American Journal of Physiology-Heart and Circulatory Physiology

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.

Age-related stiffening of large arteries is a predictor of cardiovascular morbidity and mortality, yet how pulmonary vascular stiffening integrates with right ventricular (RV) and lung functional decline--and how best to quantify "biological" cardiopulmonary aging--remains unclear. Here we map cardiopulmonary aging across the adult murine lifespan by integrating RV, proximal pulmonary artery (PA), and lung biomechanics with single-cell transcriptomics. Using ex vivo biaxial testing of the proximal PA, in vivo echocardiography, and lung mechanics, we find that cardiopulmonary aging is phase-dependent: PA circumferential stiffening and reduced distensibility progress largely linearly with age; whereas, RV remodeling and lung mechanical changes exhibit non-linear trajectories. This is consistent with early intrinsic functional decline of cells and organs followed by later, extrinsic load-dependent structural adaptation. To quantify organ-level biological aging, we apply principal component analysis to PA, RV, and lung feature sets to derive physiology-based aging scores that summarize coordinated variance within and across organs. Anchoring differential gene expression in PA single-cell RNA-seq to these continuous biological aging scores rather than chronological age reveals extensive, cell-type-specific remodeling programs (13,636 genes) that are sparse or non-informative when modeled by chronologic age. Biological aging associates across endothelia, smooth muscle cells, fibroblasts, and perivascular macrophages with increased oxidative phosphorylation signatures alongside suppression of adaptive/regulatory pathways, including impaired endothelial mechanotransduction, reduced smooth muscle Wnt signaling, altered extracellular matrix remodeling programs, and erosion of macrophage innate immune and TGF{beta}/NF-{kappa}B signaling nodes. These findings support a model in which pulmonary arterial stiffening is not merely a marker but an active contributor to cardiopulmonary aging via a biomechanical-metabolic-inflammatory uncoupling that diminishes vasoactive and mechano-adaptive reserve and promotes a positive feedback loop. Together, our work establishes physiology-derived biological aging as a powerful framework for interpreting vascular single-cell aging trajectories and identifies mechanistic pathways to target pulmonary vascular stiffening and preserve cardiopulmonary function with age.

Aging is a major risk factor for cardiac diseases, including heart failure, myocardial infarction, and arrhythmias. Activation of p38 MAPKs regulates cardiac remodeling and contributes to age-related cardiac dysfunction. However, the isoform-specific roles of p38 kinases in the aging heart remain poorly understood. Although p38{beta} has been reported to exert cardioprotective effects in models of doxorubicin-induced cardiotoxicity and ischemia-reperfusion, its role in cardiac aging remains unclear. Here, we investigated the role of p38{beta} using p38{beta} germline knockout (p38{beta}-/-) mice. Aged p38{beta}-/- mice exhibited increased LV hypertrophy, QT prolongation, calcium mishandling, heightened susceptibility to arrhythmias, increased myocardial fibrosis, and an altered inflammatory microenvironment, compared with age-matched wild-type controls. Transcriptomic profiling revealed that p38{beta} deletion reprograms the cardiac transcriptome in aged mice, suppressing innate immune and proteostasis-related pathways while promoting adaptive immune activation, developmental, extracellular vesicle-mediated, and ion-transport pathways. Collectively, these findings identify p38{beta} as a critical regulator of structural, electrophysiological, and immune homeostasis in the aging heart and demonstrate that its loss promotes maladaptive remodeling and arrhythmogenic vulnerability. NEW AND NOTEWORTHYWe identify p38{beta} as a previously unrecognized regulator of cardiac aging. Systemic loss of p38{beta} disrupts structural, electrophysiological, and immune homeostasis in the aging heart, revealing its protective role in maintaining cardiac function with age. These findings underscore the importance of isoform-specific p38 signaling and suggest that broadly targeting p38 MAPKs may have unintended consequences in age-related cardiovascular diseases.

