). To solve this equation, we formulate a minimization problem which is optimized to find the unknown ISGU/cell for the best solution. Using different formulations of the equation we can investigate the need for either cell size-dependent or independent ISGU/cell, to describe varying glucose uptake in a cell sample of various cell sizes. While the framework needs further refinement, we demonstrate that cell size-dependent uptake slightly improved the agreement between model and experimental data for some groups. Together, with further validation this could serve as a useful tool to resolve long-standing questions regarding size-dependent characteristics like adipocyte size and cellular function. Key findingsHerein we explore a method to quantify cell size-dependent glucose uptake in adipocytes

BackgroundIn critically ill patients admitted to the intensive care unit (ICU), rapid skeletal muscle atrophy frequently develops in the acute phase. This ICU-acquired weakness can significantly impair long-term physical function. Although the biceps brachii cross-sectional area (CSA) is commonly used to assess muscle atrophy, its ultrasound imaging can be technically challenging, and the flexor carpi ulnaris may offer a more accessible alternative. Therefore, this study aimed to investigate whether CSA changes of the flexor carpi ulnaris correlate with those of the biceps brachii in critically ill patients admitted to the ICU, as well as whether the flexor carpi ulnaris CSA reflects systemic muscle atrophy in the acute phase of the ICU stay. MethodsTwenty critically ill patients admitted to the ICU underwent serial ultrasound assessment of the biceps brachii and flexor carpi ulnaris CSAs on days 0, 5, 7, and 14 after admission. Longitudinal changes in CSA were analyzed using the Friedman and Wilcoxon signed-rank tests. Correlations between the biceps brachii and flexor carpi ulnaris were examined using Spearmans rank correlation, and structural equation modeling was applied to explore causal relationships between clinical variables and CSA changes. ResultsSignificant CSA reductions were observed in both the flexor carpi ulnaris (-20.6%) and biceps brachii (-16.3%) by day 14, and the relative CSA changes of the biceps brachii and flexor carpi ulnaris showed a moderate positive correlation ({rho} = 0.5489, p = 0.0122). Structural equation modeling analysis revealed that the biceps brachii CSA change had positive effect on that of the flexor carpi ulnaris ({beta} = 0.249, p = 0.0011). Moreover, body mass index was positively associated with the baseline flexor carpi ulnaris CSA ({beta} = 0.042, p = 0.0004). However, the baseline flexor carpi ulnaris CSA was not a significant predictor of subsequent CSA changes. ConclusionUltrasound measurement of the flexor carpi ulnaris CSA offers a practical alternative to that of the biceps brachii for early detection of muscle wasting in ICU patients. Given its anatomical accessibility and high sensitivity to early atrophic changes, it may serve as a feasible screening tool for ICU-acquired weakness and inform timely interventions.

BackgroundRenal sodium reabsorption occurs via both transcellular and paracellular pathways. Tight junction proteins play a key role in mediating paracellular transport. The collecting duct (CD) is critical for the fine-tuning of Na+ balance and is sensitive to changes in dietary salt intake. A low-sodium diet, which increases endogenous aldosterone secretion, stimulates transcellular sodium transport via epithelial Na+ channels (ENaC) and Na,K-ATPase. We hypothesized that a low-sodium diet also modulates paracellular Na+ permeability by regulating the expression or function of claudin-3, a major tight junction protein in the CD, in order to limit the back-leak of reabsorbed sodium and preserve sodium balance. MethodsWe used in vivo mouse models and cultured mouse CD principal cells (mCCDcl1) to assess aldosterones effects on tight junction proteins. In mCCDcl1 cells, aldosterone-induced changes in claudin-3 expression and localization were evaluated via Western blotting and immunofluorescence, and Ussing chamber assays were used to assess paracellular Na+ and Cl- permeability after modulating claudin-3 expression. Wild-type and claudin-3 knockout mice were fed low (0.01%) or normal (0.18%) sodium diets for seven days. In subsets of low sodium diet mice, spironolactone (a mineralocorticoid receptor antagonist) was administered. ResultsIn mice, a low-sodium diet upregulates renal claudin-3 expression. Concordantly, in vitro studies using mCCDcl1 cells showed that aldosterone treatment increased claudin-3 protein levels and promoted its localization to the lateral membrane. Functional analyses demonstrated that claudin-3 overexpression reduced paracellular permeability to both Na+ and Cl-, while claudin-3 silencing increased it. Claudin-3 knockout mice subjected to a low-sodium diet exhibited compensatory upregulation of the - and {gamma}-subunits of ENaC, alongside increased expression of claudin-4, claudin-8, and claudin-10. This highlights an adaptive response that maintains sodium homeostasis in the absence of claudin-3. Importantly, this compensatory mechanism persists even under spironolactone treatment, suggesting that the adaptation of claudin-3-deficient mice occurs independently of mineralocorticoid receptor activation. ConclusionsOur findings demonstrate that aldosterone enhances claudin-3 expression, reinforcing the paracellular barrier to Na+ and complementing its classical role in transcellular Na+ transport. Under low-sodium conditions, claudin-3-deficient mice adapt through complementary mechanisms aimed at increasing sodium reabsorption via ENaC activation and upregulation of claudin-4 and claudin-8, both barrier-forming claudins that restrict paracellular sodium leakage in the CD. This is associated with increased claudin-10 abundance in the thick ascending limb of Henle, a pore-forming claudin that facilitates paracellular sodium permeability. This study advances our understanding of the complex control of renal sodium handling, revealing adaptive mechanisms in response to low-salt diet and claudin-3 deficiency.

