Physiological Reports

Background Pediatric nephrotic syndrome (NS) significantly increases cardiovascular risk, primarily through activation of the renin-angiotensin-aldosterone system (RAAS) and chronic inflammation. This study aimed to evaluate the relationship between serum aldosterone and interleukin 6 (IL-6) levels with carotid intima-media thickness (CIMT) in children with steroid-sensitive (SSNS) and steroid-resistant NS (SRNS), both before and after combined treatment with angiotensin-converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB). Materials and methods A prospective cohort study enrolled 32 children aged 2-18 years with idiopathic NS at Dr. Zainoel Abidin Hospital, Banda Aceh, Indonesia (May-June 2024). Participants received lisinopril (ACEI) or valsartan (ARB) for 12 weeks. Serum aldosterone was quantified via liquid chromatography-tandem mass spectrometry, IL-6 via electrochemiluminescence immunoassay, and CIMT via high-resolution B-mode Doppler ultrasound. Data were analyzed using SPSS version 26.0, including Shapiro Wilk normality testing, Pearson/Spearman correlations, and multivariate linear regression (enter method, R2=0.022), with statistical significance set at p<0.05. Results CIMT decreased significantly post-treatment (0.13{+/-}0.04 cm to 0.06{+/-}0.01 cm; p<0.001). Serum aldosterone remained stable (5.15 to 4.0 ng/dL; p=0.933), as did IL-6 (3.5 pg/mL; p=0.156). Univariate analyses revealed weak correlations (aldosterone-CIMT: R2=0.075; IL-6-CIMT: R2=0.215). Multivariate regression (R2=0.022) identified SRNS as positively associated with CIMT change (p=0.047), while aldosterone and IL-6 showed no significant independent effects. Conclusion This study highlights that ACEI and ARB therapy improved CIMT regardless of steroid sensitivity, suggesting vascular benefits may occur through pathways independent of aldosterone and IL-6 modulation.

Introduction: The inability to measure renal sympathetic outflow clinically poses a significant problem for patient identification and intraprocedural feedback for renal denervation. Sympathetic vasomotion is a novel physiologic measure of rhythmic vascular control that reflects renal sympathetic nervous system activity, but previous measurements of sympathetic vasomotion relied on 15-minute data recordings, which may be impractical in many clinical settings. We therefore tested whether shorter data segments can provide reliable sympathetic vasomotion assessment to enhance decision making for renal denervation. Methods: Ten swine that had previously undergone unilateral surgical renal denervation underwent four rounds of catheter-based denervation of the contralateral kidney. Sympathetic vasomotion was quantified from the time-varying arterial pressure-resistive renal blood flow transfer function of recordings obtained before and after each round of ablation. Randomly placed 1- to 14-minute data segments were scored and compared with the corresponding 15-minute sympathetic vasomotion scores across 100 passes using correlation, intraclass correlation, error, and Bland-Altman agreement metrics, and by whether they reproduced the denervation decisions made from the full recordings. Results: Agreement with the 15-minute reference score improved steeply with recording length up to approximately four minutes and slowly thereafter, with the point of diminishing returns at four minutes for every group-level measure. Four-minute data segments showed an intraclass correlation coefficient of 0.94, reproduced the group-level statistical results across ablations, and preserved intraprocedural denervation decisions. Bland-Altman analysis revealed that individual-level precision continued to improve with recording length; the proportion of scores falling within a quarter of the average difference between innervated and denervated kidneys at baseline was 65 percent at four minutes and 96 percent at ten minutes. Discussion: Four-minute data segments provide a feasible minimum recording duration when the question is a group comparison or a paired, within-individual decision, such as intraprocedural confirmation that an innervated kidney has shifted toward a denervated state. When a single score must be interpreted on its own, as in preprocedural patient selection, ten-minute recordings provide greater precision. Further studies are needed to prospectively evaluate sympathetic vasomotion in clinical settings and determine whether this technology can support procedural feedback and inform patient selection.

