Physiological Reports

We have previously shown that fresh frozen plasma (FFP) and fibrinogen have protective effects in mice with hemorrhagic shock through restoration of endothelial syndecan-1 and reversal of endothelial injury. In the current study, we tested the hypothesis that a combined model of abdominal sepsis and hemorrhagic shock would induce endothelial syndecan-1 shedding and lung injury which could be attenuated by both FFP and fibrinogen. C57BL/6 mice underwent cecal ligation and puncture (CLP) followed by hemorrhagic shock (HS) and fluid resuscitation with lactated Ringers (LR), fibrinogen (5 mg/mouse), and FFP, all at 1X shed blood volume. After 24 hours, lung tissues and plasma were harvested for assays. CLP+HS induced an increase in alveolar thickness and decreases in lung syndecan-1 and lung neutrophil granule-enzymes (myeloperoxidase, neutrophil elastase, and MMP9), with reciprocal elevations in plasma syndecan-1 and plasma neutrophil granule-enzymes (myeloperoxidase, neutrophil elastase, and MMP9). All these alterations were significantly attenuated by FFP but not by fibrinogen. Additionally, CLP+HS-induced hypotension at 24 hours was partially reversed by FFP but not by fibrinogen. FFP administration inhibits CLP+HS-induced neutrophil degranulation to prevent syndecan-1 shedding and lung injury. The current study supports that FFP has therapeutic benefit in a combined septic and hemorrhage shock model.

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.

Introduction: The key role of maladaptive renal sympathetic activation in hypertension has led to the development of catheter-based renal denervation therapies. Unfortunately, the lack of practical physiological biomarkers impairs patient selection and prevents intraprocedural feedback for renal denervation. Methods: We tested whether renal arterial hemodynamic measures and the beat-to-beat variability of these measures reflect renal sympathetic innervation in two tightly controlled preclinical models of renal denervation. Bilateral renal hemodynamic data were obtained from ten conscious unilaterally denervated rabbits and ten anesthetized unilaterally denervated pigs that subsequently underwent stepwise catheter-based radiofrequency denervation of the contralateral kidney. Renal arterial mechanics were assessed by quantification of wave speed and input impedance modulus and phase shift. Variability was quantified as the within-recording standard deviation of each metric when measured on a beat-to-beat basis. Results: Wave speed trended down after surgical denervation in rabbits (P = 0.054) but not swine (P = 0.57); wave speed variability was unchanged in both models. Renal arterial input impedance modulus was not significantly affected by surgical denervation. Surgical renal denervation increased input impedance phase shift in rabbits (P = 0.0048) but not in swine (P = 0.85). The beat-to-beat variability of modulus did not differ significantly between innervated and surgically denervated kidneys in either species. Surgical renal denervation decreased phase shift variability in swine (P = 0.0048) but not rabbits. The difference in phase shift variability between kidneys in swine was eliminated after a single round of branch-vessel ablation (P = 0.42). Discussion: Renal arterial wave speed, input impedance, and their beat-to-beat variability did not consistently or dose-dependently reflect renal sympathetic innervation in these models. These findings argue against their use as direct physiological surrogates for renal sympathetic outflow.