Background Ischemic heart failure (IHF) has been shown to impair contractility and disrupt sarcomere function in the left ventricle. Left ventricular failure can cause left atrial dysfunction, which is associated with a greater risk of patient mortality. Despite this, the biochemical and biomechanical characteristics of the left atrium in IHF remain obscure. Methods Myocardial mechanical properties were measured using permeabilized muscle isolated from the left ventricle (LV) and left atrium (LA) of donors and patients with IHF. Tissue homogenates from these samples were used to measure titin and myosin isoforms as well as the phosphorylation of sarcomeric regulatory proteins. Histology was used to quantify fibrosis in the patients' left ventricle and left atrium. Results Length-dependent changes in Ca2+-sensitivity were blunted in LV myocardium from patients with IHF. LA myocardium did not show robust length-dependence of Ca2+-dependent force. The calcium sensitivity of both LA and LV myocardium was increased in IHF. The maximum force generated by LV but not LA myocardium was decreased in IHF. LA myocardial samples exhibited faster contractile kinetics than LV samples, irrespective of disease. Troponin I phosphorylation decreased in both chambers with IHF. Conclusions Left atrial IHF myocardium maintained contractile force and displayed increases in calcium sensitivity, which may allow for increased LA contraction under pathological conditions. The increases in calcium sensitivity observed in ischemic myocardium of both chambers are likely driven by decreased phosphorylation of troponin I, which alters thin filament regulation. Conversely, thick filament properties of the left ventricle, such as thick filament protein isoforms and phosphorylation of myosin binding protein-C, displayed chamber-specific differences independent of disease state. These biochemical changes may explain the chamber-specific differences in kinetics and length-dependent properties. Collectively, these biophysical and biochemical data suggest LA remodeling in IHF may assist in increasing LV end-diastolic volume to maintain adequate cardiac output.

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.

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.

Obesity is associated with an elevated risk of pathological cardiac hypertrophy, whereas exercise confers cardioprotective effects; however, the cellular mechanisms underlying these opposing influences remain incompletely defined, particularly in females. We investigated how obesity and exercise affect cardiomyocyte ultrastructure, Ca{superscript 2} release, and contractility in female Zucker Diabetic Fatty rats and their lean littermates. Animals were assigned at 12 weeks to sedentary or aerobic exercise-trained groups and maintained on a standard diet. By 18 weeks, obese rats exhibited increased body mass and myocardial hypertrophy in the absence of diabetes. Sedentary obese animals showed a reduced fraction of compact dyads and diminished stimulated and caffeine-induced Ca{superscript 2} release, while contractility remained preserved. In lean rats, exercise increased dyad density but reduced Ca{superscript 2} release, whereas in obese rats, exercise enhanced both dyad compactness and Ca{superscript 2} release. Across all groups, global cardiomyocyte ultrastructure and contractile function were similar. Type III ANOVA revealed a significant obesity x exercise interaction for dyadic structure and Ca{superscript 2} release. These findings demonstrate that obesity itself, independent of diabetes, triggers early dyadic remodeling and altered Ca{superscript 2} handling in female myocardium before detectable impairment of global cardiomyocyte structure or contractile function. Furthermore, exercise exerts beneficial effects on dyadic ultrastructure and Ca{superscript 2} signaling in obese animals. New & NoteworthyUsing a female rat model of obesity without diabetes, we demonstrate that obesity induces early remodeling of the dyadic system and impairs Ca{superscript 2} release in cardiac myocytes. We further show that the effects of aerobic exercise on dyadic structure and function are obesity-dependent, improving both dyad organization and Ca{superscript 2} signaling. These findings identify the dyadic microdomain as a vulnerable cellular site in obesity and a potential target for exercise-induced recovery.