Current evidence supports resistance training for stroke recovery but autonomic dysregulation following stroke can lead to exaggerated fluctuations in blood pressure (BP) in response to exercise, thus challenging perceptions about safety and feasibility of resistance exercise. This study compared the post-exercise BP response following a single bout of leg-press exercise between people with and without stroke. Data from 11 individuals with chronic stroke (age 39-76 years, 5 female, 1-9 years post-stroke) and 19 recreationally active controls (age 18-33 years, 11 female) were included. Brachial artery BP was measured before exercise and immediately following a single set of leg-press (8 repetitions, target rating of perceived exertion 5-7/10). The difference between pre- and post-exercise BP was calculated and compared between groups using an independent t-test of equal variance. Additionally, a two-way analysis of covariance was used to examine potential cofounding effects of activity level and baseline BP on between group differences in BP response. No group differences were observed in post-exercise change in systolic (mean difference=3.61 mmHg, 95%CI:-8.36, 15.57, p=0.54); and diastolic (mean difference=5.38 mmHg, 95%CI:-6.11, 16.86, p=0.35) BP. Results were unchanged after controlling for baseline BP, and activity levels. Our work suggests that individuals with and without chronic stroke experience similar acute BP responses immediately following resistance exercise; however, work with more appropriately matched controls and greater sample sizes is needed to support these findings. Future studies may elucidate mechanisms related to the acute cardiovascular response to resistance exercise in individuals with stroke.

Axonal action potential propagation critically depends on tightly regulated intra and extracellular electrolyte concentrations and acid base homeostasis. Experimental studies have demonstrated that changes in extracellular sodium, potassium, and calcium concentrations alter axonal excitability and conduction properties. However, it remains unknown whether small variations in electrolyte concentrations within the physiological range are associated with measurable changes in nerve conduction velocity in humans. In this study, data from neurologically healthy children who underwent both nerve conduction studies and blood gas analysis were analyzed to investigate the relationship between electrolyte concentrations and median motor nerve conduction velocity. Nerve conduction velocity was significantly negatively correlated with ionized calcium concentration and positively correlated with sodium concentration, whereas no significant correlation was observed for potassium. In an a priori defined multiple linear regression model, ionized calcium, sodium, and bicarbonate remained significantly associated with nerve conduction velocity after correction for multiple testing. These findings indicate that even small variations in electrolyte concentrations within the physiological range are associated with measurable differences in peripheral nerve conduction velocity and suggest an additional contribution of acid base status to nerve conduction.

Purpose We examined whether drinking a small volume of cold water during orthostatic stress attenuates the fall in systolic blood pressure (SBP). Methods In a randomized crossover study, 19 healthy young adults underwent 15 min of 60 degree head-up tilt under three conditions on separate days: no water, 200 mL of body-temperature water (37 degrees Celsius), and 200 mL of cold water (4 degrees Celsius). Drinking began 5 min after tilting and lasted 1 min. Hemodynamics and tympanic temperature were analyzed using covariate-adjusted linear mixed models with within- and between-condition comparisons. Results During drinking, SBP increased from pre-drinking values by 9.54 mmHg (95% confidence interval [CI] 5.58 to 13.50) with body-temperature water and 14.70 mmHg (10.74 to 18.66) with cold water. After cold water ingestion, SBP remained higher by 5.95 mmHg (95% CI 1.99 to 9.91) over the first 4 min and 4.84 mmHg (0.88 to 8.80) over the subsequent 5 min. No clear post-drinking increases occurred with body-temperature water. SBP was higher with cold water than with both other conditions during the first 4 min after drinking, and higher than with body-temperature water during the subsequent 5 min. After cold water ingestion, stroke volume was higher and heart rate lower than before drinking, and tympanic temperature declined between the two post-drinking periods. Conclusion Drinking 200 mL of cold water after blood pressure had fallen attenuated the reduction in SBP during continued orthostatic stress in healthy young adults.

Mitochondrial reactive oxygen species (ROS) are implicated in cardiac dysfunction, but complex dynamic interactions between ROS, intracellular calcium, and electrophysiology make it difficult to resolve mechanisms experimentally. We developed the Zukowski excitation-contraction-mitochondrial-ROS (ECM-ROS) model, a computational model that couples human ventricular electrophysiology with mitochondrial calcium handling, energetics, ROS production, and ROS dependent modulation of the ryanodine receptor, SERCA, L-type calcium current, and late sodium current. Model predictions were evaluated against independent experimental measurements of ROS induced changes in action potential duration and intracellular calcium. We then used the model to characterize ROS calcium feedback across physiological pacing rates and applied it to doxorubicin induced oxidative stress. Increasing cytosolic calcium produced nonlinear amplification of mitochondrial activity and cytosolic ROS, with greater amplification at faster pacing rates. Moderate ROS elevation initially enhanced calcium release, whereas greater oxidative stress depleted sarcoplasmic reticulum calcium stores. During simulated doxorubicin exposure, faster pacing lowered the level of oxidative stress required to produce calcium dysregulation and action potential prolongation. Mitochondrial ROS and calcium accumulation preceded detectable electrophysiological remodeling. In populations incorporating variability in ion channel expression, neither doxorubicin induced oxidative stress nor subtle pharmacological IKr block alone produced early afterdepolarizations. However, early afterdepolarizations were predicted to emerge with combined perturbations. These findings establish a computational framework for investigating bidirectional coupling among mitochondrial function, ROS, calcium handling, and human cardiac electrophysiology. The model generates testable predictions regarding the early detection of oxidative cardiac injury and the physiological conditions that increase susceptibility to arrhythmia.