BackgroundHypertension, the persistent elevation of blood pressure (BP), is characterized by chronic low-grade inflammation and systemic cytokine release. Circulating cytokines contribute to the development of hypertension and end-organ damage. However, the specific immune profile associated with the progression of hypertension remains unclear. We hypothesize that a plasma cytokine signature reflects early BP changes in older adults. MethodsSeventy participants aged 57-81 years were categorized as normotensive (n = 17), elevated BP (n = 10), or hypertensive (n = 43) based on 24-hour ambulatory BP monitoring and antihypertensive treatment status. Plasma IL-1{beta}, IL-6, IL-10, IL-17A, IL-21, IL-22, IL-23, and TNF- were quantified using immunoassays. Partial Pearson correlations adjusted for demographic and biochemical covariates were used to assess associations between cytokines, BP, and cytokine-cytokine networks. ResultsIn untreated hypertensive individuals, plasma IL-23 was positively correlated with 24-hour diastolic BP. Antihypertensive treatment was associated with reduced IL-17A concentrations, which are negatively associated with 24-hour systolic BP. In the elevated BP group, IL-21 concentrations were higher than in normotensive individuals. To further characterize the cytokine signature, cytokine-cytokine correlations were examined. IL-23 and IL-17A were positively correlated with most interleukins, whereas TNF- showed few associations. IL-1{beta} exhibited strong correlations with both IL-23 and IL-17A, particularly in untreated participants. ConclusionIL-23 and IL-17A are associated with BP status and are broadly interconnected with other inflammatory cytokines, highlighting the potential importance of the IL-23/IL-17A axis in the hypertension of development. Early alterations in IL-21 in elevated BP may reflect immune changes that precede the onset of hypertension.

An administration of 3 hydroxy-butyrate (3-OHB) in experimental animals or humans is associated with an increase of cardiac output (CO), but whether this hemodynamic benefit reflects direct myocardial inotropy or heart unloading due to peripheral vasodilation remains unclear. It is also unknown if myocardial effects are due to engagement of receptor-mediated inotropic response, or due to improvement of myocardial bioenergetics. To address these questions, we studied integrated cardiovascular response (pressure-volume analysis of the left ventricle) and isolated left ventricular (LV) trabeculae from normotensive male HanSD rats (age 20-30 weeks) after administration of 3-OHB (1 mmol/l) and drugs targeting adrenergic and cAMP-dependent signaling - metoprolol, adenylyl cyclase inhibitor 2,5-dideoxyadenosine and isoprenaline, during Tyrode solution perfusion and 1 Hz pacing. Pressure-volume analysis demonstrated increased left ventricular end-systolic elastance (+34%), improved ventriculo-arterial coupling (+65%) and increased LV efficiency (+28%), indicating direct inotropic effect of 3OHB. In isolated LV trabeculae, 3-OHB increased twitch force in both groups, but increase was greater in healthy hearts (+55.8%) than in the HF group (+35.6%) (p = 0.014). The inotropic effect of 3-OHB was not further augmented by coadministration of isoprenaline and was not attenuated by beta-1 adrenergic blockade or cAMP inhibition. Results suggest that 3-OHB directly improves LV inotropy and by a mechanism that is independent of receptor-mediated beta-adrenergic signaling, most consistent with a direct bioenergetic pathway. These findings support 3-OHB as a candidate inotropic agent with a mechanistic profile distinct from conventional cAMP-dependent inotropes.

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.