BackgroundModerate to severe tricuspid regurgitation (TR) affects approximately 1.6 million Americans, yet more than 90% of patients with significant TR remain untreated. Women exhibit higher TR prevalence and more rapid disease progression than men, but the valve-intrinsic mechanisms underlying these sex disparities remain unclear. We hypothesized that sex and circulating testosterone influence tricuspid leaflet remodeling during right-sided pressure overload. MethodsFemale, castrated male (C-Male), and non-castrated male (NC-Male) adult Dorset sheep (n = 45) underwent pulmonary artery banding (PAB) and were followed for 13 {+/-} 1.5 weeks. Tricuspid leaflets were evaluated using morphometry, 3D profilometry, biaxial mechanical testing, histology, and bulk RNA sequencing. Sex-stratified differential gene expression was performed, and pathway enrichment of key biological processes were compared between sexes. ResultsPAB produced a uniform hemodynamic stimulus and equivalent moderate-to-severe TR across sex groups. Despite similar TR burden, leaflet remodeling diverged substantially by sex and castration status. C-Males developed the broadest remodeling phenotype, characterized by diffuse multi-leaflet growth, thickening, increased nuclei count, and low-strain stiffening. Females demonstrated more restricted leaflet and region-specific structural and cellular changes, along with circumferential low-strain stiffening. NC-Males exhibited preferential septal remodeling characterized by growth, thickening, increased nuclei count, and radial high-strain stiffening. Transcriptomic analysis revealed that females upregulated a focused matricellular remodeling program enriched for extracellular space organization (67 DEGs; FDR=0.025), whereas C-Males activated coordinated extracellular matrix and apoptosis-regulatory programs (388 DEGs; FDR=0.009). In contrast, NC-Males exhibited broad transcriptional response (406 DEGs) without significant pathway enrichment. ConclusionsTricuspid leaflet maladaptation during pressure overload is sex-dependent and testosterone-sensitive, involving distinct structural, mechanical, and transcriptional remodeling programs. These findings identify sex and testosterone status as previously under-recognized modulators of tricuspid valve remodeling and may help explain clinical sex disparities in TR progression. NOVELTY AND SIGNIFICANCE What is known?O_LIPulmonary hypertension and right ventricular pressure overload are linked to tricuspid leaflet remodeling through leaflet thickening, enlargement, and altered mechanical properties. C_LIO_LISex and sex-steroid hormones regulate fibrosis and extracellular matrix remodeling in cardiovascular tissues, but their role in tricuspid leaflet remodeling remains poorly understood. C_LI What new information does this article contribute?O_LISex and circulating testosterone status influence the magnitude, spatial distribution, biomechanical behavior, and transcriptional organization of tricuspid leaflet remodeling during pressure overload. C_LIO_LIFemales, castrated males, and non-castrated males develop distinct remodeling programs characterized by focused matricellular remodeling, coordinated extracellular matrix/apoptosis signaling, and diffuse transcriptional activation, respectively. C_LIO_LIThese findings identify sex and hormonal status as biological regulators of tricuspid valve maladaptation during functional tricuspid regurgitation. C_LI SummarySex differences in tricuspid regurgitation progression are recognized clinically, yet the mechanobiological basis underlying these disparities remains poorly understood. Using a controlled ovine model of pressure overload-induced secondary tricuspid regurgitation, we demonstrated that tricuspid leaflet maladaptation is a sex-specific and testosterone-sensitive process spanning structural, mechanical, and transcriptional scales. Under comparable hemodynamic overload, all animals developed significant tricuspid regurgitation, but leaflet remodeling patterns diverged substantially across sexes. Castrated male sheep exhibited the broadest maladaptive phenotype, characterized by diffuse multi-leaflet growth and thickening, increased low-stretch stiffness, and coordinated extracellular matrix and apoptosis-regulatory transcriptional programs. Female sheep developed more spatially restricted remodeling accompanied by a focused matricellular and extracellular matrix secretory response, whereas non-castrated male sheep demonstrated selective leaflet remodeling with broad, but less coordinated, transcriptional activation. Different remodeling patterns emerged in females and castrated males despite comparable testosterone levels, suggesting that testosterone depletion alone does not fully explain these tricuspid valve remodeling phenotypes. These findings establish sex and testosterone status as previously underrecognized biological regulators of tricuspid leaflet maladaptation and support the emerging view that valve leaflets are active, mechanobiologically responsive, participants in functional tricuspid regurgitation progression.