Quantitative measurement of fetal hemoglobin (HbF) using high-performance liquid chromatography (HPLC) is standard but expensive and widely unavailable in low-income settings. The newer Gazelle method, which employs single-use alkaline electrophoresis cartridges, provides a portable and relatively inexpensive potential alternative. In the current study, we tested 3055 venous blood samples collected from 1780 children with sickle cell anemia who were enrolled into the H-PRIME trial by both Gazelle and the Bio-Rad D-10 HPLC instrument, before and during hydroxyurea treatment. Data reinterpretation was required to quantify accurately high HbF values derived using the D-10. Mean (standard deviation) baseline HbF values by HPLC and Gazelle (N=2042) were 11.4% (SD 5.9%) versus 16.3% (SD 7.5%), respectively, while mean on-treatment HbF values (N=1013) were 18.9% (SD 9.0%) versus 25.4% (SD 11.5%), respectively. While overall agreement was good (Lins concordance 0.76, 95% CI 0.75-0.78), HbF values derived using Gazelle were modestly higher than HPLC values (Bland-Altman agreement mean +5.4%, 95%CI -2.4% to +13.2%), with the difference between measures increasing at higher HbF percentages. With strong concordance and more easily interpretable HbF values, Gazelle represents an attractive and inexpensive alternative to HPLC for HbF quantification in the low-income settings where most patients with sickle cell anemia live.

Background: Prone cardiopulmonary resuscitation (CPR) may be necessary when turning a prone patient supine would delay chest compressions. Although prone compressions can generate arterial pressures comparable with or greater than supine CPR, the pathway of pressure transmission is uncertain. We examined synchronized intrathoracic, intra-abdominal, and central arterial pressures in both supine and prone positions. Methods: Two thawed fresh-frozen adult cadavers underwent three, 2-minute mechanical CPR trials per position in a counterbalanced crossover sequence. Solid-state catheters recorded pleural, peritoneal, and central arterial pressures simultaneously. Trial-level outcomes included peak pressure, mean pressure, pressure-time area, and the mean peritoneal-to-pleural pressure gradient. Exploratory fixed-effects models included position, cadaver, and their interaction. Results: Prone CPR increased peak intrathoracic pressure by 7.04 mmHg, peak intra-abdominal pressure by 21.69 mmHg, and peak arterial pressure by 15.40 mmHg. Mean intra-abdominal and arterial pressures increased by 16.22 and 9.90 mmHg, respectively. The mean peritoneal-to-pleural gradient reversed direction from -8.46 mmHg supine to 4.85 mmHg prone. Intrathoracic pressure-time area increased 3.4-fold, from 1.62 to 5.46 mmHg{middle dot}s, and arterial pressure-time area increased 2.2-fold, from 2.96 to 6.42 mmHg{middle dot}s. Conclusions: Compared to supine, prone mechanical CPR generated higher arterial pressures and reversed the pressure relationship across the thoracoabdominal boundary in both cadavers. Higher abdominal pressure coincided with a larger intrathoracic pressure-time area, a pattern compatible with reduced caudal pressure dissipation.