Globally, hypercholesterolemia affects over 20% of the population; and while many studies have examined its impact on cardiovascular health, little is known about its effects on the lymphatic system. In mice, hypercholesterolemia has been linked to multiple aspects of lymphatic dysfunction; and a recent study demonstrated that cholesterol depletion by cyclodextrins promoted lymphatic vessel regeneration and restored lymphatic drainage in mouse models of lymphedema. Collecting lymphatic vessels rely on the spontaneous and highly entrained contractions of lymphatic muscle cells (LMCs) and competent unidirectional on-way valves to propel lymph forward. Critical to lymphatic pacemaking and contractility is the proper functioning of ion channels, which are known to be modulated by the cholesterol content in the plasma membrane. Therefore, we sought to understand the role cholesterol plays in regulating lymphatic contractility. The effects of cholesterol depletion by the cyclodextrins M{beta}CD and HP{beta}CD were assessed in cannulated and pressurized inguinal-axillary collecting lymphatic vessels (CLVs) from C57BL6/J (WT) mice. Noteworthy, studies have shown that HP{beta}CD is safe for human use, and in fact, it is commonly used as a drug excipient. Acute treatment with both cyclodextrins significantly increased the pumping capacity of CLVs, as demonstrated by the increased contraction amplitudes by [~]50{+/-}12% and calculated fluid volume displacement by each contraction by [~]35{+/-}11%. Calcium imaging demonstrated that HP{beta}CD increased the amplitude and duration of the large Cav1.2-mediated calcium events (termed calcium flashes. In contrast, cholesterol supplementation by incubation with BODIPY-cholesterol, which presumably incorporates cholesterol into the cell membrane, significantly impaired the contractile activity of CLVs compared to controls by decreasing contraction amplitude (control: 42{+/-}2 {micro}m versus BODIPY-cholesterol: 20{+/-}7{micro}m) and calculated fluid volume displacement (control: 9.2{+/-}3.9nL versus BODIPY cholesterol: 3.3{+/-}1.2nL) which were significantly restored with subsequent cholesterol depletion using HP{beta}CD (amplitude: 36{+/-}11{micro}m, volume displacement: 5.5{+/-}2.4nL). Similarly, treatment with HP{beta}CD significantly improved the contractile capacity of dysfunctional CLVs isolated from hypercholesterolemic ApoEKO mice. In conclusion, changes to cell membrane cholesterol content acutely and significantly altered CLV contractility with depletion improving contractility associated with recruitment of voltage-gated Cav1.2 channels in lymphatic muscle cells (LMCs). Future studies from our lab will determine whether pharmacological depletion of membrane cholesterol can be therapeutic strategy to improve and/or restore lymphatic contractile function in secondary lymphedema, including obesity/hypercholesterolemia-induced and cancer-related lymphedemas.

Left ventricular hypertrophy, common in cardiometabolic and renal disease, is a major risk factor for cardiovascular morbidity and mortality. Left ventricular mass is a highly heritable, polygenic trait. Linkage studies in WKY and SHRSP rats have identified a quantitative trait locus for left ventricular mass index on chromosome 14. Congenic strains, where trait-associated genetic loci are introduced into a control strain, can identify causal genetic mediators relevant to human disease. Chromosome 14 congenic (WKY.SPGla14a), WKY, and SHRSP strains underwent cardiac phenotyping and transcriptome profiling at; 1-3 days (neonate), 5 weeks, and 16-weeks. Compared to WKY, LVMI was significantly increased in SHRSP and WKY.SPGla14a at 5 weeks (LVMISHRSP-WKY=0.26g/kg, LVMIWKY.SPGla14a-WKY=0.30g/kg), prior to measured hypertension in this model. SHRSP blood pressure was significantly greater than WKY.SPGla14a, and WKY from 12-20 weeks (AUCdiff=497 vs WKY, AUCdiff=412 vs WKY.SPGla14a). Cardiac transcriptome analysis of neonate, 5-week, and 16-week hearts identified significantly increased expression of secreted phosphoprotein 1 (Spp1/osteopontin) in SHRSP and WKY.SPGla14a compared to WKY, which is positioned within the transferred congenic region. Overexpression of Spp1 mRNA significantly increased H9c2 cell size and was shown to be transferred in small extracellular vesicles (sEV). Overexpression of Spp1 in neonatal chromosome 14 congenic and SHRSP strains preceded development of increased cardiac mass and onset of hypertension. The congenic strategy identified Spp1 as a positional and functional candidate gene determining increased LVMI in the SHRSP model of human cardiovascular disease.

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.