AimsHeart failure (HF) due to diastolic dysfunction (DD) but normal left ventricular (LV) ejection fraction (EF) is termed HF with preserved EF (HFpEF). Given its high prevalence in post-menopausal women, we hypothesized that 17{beta}-estradiol (E2) function is mechanistically linked to DD HF and investigated E2-deficiency in the etiology of DD in a mouse model. MethodsFemale C57BL/6J mice were divided into one group with hypertension induced by N({omega})-nitro-L-arginine methyl ester (L) and cardiac pressure overload induced by angiotensin II (A), collectively inducing DD, and one group with sham treatment. Groups were subdivided to receive an ovariectomy (OVX) to induce estrogen-deficiency and emulate menopause or sham surgery. During the next 21 days, mice were tested for cardiac function, food intake, response to E2 agonists, gene profiling, cardiomyocyte-contractility and elasticity, and cardiac mitochondrial function. ResultsOVX-associated E2-deficiency exacerbated DD in a time-dependent way without affecting EF or stroke volume, emulating a severe DD phenotype. These changes paralleled those for mitochondrial dysfunction, i.e., upregulation of genes associated with stress, energy metabolism, and fibrosis, as well as functional and structural defects in cardiomyocytes. Treatment of OVX + (L + A) with E2 or a G-protein-coupled estrogen receptor agonist normalized diastolic function, whereas estrogen receptor beta agonists did not. The OVX DD mice exhibited moderately impaired mitochondrial function, which delayed cardiomyocyte relaxation but not contraction, altered cardiac substrate utilization, reduced cardiomyocyte elasticity, increased production of reactive oxygen species, and potentiated extracellular fibrosis. ConclusionsOVX-induced E2-deficiency generates metabolic, structural, and functional changes in cardiomyocytes and the adjacent extracellular matrix, exacerbating the effects of L and A on diastolic function. This robust DD model revealed a role for E2 via ER in diastole-regulation in female mice and raised questions about similar mechanisms operative in postmenopausal women. HIGHLIGHTSO_LIOvariectomy (OVX)-induced estrogen-deficiency exacerbated diastolic dysfunction (DD) induced by hypertension and pressure overload with preserved ejection fraction in female C57BL/6J mice. C_LIO_LIIn post-OVX-treated DD mice, heart weight and fibrosis preceded other metrics of cardiac dysfunction. C_LIO_LIOVX-induced estrogen-deficiency increased the expression of genes associated with stress, energy metabolism, and especially fibrosis. C_LIO_LIMetabolic imaging of the heart by positron emission tomography revealed that DD with preserved ejection fraction was associated with mitochondrial dysfunction presenting as increased accretion of cardiac energy substrates, [18F]deoxyglucose and [11C]palmitate, effects that worsened following OVX. C_LIO_LIOVX increased cardiomyocyte stiffness and fibrosis and reduced cardiomyocyte lengthening and compliance in the context of female DD. C_LIO_LIImproved diastolic function following delivery of GPER agonists or estradiol to OVX DD female mice implicates estrogen receptor in the maintenance of normal cardiac function. C_LI

Advances in mass spectrometry have seen the identification of hundreds of new lipid species, some of which have been found to be associated with adverse cardiac remodeling. Key among these are GM3 gangliosides, which have been associated with metabolic disease, and more recently, adverse cardiac remodeling. Whether GM3s have a direct pathophysiological effect in the heart remains unclear. The present study investigated the effects of cardiomyocyte-specific knockout of GM3 synthase (GM3S, enzyme responsible for the synthesis of GM3) in the heart under basal settings and in response to ischemia-reperfusion (I/R) injury. A new cardiomyocyte-specific GM3S knockout (KO) model was generated, with knockout confirmed via lipidomic profiling. Under basal conditions, male GM3SKO mice exhibited reduced heart weight to tibia length (HW/TL) ratios with no evidence of pathological remodeling, while female mice showed no significant morphological differences. Male GM3SKO mice subjected to 1 hour ischemia and 4 weeks reperfusion demonstrated reduced HW/TL ratio compared to control mice subjected to I/R. However, no significant differences were observed in cardiac function, heart failure and fibrotic markers. Lipidomic profiling (49 classes, [~]850 species) revealed significant accumulation of dihexosylceramide, a metabolic precursor of GM3 in the male heart under basal and post-I/R conditions. In male GM3SKO I/R hearts, GM3 reduction was associated with decreases in odd- and branch-chained phospholipids, together with distinct changes in circulating ether lipid species. Collectively, cardiomyocyte-specific GM3 depletion contributed to sphingolipid remodeling but did not confer protection against I/R-mediated injury. These findings suggest that elevated GM3 levels observed in settings of cardiac pathology are not cardiomyocyte driven, highlighting the importance of understanding cell-type specific contributions to adverse cardiac remodeling.