BackgroundDeoxycorticosterone acetate (DOCA)-salt induces greater increases in blood pressure (BP) and a more pro-inflammatory T cell profile in males compared to females. T cells contribute to DOCA-salt hypertension, however, the mechanisms driving T cell activation remain unclear. The NLRP3 inflammasome has been implicated in DOCA hypertension in male mice. Little is known regarding NLRP3 in females. The goal of the current study was to test the hypothesis that NLRP3 contributes to greater increases in BP and renal inflammation with DOCA in males vs. females. MethodsRenal NLRP3 protein levels were measured in normotensive and hypertensive male and female subjects and in male and female Sprague Dawley uni-nephrectomized (UNX) control and DOCA-salt rats. Additional 11-wk-old Sprague Dawley rats were UNX and randomized to: 1) DOCA + vehicle or 2) DOCA + the NLRP3 inhibitor MCC950 (10 mg/kg/day in saline) from 11-14 wks of age. At 14-wks-of-age rats were euthanized, terminal plasma samples and remaining kidneys were collected for flow cytometric analysis of T cells. ResultsRenal NLRP3 levels were significantly greater in hypertensive males and females vs. normotensive controls. DOCA increased BP in both sexes, with greater elevations in males. MCC950 attenuated DOCA-induced increases in BP in male, but not female rats. MCC950 decreased circulating and renal CD4 and Th17 cells in both sexes, although the effect was greater in males. ConclusionDespite both males and females exhibiting an increase in NLRP3 in hypertension, NLRP3 contributes to BP elevations only in DOCA-salt males.

Concentrations of estradiol (E2) and progesterone (P4), the main female sex hormones, exhibit large fluctuations across the menstrual cycle. Due to their receptors throughout the central nervous system, both hormones have the potential to influence motor function by influencing ionotropic and metabotropic inputs to motor pools, which can be estimated through the neural codes extracted from motor unit discharge patterns. To address key methodological limitations in prior menstrual cycle research on motor output, we established the Motor Units and Sex Hormones (MUSH) collaboration. The objective of this multi-site investigation was to determine whether endogenous fluctuations in estradiol and progesterone influence human motor unit activity. We hypothesized that motor unit discharge rates and persistent inward current (PIC)-related contributions to discharge would be greatest during the late follicular phase, when estradiol concentrations were highest. Fifty females completed a comprehensive protocol involving menstrual cycle and ovulation tracking, serum hormone measurement, and high-density surface electromyographic recordings during isometric contractions to quantify motor unit activity in the early follicular, late follicular, and mid luteal phases. After exclusion of 10 females with either atypical hormone concentration profiles or insufficient motor unit yield, 40 remained in the final analysis. There were significant changes in several motor unit discharge variables between menstrual cycle phases and significant associations with hormone concentrations. Increased estradiol was associated with higher peak discharge rates and ascending discharge rate nonlinearity, while increased progesterone was associated with higher peak discharge rates, more discharge rate hysteresis and ascending discharge rate nonlinearity. Despite reaching statistical significance, the magnitudes of these effects (i.e., effect sizes) were small. Overall, these findings indicate that fluctuations in sex hormones influence motor unit behavior, but the effects are subtle, highlighting the need for well-powered and methodologically rigorous menstrual cycle research. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/699975v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@2eb2c0org.highwire.dtl.DTLVardef@1d98359org.highwire.dtl.DTLVardef@13e772borg.highwire.dtl.DTLVardef@1bb27_HPS_FORMAT_FIGEXP M_FIG C_FIG KEY POINTSO_LIThere are small but detectable differences in motor unit discharge rates between menstrual cycle phases, which are predicted by within-participant fluctuations in estradiol and progesterone. C_LIO_LIDischarge rate patterns that provide estimates of neuromodulatory and inhibitory input suggest that estradiol and progesterone can influence spinal cord circuitry differently than has previously been documented in the brain, highlighting an understudied aspect of female neurophysiology. C_LIO_LIVariability in menstrual cycles and associate hormones makes large-scale, rigorous studies especially valuable in female neuromuscular research. C_LI

Whole-cell patch-clamp studies often fail to observe the expected effect of melatonin on the IK1 current in cardiomyocytes, which may be due to cytoplasmic dialysis and the loss of key components of the intracellular signaling system. The aim of this study was to develop a simple theoretical model to estimate the expected effect on the IK1 inward-rectifying potassium current in an experiment with intact melatonin signaling. The modeling was performed using a well-established model of rat cardiomyocyte electrophysiology (Pandit et al., 2001). The maximum conductance of IK1 (gK1) channels was chosen as the target for modulation, consistent with the established mechanism of direct receptor-mediated increase in potassium conductance under the action of melatonin.Realistic modulation values were used for the modeling. The -50% value for the antagonist effect of 1 M luzindole was obtained by direct calculation from our experimental data. The +20% value for the agonist effect (melatonin) was determined by generalizing literature data and reflects the typical expected strength of signaling pathway modulation, rather than being strictly tied to a specific concentration.It was shown that modulation of gK1 in the specified ranges leads to significant changes in IK1 amplitude in the physiologically important range of resting potentials. The developed model serves as a "computational benchmark" for validating experimental protocols, allowing one to distinguish methodological artifacts from a true lack of effect.