Background Limited heart rate rise contributes to reduced exercise tolerance for people who have heart failure with reduced ejection fraction (HFrEF), yet rate-adaptive pacing does not improve functional capacity due to an attenuated force-frequency relationship (FFR). How the FFR relates to total peripheral resistance and sympathetic tone in HFrEF is unknown. Methods In a prospective, observational study, participants with HFrEF and controls underwent an incremental pacing protocol, during which heart rate was increased from 50 to 140 beats per minute. At each heart rate increment LV contractility was measured by echocardiography to determine the FFR, as well as continuous beat-to-beat measurement of systolic and diastolic blood pressures with a plethysmography device to determine cardiac output, total peripheral resistance and blood pressure variability (BPV). A microneurography study was then conducted to measure muscle sympathetic nerve activity (MSNA) during incremental pacing. Results A total of 157 participants with HFrEF and 55 controls (mean age 71.1{+/-}1.4 years, 172 (81.1%) male) underwent the pacing protocol. We observed single units in seven of 11 participants who participated in the microneurography study. In both groups, LV contractility and cardiac output increased until the peak of the FFR, after which these declined. We observed a reduction in total peripheral resistance, blood pressure variability, MSNA frequency and incidence coinciding with the peak of the FFR, beyond which these increased. Whilst these relationships were present in both groups, they were more evident in participants with HFrEF. Conclusions For people with HFrEF there is a bidirectional relationship between heart rate and sympathetic activation, with a nadir of sympathetic tone occurring at the peak of the FFR. Both excessively low and high heart rates are accompanied by greater sympathetic activation. Taken together, these data suggest that optimal heart rate targets for HFrEF are likely to be individual.

BackgroundHydrogen sulfide (H2S) is an important endothelial-derived vasodilator, but the signaling mechanism remains incompletely understood. We previously demonstrated that H2S-mediated vasodilation requires transient receptor potential vanilloid type 4 (TRPV4) channels. Because H2S has been reported to enhance heme oxygenase (HO) activity and HO-derived carbon monoxide (CO) regulates endothelial signaling, we hypothesized that H2S-mediated vasodilation requires HO-2-derived CO. MethodsPressure myography was performed in isolated rat mesenteric arteries to determine the contribution of HO, TRPV4, eBK, and SK/IK channels to H2S-mediated vasodilation. HO-2 sulfhydration was assessed using a maleimide assay, and spatial association among HO-2 and TRPV4 was examined using proximity ligation assays in human aortic endothelial cells. ResultsH2S Selicited concentration-dependent vasodilation that was abolished by HO inhibition. Repletion of CO restored H2S-mediated vasodilation in the presence of HO inhibition. CO-mediated vasodilation was abolished by TRPV4 and SK/IK inhibition but was unaffected by eBK inhibition. H2S increased HO-2 sulfhydration and enhanced HO activity. In endothelial cells, HO-2 and TRPV4 exhibited close spatial association. ConclusionsThese findings support a model in which H2S stimulates HO-2-derived CO production, leading to TRPV4-dependent endothelial signaling, SK/IK activation, and vasodilation. Together, the data support the existence of an endothelial HO-2/TRPV4/SK/IK signaling domain that contributes to H2S-mediated vascular reactivity.