Apolipoprotein E4 (E4) increases the risk of Alzheimers disease (AD) by up to 12-fold. However, understanding of the mechanisms underlying this increased risk has been limited by a lack of preclinical models that accurately reflect the effects of E4 in the presence of humanized non-mutant amyloid-{beta} precursor protein (hA{beta}PP). Therefore, we studied novel humanized APOE and hA{beta}PP mice to investigate the contributions of the E4 genotype to cognitive, inflammatory, and vascular dysfunction, specifically comparing male and female E3/hA{beta}PP and E4/hA{beta}PP mice. E4/hA{beta}PP mice exhibited impaired nest-building behavior and novel object recognition compared with E3/hA{beta}PP mice. Microglial content was higher in E4/hA{beta}PP mice, whereas astrocyte content was not different across groups. E4/hA{beta}PP mice had greater carotid and cerebral artery stiffness, and higher collagen I content in cerebral arteries than E3/hA{beta}PP mice. Under static pressure, cerebral artery endothelium-dependent and endothelium-independent vasodilation were similar across genotypes. However, high pulse pressure selectively impaired cerebral artery endothelial function in E4/hA{beta}PP mice, with the greatest impairment observed in females. The E4/hA{beta}PP mice also exhibited higher cortical expression of Nox2 and Sod1 and elevated cerebral artery Il1b expression. As such, E4/hA{beta}PP mice exhibit convergent cognitive, inflammatory, and vascular abnormalities that recapitulate several features of AD. Elevated pulse pressure revealed an E4-dependent vulnerability of the cerebral vasculature, suggesting that vascular stress may be an important contributor to disease risk. Together, our findings support the use of the APOExhA{beta}PP model to investigate the mechanisms by which E4 promotes vascular dysfunction, neuroinflammation, and cognitive impairment in AD.

Heart Failure with Preserved Ejection Fraction (HFpEF) is more prevalent in females and is associated with altered cardiac glucose metabolism. However, whether these metabolic alterations are conserved across sexes and between humans and widely used cardiometabolic mouse model of HFpEF remains unclear. We investigated species-, sex-, and ventricle-specific conserved and divergent features of HFpEF. Cardiometabolic HFpEF was induced in mice using the 'two-hit' model (high-fat diet + L-NAME), followed by assessment of cardiac function, RNA sequencing, and protein expression in the right (RV) and left (LV) ventricles. Published human HFpEF RV and LV RNA-seq datasets were reanalyzed and compared with our mouse data. Only male HFpEF mice recapitulated human phenotype of increased RV GLUT1 protein. In contrast, mouse GLUT1 was downregulated in RV of females and in the LV of both sexes, whereas GLUT4 protein remained unchanged. Cardiac PDK4 transcript and protein levels increased in the RV and LV of mice. Conversely, human PDK4 mRNA levels were reduced in the RV with HFpEF and unchanged in LV. Cardiac transcriptome analysis in mice revealed extensive alterations in LV, particularly in females, with enrichment of inflammatory pathways. Cross-species analysis demonstrated greater conservation of HFpEF-associated signatures in the RV than the LV. Furthermore, number of differentially expressed transcripts in human LV increased substantially after excluding patients with atrial fibrillation or diabetes. Overall, the RV of the 'two-hit' model more closely resembles human HFpEF. The cardiac transcriptome reflects sexual dimorphism, and conserved signatures are primarily associated with metabolic alteration, mitochondrial dysfunction, and cellular stress.

Cardiac electrophysiology adapts throughout pediatric development, driven by factors including age-associated ion channel expression changes and decreasing heart rate. Our prior transcriptomic-guided simulations of human atrial cardiomyocytes predicted developmental-associated changes in electrophysiology biomarkers at a fixed pacing rate, leaving the contribution of age- and patient-specific heart rate unresolved. In this study, we incorporated intrinsic heart rate into gene expression-guided computational models to predict the interaction between developmental maturation and pacing rate to shape atrial electrophysiology. Virtual patient-specific populations of atrial cardiomyocytes were generated from the right atrial cardiomyocyte gene expression data from 117 patients, spanning neonates to young adults. We simulated each population at pacing rates corresponding to each patients intrinsic ECG-based heart rate and at fixed rates corresponding to the patient cohort minimum, median, and maximum. Action potential and calcium transient biomarkers were quantified, and partial least squares regression assessed key biomarker dependencies. For intrinsic-rate pacing conditions, action potential duration at 50% and 90% repolarization increased with age, whereas early repolarization shortened; maximum upstroke velocity increased, resting membrane potential became more negative, and alternans prevalence decreased. Developmental differences persisted during fixed-rate pacing conditions, indicating that differences were not explained solely by the faster heart rates of younger patients. Notably, intrinsic-rate simulations exhibited stronger age associations for upstroke velocity and alternans than fixed-rate simulations. Sensitivity analyses indicated that electrophysiological phenotypes arose from interactions among ionic conductances, calcium handling, age, and heart rate. Collectively, we find that pediatric atrial electrophysiology reflects both intrinsic developmental remodeling and rate-dependent modulation.