Background: Murine models are widely used to investigate cardiovascular disease (CVD), yet many established models rely on invasive surgery or genetic manipulation to induce hyperlipidemia and therefore may not fully capture the gradual cardiovascular consequences of mutant hematopoiesis. Tie2-Cre/-FF1/- (Tie2FF1) mice express JAK2V617F mutation in both hematopoietic and endothelial cells and develop spontaneous CVD on a standard chow diet. Methods: We applied a comprehensive, non-invasive cardiovascular phenotyping protocol to 1-year-old Tie2FF1 mice and age-matched controls. Assessments included transthoracic echocardiography, coronary flow reserve by Doppler ultrasound, quantitative analysis of coronary arteriolar structure and perivascular fibrosis, endocardial histology and subendocardial collagen deposition, and histologic evaluation of pulmonary and hepatic congestion. Results: Tie2FF1 mice demonstrated left ventricular dilation, increased cardiac mass, and mildly reduced systolic function without obstructive epicardial coronary disease. Coronary microvascular remodeling was evident by arteriolar narrowing and increased perivascular fibrosis, while coronary flow reserve was markedly reduced in an exploratory cohort. Endocardial endothelial disruption and increased subendocardial fibrosis were also observed. In addition, Tie2FF1 mice showed increased lung and liver weights, pulmonary fluid accumulation, alveolar septal thickening, pulmonary vascular fibrosis, hepatic sinusoidal dilatation, and centrilobular hepatocyte swelling, providing objective evidence of clinically relevant heart failure. Significance: These findings establish Tie2FF1 mice as a spontaneous, non-invasive model with features of coronary microvascular and endocardial endothelial dysfunction and heart failure with mildly reduced left ventricular ejection fraction. The integrated approach described here provides a practical framework for comprehensive murine cardiovascular phenotyping beyond traditional atherosclerosis- and surgery-based models.

While absolute blood biomarkers have long guided diagnosis, their rate of change often provides greater diagnostic value. For the Limb Girdle Muscular Dystrophies studied here, increased muscle weakness is associated with abnormal sarcolemma repair. Time course analyses were made to changes in muscle-derived plasma enzymes after modest physical activity of ambulatory inpatients. Blood samples at 20 selected time points at variable intervals were obtained at rest, and before and after routine morning activities and motor function testing. After these activities, plasma enzyme levels increased in a similar way, suggesting damage and leakage from skeletal muscle. The levels then decreased in a similar way, suggesting that muscle cells stopped leaking and enzymes were subsequently cleared from the blood. The complex temporal changes were modeled using both continuous time and data features to reduce the number of parameters required to describe the irregularly sampled, interrupted, time series data. Unlike individuals with cardiac muscle ischemia or following intense exercise, there was no extended post-injury rising phase of enzyme levels. In contradistinction, levels fell once activity ceased. Time series analyses of blood biomarkers most likely provide an assay and index of sarcolemma fragility and by extension skeletal muscle damage.

Background Takotsubo cardiomyopathy (TTC) is a stress-induced cardiomyopathy characterized by acute cardiac dysfunction, inflammation, and hypertrophy, yet no therapy directly targets the underlying myocardial injury. Clinical studies have reported elevated circulating Angiotensin II (Ang II) levels in TTC patients, suggesting dysregulation of the renin-angiotensin system (RAS). Given the established cardioprotective effects of Angiotensin-(1-7) [Ang-(1-7)] in other cardiovascular diseases, we hypothesized that oral administration of Ang-(1-7) could attenuate acute cardiac remodeling during TTC. Methods Female C57BL/6J mice were subjected to a single high-dose isoproterenol (ISO) injection (300 mg/Kg, intraperitoneal) to induce TTC and received daily oral treatment with Ang-(1-7) complexed with hydroxypropyl {beta}-cyclodextrin [Ang-(1-7)-CD] or vehicle (CD), for 4 days, beginning one day before ISO administration. Cardiac proteome alterations were characterized by tandem mass tag (TMT)-based quantitative proteomics. Cardiac hypertrophy was assessed by heart weight normalized to tibial length and cardiomyocyte cross-sectional area. Myocardial inflammation was evaluated by histopathology, CD68 immunostaining, and flow cytometric analysis of infiltrating leukocyte populations. Results We observed a distinct cardiac proteome signature in Ang-(1-7)-treated ISO mice, predicting inhibition of pathways associated with cardiac hypertrophy and adverse remodeling. These molecular changes were accompanied by reduced heart weight normalized to tibial length and smaller cardiomyocyte cross-sectional area compared with vehicle-treated ISO mice. Oral Ang-(1-7) also attenuated myocardial inflammation, as evidenced by lower inflammatory scores, reduced CD68+ macrophage infiltration, decreased numbers of CD45+ leukocytes, and marked suppression of Ly6G+ neutrophil and CCR2+ monocyte recruitment to the heart. These protective effects were associated with improved survival following ISO challenge. MS data are available via ProteomeXchange (PXD082872). Conclusions Oral administration of Ang-(1-7) attenuated acute cardiac hypertrophy and inflammation in experimental TTC while inducing cardiac proteome changes consistent with reduced adverse remodeling. These findings identify activation of the protective RAS axis as a promising therapeutic strategy for limiting myocardial injury during TTC.