Background: Heart failure (HF) is classified by ejection fraction: reduced EF (<40%) is HFrEF and preserved EF (>50%) is HFpEF. Unlike HFrEF, no therapeutic agent improves mortality in HFpEF. The molecular mechanism that produces HFpEF is not completely understood, but the cascade of pathology that produces HFpEF is thought to begin with changes in vascular reactivity, including a decrease in NO mediated vasodilatation, which coupled with subsequent changes in contractility, energetics and coronary blood flow produce HFpEF. If abnormal vascular reactivity is the initial step in the pathological cascade that produces HFpEF, restoring and/or improving vascular reactivity could represent a novel treatment strategy. Vascular reactivity is primarily regulated by myosin light chain phosphatase, which has catalytic, myosin targeting (MYPT1) and 20kDa subunits. Alternative mRNA splicing of exon24 (E24) of the MYPT1 transcript produces MYPT1 isoforms that differ by the presence or absence of a COOH-terminal leucine zipper (LZ+/LZ-); E24 exclusion produces an NO responsive LZ+ MYPT1, while E24 inclusion produces an NO unresponsive LZ- MYPT. Methods: We used the mouse two-hit model of HFpEF (high fat diet and L-NAME) and treated mice with an antisense octo-guanidine targeting the 5' splice site of E24 (ASO-E24) to increase the expression of the NO responsive, LZ+ MYPT1 isoform in vascular smooth muscle. Invasive and noninvasive hemodynamics were used to determine LV function. Results: Compared to mice with HFpEF, ASO-E24 treatment maintains LZ+ MYPT1 expression (4.7{+/-}0.7au v 1.0{+/-}0.4au v 2.0{+/-}0.4au, control v HFpEF v ASO-E24 Rx, p<0.05), improves diastolic function; LVEDP (10{+/-}1mmHg v 20{+/-}4mmHg v 14{+/-}3mmHg, p<0.05), dP/dtmin (-8000{+/-}300mmHg/s v 6000{+/-}500mmHg/s v 8500{+/-}700mmHg/s, p<0.05), both early (E; 0.60{+/-}0.05m/s v 0.42{+/-}0.06m/s v 0.64{+/-}0.06m/s, p<0.05) and late diastolic filling (A; 0.38{+/-}0.03m/s v 0.24{+/-}0.02m/s v 0.47{+/-}0.04m/s, p<0.050 and also prevents the increase in lung weight (167{+/-}5g v 175{+/-}7g v 166{+/-}5g, p<0.05). Further, mice treated with ASO-E24 maintained normal relaxation to 8Br-cGMP (65{+/-}5% v 44{+/-}9% v 72{+/-}9%, p=0.05). Conclusion: These data demonstrate that maintaining normal LZ+ MYPT1 expression and vascular reactivity prevent the development of HFpEF. These results are consistent with the hypothesis that abnormal vascular reactivity is the initial and primary step in the pathological cascade that produces HFpEF and ASO-E24, which is designed to preserve normal LZ+ MYPT1 expression and vascular reactivity, could represent a novel and effective treatment strategy for HFpEF.

BackgroundInsular damage leads to marked cardiovascular alterations and the mechanisms need to be understood. Mouse models provide unique opportunities to gain insights into pathophysiological mechanisms. Here, we evaluated the effects of rilmenidine, a centrally acting antihypertensive drug, on the cardiac functional parameters and cardiac inflammatory cell infiltration in a newly developed mice model of insular hemorrhagic stroke. MethodsC57BL/6J mice were instrumented for injection of blood or vehicle into the insular cortex (IC). Immediately after IC stroke induction, separate groups received intraperitoneal treatment with vehicle (0.9% NaCl, 0.1 mL/100 g) or rilmenidine (10 g/kg) for three days. Electrocardiogram recording,cardiac catecholamine levels and myocardial accumulation of immune cells were evaluated. ResultsMice subjected to hemorrhagic stroke exhibited higher baseline heart rate (HR) (control: 296 {+/-} 33 bpm vs. stroke: 349 {+/-} 38 bpm; P < 0.01) and prolonged QTc interval (control: 89 {+/-} 11 ms vs. stroke: 100 {+/-} 7 ms; P < 0.01). Stroke also increased cardiac norepinephrine levels (control: 9 {+/-} 4 ng/mg vs. stroke: 25 {+/-} 14 ng/mg; P < 0.05), as well as the number of myocardial CD68+ macrophages (control: 7 {+/-} 4 vs. stroke: 16 {+/-} 6 cells/field; P < 0.0001) and Ly6G+ neutrophils (control: 0.5 {+/-} 0.7 vs. stroke: 1.5 {+/-} 1 cells/field; P < 0.001). Rilmenidine treatment markedly prevented all major stroke- induced myocardial functional and inflammatory changes ConclusionsInsular hemorrhagic stroke in mice induces centrally mediated cardiac noradrenergic hyperactivation accompanied by myocardial accumulation of immune cells. These findings support the relevance of this murine model for investigating mechanisms associated with insular stroke.