Tricuspid valve regurgitation is a frequent valve lesion and, if severe, an independent predictor of mortality. In most patients, the valve itself has historically been considered intact. Yet, we have previously shown that the valve may not be an innocent bystander. In multiple sheep models, we have shown that the tricuspid valve thickens and stiffens. This remodeling may contribute to valve disease. Our goal is to extend our investigation of tricuspid valve remodeling to a rodent model, potentially opening scientific opportunity and enabling scaling our studies. To this end, we used pulmonary artery banding (PAB) in male rats to induce pressure overload and right ventricular remodeling. After excising the tricuspid valve, we quantified anterior leaflet morphology, mapped anterior leaflet thickness using optical coherence tomography, and evaluated anterior leaflet belly mechanics using a custom bulge testing system. Compared with SHAM controls, PAB increased anterior leaflet area. Moreover, anterior leaflets in PAB animals exhibited region-specific thickening, with the largest increases near the annulus. Finally, anterior leaflets in PAB animals were significantly less compliant. However, leaflet stiffening stemmed from aforementioned thickening, i.e., structural stiffening, not constitutive stiffening. Our findings demonstrate that we can reliably quantify leaflet area, thickness, and stiffness in the minuscule tricuspid valves of rats. We also show that tricuspid valve remodeling is not ovine-specific, but also affects the tricuspid valves of rats. Together, our findings support our hypothesis that tricuspid valves are not innocent bystanders in regurgitation, and that rats may serve as a scalable model system for future investigations. NEW & NOTEWORTHYUsing a rat pulmonary artery banding model of pulmonary hypertension, we show that chronic right ventricular pressure overload induces leaflet enlargement and region-specific thickness remodeling of the tricuspid valve. Although structural mechanical metrics change under pressure loading, normalization by thickness reveals that geometric remodeling rather than intrinsic material stiffening predominates. These findings highlight leaflet structural (mal)adaptation as a potential contributor to functional tricuspid regurgitation and underscore the importance of considering leaflet geometry in therapeutic strategies.