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

Certain glomerulopathies (GN) feature impaired protein homeostasis in the endoplasmic reticulum (ER) of podocytes. Protein misfolding causing ER stress activates the unfolded protein response (UPR), a protective signaling network. Mesencephalic astrocyte-derived neurotrophic factor (MANF) and ERdj3/DNAJB11 are ER chaperones that may be secreted from cells during the UPR. In rats with membranous nephropathy (MN) and focal segmental glomerulosclerosis (FSGS), secretion of MANF or ERdj3 from podocytes into urine identified glomerular ER stress/UPR activation. We address the biomarker potential of MANF and ERdj3 in human MN and FSGS. Glomerular gene expression of MANF and ERdj3 was increased in human MN and FSGS. Urinary MANF and ERdj3 were increased in proteinuric MN and FSGS compared to MN and FSGS in remission (low proteinuria), as well as other GNs, polycystic kidney disease, hypertension and healthy controls. In individuals with proteinuric MN or FSGS, amounts of urinary MANF and ERdj3 correlated weakly with proteinuria; correlation of MANF with ERdj3 was moderate. Males represented ~60% of persons in each group. eGFR in proteinuric MN and FSGS was >40 ml/min/1.73 m2. In MN, there were no significant differences in urinary MANF and ERdj3 between anti-PLA2R positive and negative individuals. In conclusion, proteinuric MN and FSGS show increased glomerular expression of MANF and ERdj3. High urinary levels of these ER chaperones correlate only weakly with proteinuria. In these GNs, urinary ER chaperones are thus non-invasive biomarkers of glomerular/podocyte ER stress. Such biomarkers may eventually be useful in identifying patients for therapies targeting ER protein misfolding.

Many hormones and substances acting through G-protein coupled receptors and protein kinase A (PKA) activation inhibit the salt-inducible kinases (SIKs) by phosphorylation. SIKs tonically phosphorylate CREB-regulated transcriptional coactivators (CRTC1, 2 and 3), sequestering them in the cytoplasm and, thus, preventing their translocation into the nucleus. Once in the nucleus, CRTCs bind CREB family member transcription factors and enhance their activity. We and others have shown that parathyroid hormone (PTH) activation of PKA and resultant SIK2/3 inhibition allows CRTC2/3 nuclear translocation. One of the major actions of CRTC2/3 in the osteoblast lineage is the regulation of transcription of Rankl, as well as other PTH-controlled genes. However, little is known about the role of these co-activators in the osteoblast lineage in vivo. Here, we have investigated whether there are basal effects in vivo on bone examined at 2 different ages of conditional deletion of these two co-activators in the osteoblast lineage using Col2.3-Cre. We found significant increases in body weight, length, bone mineral density, bone volume/total volume, trabecular thickness and number with decreased trabecular separation in young (2 months old) male mice, all of which dissipated by 6 months of age. Female mice showed minimal changes in the bone phenotype at either age. Nevertheless, there were gene expression changes in bones of both sexes at both ages, and in particular decreases in Rankl, Runx2 and Sost, and accompanying changes in Wnt pathway genes. These effects may explain the changes in the bone phenotype in the young male mice, but it is notable that there is a sexual dimorphism in the action of CRTC2 and CRTC3. Overall, the work supports the data from research in vitro and forms a basis for investigation of the role of these co-activators in PTH action in vivo.