Coronary flow waveforms have a distinct diastolic-dominant shape with periods of low or retrograde flow during systole. While the general waveform shape has been attributed to complex interactions between cardiac and vascular mechanics, there is limited research into the variability in coronary flow waveforms and what this variability may reveal about cardiac function. This work presents a shape analysis of left anterior descending artery (LAD) flow waveforms using Fourier transforms and Singular Value Decomposition (SVD) performed on baseline data collected from 32 pigs. Pigs included in the study reflect two breeds (Ossabaw and Yorkshire) and three different experimental conditions (lean-control, lean-paced, and obese-paced). Fourier transforms were used to decompose the waveforms into 15 harmonics for each pig. An SVD analysis is then used to extract temporal patterns of the waveforms. Correlations between pig-specific coefficients for the SVD modes and clinical metrics were used to investigate physiological explanations of LAD waveform variability. Temporal LAD flow patterns of the second SVD mode are significantly correlated with heart rate. The third SVD mode significantly correlates with mean blood pressure and maximum hyperemic flow. Furthermore, the fourth SVD mode is weakly correlated with left-ventricular end diastolic pressure and endocardial-epicardial flow ratios. This work demonstrates that LAD flow waveforms can be broken down into temporal patterns that correlate with physiological features. Furthermore, this shape-analysis method allows for waveform reconstruction and simplifies visualization of the temporal patterns identified using SVD, an advantage over existing methods that focus on characterizing flow waveforms by points of interest.

Background: Venous thromboembolism (VTE), which encompasses deep vein thrombosis (DVT) and pulmonary embolism (PE), is a frequent disease associated with thrombus formation and vein wall remodeling. Hence, fibrosis might result from endothelial-to-mesenchymal transition (EndMT), characterized by the loss of endothelial markers and the acquisition of mesenchymal markers. In chronic thromboembolic pulmonary hypertension, transforming growth factor (TGF{beta}), the most potent inducer of EndMT, impairs thrombus resolution. However, the molecular mechanisms implicated in TGF[beta] signaling in the context of VTE are unknown. We hypothesized that epigenetic processes regulate the TGF{beta} signaling pathway in endothelial cells promoting EndMT and vascular fibrosis. Aims: To determine if the histone deacetylase 6 (HDAC6) regulates the TGF{beta} signaling pathway in endothelial cells promoting EndMT and delays venous thrombosis. Methods: To study the role of HDAC6 in EndMT, endothelial cells were treated with a pharmacological inhibitor (TCS20b) and incubated with TGF{beta} and thrombin for 2, 3, and 5 days. Real time PCR and Western blot were performed to analyze endothelial and mesenchymal marker expression and TGF{beta} signaling. An experimental model of VTE was used to study the role of HDAC6 on thrombus size overtime. Animals were treated or not with a specific HDAC6 inhibitor (tubastatin A) for 7 to 21 days. Analysis of RNAseq data sets publicly available were used to confirm our main results. Within group and treatment differences were analyzed using two-way ANOVA and Tukeys multiple comparisons. Results: Expression of the mesenchymal markers, calponin and transgelin, was increased by TGF{beta} and thrombin. Interestingly these changes were inhibited in presence of TCS20b. TGF{beta} mediated these effects through ERK1/2 and HDAC6 activation. Inhibition of HDAC6 in vivo reduced thrombus size 7 days after surgery compared to controls. This was associated with reduced expression of the EndMT marker transgelin in endothelial cells compared to the control animals. We found that FN1-EDA expression was associated with EndMT and regulated by HDAC6 in vitro. This marker was also associated with thrombosis in the RNAseq data set that we analyzed and potentially in patients with recurrent DVT. Conclusion: We found that HDAC6 regulates EndMT in venous thrombosis and impairs thrombus resolution. HDAC6 also regulates expression FN1-EDA that appears to be a strong marker associated with DVT and DVT recurrence. Thus, HDAC6 might represent an attractive therapeutic target for patients with a high risk of recurrent VTE.