Objective(s)Tricuspid valve (TV) remodeling and functional tricuspid regurgitation (FTR) progression during right ventricular (RV) pressure overload and reverse remodeling after resolution of RV afterload is poorly understood. We set out to investigate tricuspid leaflet tissue response to induction and subsequent alleviation of pressure overload in a large animal model of RV failure with FTR. MethodsFifteen healthy adult male Dorset sheep (72{+/-}4 kg) underwent pulmonary artery banding (PAB) to induce RV failure and FTR. After 8 weeks, 7 sheep (PAB, n=7) were terminated, and remaining 8 had the PAB removed (rPAB, n=8) and were followed for another 8 weeks before termination. Both groups underwent epicardial echocardiography and hemodynamic assessment during banding surgery and at terminal operation. Ten healthy sheep served as a control group (CTL, n=10) and underwent terminal procedure only. In all animals, TV leaflets and right ventricular (RV) tissue were harvested at terminal procedure and analyzed histologically and transcriptionally. ResultsTV leaflets in PAB animals showed increased cross-sectional area and ECM alterations, some of which persisted after resolution of RV pressure overload. rPAB valves exhibited distinct ECM composition, with notably altered mucin and fibrin content, suggesting a shift toward matrix stabilization, dissimilar to control and PAB. RNA sequencing uncovered a unique molecular state in rPAB valves, with persistent changes in PRG4, PDE3A, CXCL8, and HLA transcripts. RV tissue also demonstrated a separate remodeling trajectory, with sustained expression of stress-related genes including PDE3A, NAV2, ANFB, and ACTS. These findings indicate that both valve and ventricular tissues retain a persistent remodeled phenotype post-unloading. ConclusionsTV leaflets actively remodel in response to hemodynamic stress and do not fully revert to a normal state after relief of pressure overload. This persistent altered phenotype may represent a biological contribution of the TV leaflets to recurrent TR with implications for long-term outcomes following treatment of FTR. Clinical Perspective What is new?O_LIRelief of right ventricular pressure overload, in a large animal model, resulted in substantial reverse remodeling of the right heart and reduction of tricuspid regurgitation severity, but tricuspid valve leaflets did not return to a normal state. C_LIO_LIReverse remodeled leaflets remained enlarged despite normalization of hemodynamics with an altered extracellular matrix. C_LIO_LICellular proliferation and immune cell infiltration observed during pressure overload resolved after unloading, yet transcriptomic analysis identified a distinct molecular phenotype that differed from both healthy and diseased valves. C_LIO_LITricuspid valve leaflets are active biological participants in the remodeling process and exhibit persistent adaptation or maladaptation after resolution of the initiating hemodynamic stress. C_LI What Are the Clinical Implications?O_LISecondary tricuspid regurgitation should be considered a disease involving both right heart geometry and leaflet biology. C_LIO_LIResolution of the underlying cause of tricuspid regurgitation may not restore leaflet structure and molecular homeostasis. C_LIO_LIPersistent leaflet remodeling may contribute to residual or recurrent tricuspid regurgitation despite successful treatment of pulmonary hypertension or other inciting conditions. C_LIO_LITherapies directed at leaflet remodeling may ultimately complement surgical and transcatheter strategies currently focused on annular and ventricular geometry. C_LI

Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal storage disease caused by pathogenic variants in NPC1 or NPC2. Sudden death can occur due to seizures, but cardiac involvement has not been well defined. We performed 12-lead electrocardiograms (ECG) in 14 adult NPC patients (8 male, 6 female). Cardiac structure and function were examined in Npc1-/- adult mouse hearts, alongside wild-type controls. Glycosphingolipid accumulation was quantified by high-performance liquid chromatography, fibrosis and collagen deposition were quantified using Massons Trichrome (M&T) and Picrosirius Red (PR) staining. Whole-heart morphology, including chamber size and wall thickness, was assessed. Ex vivo ECG recordings assessed conduction abnormalities and arrhythmias. RNA-seq transcriptomics characterised molecular pathways altered in Npc1-/- hearts. 8/14 patients showed ECG abnormalities including abnormal QRS transitions (N=8), increased QRS amplitude (N=4), fascicular block (N=2), and abnormal T wave inversion (N=1). 13 patients also had transthoracic echocardiograms identifying mildly impaired LV systolic function (N=2) and increased wall thickness/LV mass (N=4). In Npc1-/- mice, age-related glycosphingolipid accumulation was associated with pronounced ventricular fibrotic remodelling. There was a significant increase in stained connective tissue area and connective tissue to cardiac tissue ratio in both MT and PR staining. ECG from Langendorff-perfused Npc1-/- hearts showed QT prolongation and atrioventricular conduction abnormalities under isoprenaline stress. Transcriptomics revealed major changes in Npc1-/- hearts, consistent with histological fibrosis and linking NPC to inflammation-driven remodelling and arrhythmogenesis. These findings support routine cardiac screening in NPC patients and highlight the need for further studies to improve management and treatment.