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

Background: KCNH2 encodes Kv11.1 channel proteins that conduct the rapidly activating delayed-rectifier K+ current (IKr), which is critical for cardiac repolarization. KCNH2 encodes two functional isoforms, Kv11.1a and Kv11.1b, via alternative transcription start sites. Kv11.1a is the principal determinant of cardiac IKr and ventricular repolarization. The circadian clock, a transcriptional-translational feedback loop that cycles with a period of ~24 hours and drives the circadian expression of many genes, including Kcnh2 in the mouse heart. Because daily body temperature rhythms provide a systemic signal that synchronizes cardiac circadian clocks, we tested whether physiological temperature cycles drive the circadian promoter activity of the cloned human KCNH2 (hKCNH2) promoter. Hypothesis: hKCNH2 is a direct transcriptional target of the circadian clock, with temperature driving its promoter activity through BMAL1:CLOCK acting at a conserved tandem E-box. Methods: We cloned the conserved proximal promoter of KCNH2 (-1631 bp upstream of Kv11.1a exon 1) to generate hKCNH2 promoter luciferase reporter constructs. Constructs were transfected into C2C12 myotubes and synchronized by serum shock (static 37{degrees}C) or temperature cycling (36.5-38.5{degrees}C). Bioluminescence was recorded and assessed for period, phase, and amplitude. BMAL1:CLOCK dependence was tested via dominant-negative CLOCK{Delta}19 co-expression. Results: Temperature cycling did not exhibit the rapid damping characteristic of serum-shock-synchronized oscillations, consistent with continuous entrainment by an external zeitgeber rather than a free-running oscillator. Deletion analysis identified a conserved tandem E-box required for oscillation under both serum shock and temperature cycling, and for BMAL1:CLOCK-dependent transactivation (1.75 {+/-} 0.21 vs. 0.86 {+/-} 0.06 RLU, p = 0.0038). CLOCK{Delta}19 reduced hKCNH2 promoter amplitude under temperature cycling without altering period. Conclusion: The circadian clock regulates KCNH2 promoter activity through daily temperature rhythms.

Introduction: Cerebral ischemia induces a reduction in brain pH. GPR4, a proton-sensing G protein-coupled receptor is highly expressed in vascular endothelial cells. Pharmacological inhibition of GPR4 with NE52-QQ57 (NEQQ) reduces ischemia-induced blood-brain barrier (BBB) disruption and brain injury. To facilitate targeting GPR4 in vivo, it is important to better define the effect of NEQQ on post-ischemia signaling and the pharmacokinetics of NEQQ in vivo. Methods: We performed biochemical analysis following transient middle cerebral artery occlusion (tMCAO). We used Liquid Chromatography-Mass spectrometry (LC-MS/MS) to quantify the concentration of NEQQ in serum, brain, and peripheral tissues. Results: NEQQ administration decreased the phosphorylation of PKA substrates, PKC substrates, ERK1/2, and CREB. LC-MS/MS analysis detected NEQQ in both ipsilateral and contralateral hemisphere of the brain at 5 h and 24 h after reperfusion, demonstrating that NEQQ crosses the BBB. In peripheral tissues, NEQQ exhibited highest level in kidney and lowest in the heart. In sham controls, average serum concentrations were ~4 and 0.9 g/g at 5 and 24 h, respectively. Following tMCAO, ipsilateral brain exhibited higher levels as compared to the contralateral side at 5 h while the difference diminished at 24 h. Conclusions: NEQQ attenuated the ischemia-induced activation of multiple signaling pathways. NEQQ exhibited broad distribution in peripheral organs, and its brain level was sufficient for GPR4 inhibition at 24 h after injection. These findings support the use of NEQQ as a pharmacological inhibitor of GPR4-mediated signaling following ischemic stroke.

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

Background: Arrhythmogenic cardiomyopathy (ACM) is a genetic and phenotypically heterogeneous myocardial disorder characterized by progressive cardiomyocyte loss, immune cell infiltration, and fibro-fatty replacement of the ventricular myocardium, with an increased risk of sudden cardiac death (SCD), predominantly caused by mutations in cardiac desmosomal genes including PKP2, DSP, and DSG2. We previously described the pivotal role of NF{kappa}B and CCR2+ cardiac macrophage-triggered inflammation in ACM pathogenesis. Methods: WT or Dsg2mut/mut mice were treated with either isotype control or anti-CD14 antibody once a week for the described treatment period (4 or 8 weeks). At the end of the treatment period, disease characteristics were assessed by echocardiography, electrocardiography, histology, immunofluorescence, positron emission tomography (PET) imaging and single-cell transcriptomics. Results: Here, we demonstrate the efficacy of neutralizing CD14 signaling, an important mediator of inflammatory macrophage activation, in attenuating ACM disease in vivo. Additionally, we elucidate shifts in the cellular and transcriptomic landscapes that occur following anti-CD14 antibody treatment in a well-established mouse model of ACM and identify substantial reductions in inflammatory macrophage and pro-fibrotic fibroblast populations. Conclusion: This study provides evidence that blockade of CD14 can attenuate the ACM disease phenotype. It may do so by inhibiting macrophage-based activation and inflammation, while also altering the transcriptional state of other important cell types. Collectively, these findings suggest that CD14 inhibition may be an effective option for ACM, which should be explored in early-phase clinical studies.

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