Modulation of renal nerves to improve blood pressure (BP) control has become a topic of intense investigation over the last 10-15 years. Given that renal innervation is composed of mixed nerve fibers containing both afferent (sensory) and efferent (sympathetic) fibers, subsequent preclinical studies have been investigating their respective roles in hypertension pathobiology in different genetic and salt-sensitive rat models. Here we set out to investigate how renal afferent and efferent nerves regulate hypertension development in the chronic mild stress model (CMS). We show that in male CMS rats, ablation of afferent renal nerves (ARDNx) and all renal nerves (TRDNx) resulted in similar BP (104 {+/-} 2 mmHg vs. 101 {+/-} 3 mmHg, respectively), both reduced compared to the SHAM group (118 {+/-} 1 mmHg, p = 0.003 and p < 0.001, respectively) arguing for a prominent role of afferent renal nerves in CMS hypertension. Additionally, we show a reduction of vasopressin (AVP) V1b but not V1a receptor abundance in ARDNx CMS males but not females, suggesting that afferent renal nerves are involved in increase in BP via V1b AVP receptor. We additionally show that despite normal BP, female CMS rats display increased renal sympathetic nerve activity (RSNA; 2.39 {+/-} 0.23 bursts/beat vs. 1.44 {+/-} 0.12 bursts/beat, p < 0.005) measured directly with implanted telemetry in conscious rats over one week and aortic stiffness, as evidenced by increased aortic pulse wave velocity (173.2 {+/-} 50.9 mm/s vs. - 10.7 {+/-} 54.6 mm/s in controls, p = 0.0393). NEW & NOTEWORTHYWe show that renal denervation mitigates the rise in blood pressure (BP) in a model that is not genetic nor diet-dependent, the chronic mild stress model (CMS). Specifically, we demonstrate the role of afferent, rather than efferent, renal nerves in mediating the rise in BP in male CMS rats. Finally, we report that renal sympathetic nerve activity, but not BP, is elevated in female CMS rats measured by telemetry over seven days in conscious rats.

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.

BACKGROUD: Sepsis is a life-threatening condition characterized by a dysregulated host response to infection. It frequently precipitates sepsis-associated acute kidney injury (SA-AKI). 20-Hydroxyeicosatetraenoic acid (20-HETE), is an important regulator of vascular tone. We aim to investigate whether disruption of 20-HETE signaling contributes to septic renal dysfunction. METHODS: In the animal study, Sprague-Dawley rats underwent the cecal ligation and puncture to induce sepsis and SA-AKI. Comprehensive cardiac and renal ultrasonographic assessments were performed in all animals. Blood samples were collected for biochemical assessment of renal function and quantification of circulating 20-HETE levels. Kidney tissues were harvested for analysis of the 20-HETE-GPR75-PLC/PKC signaling pathway and histological evaluation by hematoxylin and eosin staining. In the clinical study, circulating 20-HETE levels and renal hemodynamic parameters derived from point-of-care ultrasound were evaluated in healthy individuals and patients with sepsis. Venous blood samples were collected for quantification of serum 20-HETE concentrations. RESULTS: Firstly, CLP-induced septic rats produced a biphasic hemodynamic response, with early compensation followed by late cardiovascular decompensation, accompanied by progressive renal dysfunction, tubular injury, depletion of circulating and renal 20-HETE, and reduced renal GPR75 expression. Secondly, Restoration of 20-HETE signaling with a pharmacological analog improved systemic and renal hemodynamics, increased MAP, reduced serum creatinine, blood urea nitrogen, and ameliorated renal pathological injury. These protective effects were partially attenuated by PKC inhibition, implicating PKC-dependent signaling in 20-HETE-mediated renal protection. Moreover, in septic patients, circulating 20-HETE concentrations were reduced compared with healthy individuals and were lowest among those with SA-AKI, paralleling alterations in renal hemodynamic parameters measured by point-of-care ultrasound. CONCLUSIONS: Sepsis-associated depletion of 20-HETE accompanies impaired systemic and renal hemodynamic regulation and is associated with the development of SA-AKI. Restoration of 20-HETE signaling improves hemodynamic function and attenuates renal injury, potentially involving GPR75-associated PLC/PKC signaling. KEY WORDS: sepsis-associated acute kidney injury, 20-hydroxyeicosatetraenoic acid, GPR75, renal hemodynamics, point-of-care ultrasonography

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.