BackgroundAldosterone promotes endothelial dysfunction and cardiovascular injury through mineralocorticoid receptor (MR) activation. Autophagy is essential for endothelial homeostasis, yet its role in aldosterone-mediated vascular dysfunction remains unclear. We tested whether aldosterone impairs autophagic flux and whether restoring autophagy via Beclin1 (BCN1) activation protects vascular and cardiac function. MethodsEndothelial and vascular responses to aldosterone were assessed in wild-type mice, BCN1 gain-of-function mice (Becn1), and mice treated with spermidine or a BCN1- activating TB-peptide. Vascular function, nitric oxide (NO)/reactive oxygen species (ROS) production, autophagy markers, endothelial migration, and cardiac fibrosis were evaluated using wire myography, fluorescence assays, Western blotting, confocal microscopy, migration assays, and histology. ResultsAldosterone impaired endothelium-dependent relaxation, decreased NO, increased ROS, and disrupted autophagic flux in an MR-dependent manner, indicated by LC3 accumulation and reduced p62 and BCN1 expression. Spermidine restored endothelial function and normalized NO and ROS levels. BCN1 gain-of-function mice were protected from aldosterone-induced endothelial dysfunction and exhibited reduced coronary and myocardial fibrosis. TB-peptide activation of BCN1 enhanced autophagic flux, improved vascular function, decreased cardiac fibrosis, and rescued endothelial migration impaired by aldosterone. ConclusionsAldosterone induces endothelial dysfunction by suppressing autophagic flux through MR activation. Genetic or pharmacologic enhancement of BCN1-dependent autophagy restores endothelial homeostasis and prevents vascular and cardiac injury, identifying autophagy activation as a promising therapeutic approach for cardiovascular diseases associated with mineralocorticoid excess.

Objective: One of the strongest genetic associations with coronary artery disease (CAD) risk maps to the metalloproteinase 'a disintegrin and metalloproteinase with thrombospondin motifs 7' (ADAMTS-7) locus. The protein was shown to promote plaque formation and instability. We aimed to generate and evaluate an antibody-based strategy targeting ADAMTS-7 therapeutically to reduce atherosclerotic plaque formation. Approach and Results: A truncated form of human ADAMTS-7 was produced in Nicotiana benthamiana and used as antigen for antibody generation by hybridoma technology. Eight monoclonal antibodies (mAbs) were screened, among which ADAMTS-7-mAb32 (mAb32) demonstrated the highest affinity, as confirmed by surface plasmon resonance analyses and immunoblotting against full-length ADAMTS-7. In vitro, mAb32 inhibited interactions of ADAMTS-7 with its substrates TIMP-1 and SVEP1 in a dose- and time-dependent manner, as assessed by time-resolved Forster resonance energy transfer assays. To assess therapeutic efficacy in vivo, Apoe-/- mice were fed a Western diet for ten weeks and treated with weekly injections of mAb32 or control IgG over the last six weeks. En face aortic Oil Red O staining revealed significantly reduced plaque area in the treatment group, without changes in plasma cholesterol levels or body weight. No evidence of liver or kidney toxicity was observed. Conclusion: Monoclonal antibody-based inhibition of ADAMTS-7 reduced atherosclerotic burden in vivo without affecting lipid metabolism, supporting ADAMTS-7 as a viable therapeutic target in CAD. Further development of mAb32 may provide a cholesterol-independent treatment strategy for atherosclerosis.

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.

Purpose: To determine whether the velocity reflection index (VRI) is the carotid Doppler waveform feature most strongly associated with chronological age after adjustment for sex and exercise habit, and whether its feature ranking remains stable across cross-validated and cohort-sensitivity analyses. Methods: Eight waveform-derived features were analysed in 197 participants meeting the study eligibility criteria and measured using a validated continuous-wave carotid Doppler system. Pearson and partial correlations and multivariable regression evaluated associations with chronological age. Random Forest regression with repeated 10-fold cross-validation, held-out permutation importance and bootstrap resampling assessed feature ranking. Sensitivity analysis evaluated the influence of cohort construction. Results: VRI showed the strongest association with chronological age (r = 0.738, 95% CI [0.667, 0.796]) and remained strongly associated after adjustment for sex and exercise habit (partial r = 0.798). VRI ranked first by both impurity-based (0.536) and held-out permutation (0.765) importance; repeated cross-validation yielded MAE = 6.87 +/- 1.26 years and R^2 = 0.572 +/- 0.153. Its leading ranking was stable in 85.3% of bootstrap resamples and the age-VRI correlation was essentially unchanged in the cohort-sensitivity analysis. The exercise association was significant after age adjustment (B = -0.043, p = 0.018) but attenuated after additional adjustment for sex (B = -0.026, p = 0.098). The sex association remained significant after adjustment for age and height. Conclusion: VRI was robustly associated with chronological age and retained the leading feature-importance ranking across adjusted statistical and cross-validated machine-learning analyses. Validation against an established arterial-stiffness measure in an independent cohort is required before VRI can be considered a clinical vascular-aging biomarker.