American Journal of Physiology-Heart and Circulatory Physiology

AimsGenome-wide association studies have identified numerous cardiac transcription factors in association with atrial fibrillation. Amongst these transcription factors, the paired-like homeodomain transcription factor 2 (PITX2) is the strongest genetic risk variant associated with atrial fibrillation. However, the downstream mechanisms of PITX2 are not completely understood. Here, we explore the role of PITX2 in oxidative metabolism and stress as a unifying mechanism of arrhythmogenesis. Methods and resultsTo identify PITX2 mechanisms, we performed transcriptomic analysis in Pitx2c-deficient neonatal rat atrial myocytes. We identify oxidative phosphorylation as the top dysregulated pathway and direct transcriptional targets lie in mitochondrial electron transport chain complexes I and IV. Using the Seahorse Extracellular Flux Analyzer, we identified a functional decrease in oxidative metabolism in Pitx2c-deficient cardiomyocytes. As electron transport chain complexes I and IV may generate reactive oxygen species (ROS) under mitochondrial dysfunction, we quantified mitochondrial specific ROS using MitoSOX and observed an increase in mitochondrial specific ROS in Pitx2c-deficient cardiomyocytes. We additionally assessed spontaneous cardiomyocyte calcium cycling using Fluo-8AM and observed an increased frequency of pro-arrhythmogenic mechanisms including early and delayed afterdepolarizations as inferred through calcium traces. Further, we identified sarcomere disassembly including a potential role of PITX2 in regulating Titin, where Pitx2c-deficient cardiomyocytes display Titin mis-localization within the sarcomeres. To assess whether ROS drives these phenotypes, we treated neonatal rat atrial myocytes with N-acetylcysteine, a potent ROS scavenger, and observed decreased early and delayed afterdepolarizations, as well as restoration of Titin localization. ConclusionPITX2C maintains atrial metabolism and redox balance; the loss of PITX2C results in reduced oxidative metabolism and an elevation in oxidative stress that ramifies cardiomyocyte dysfunction. Treatment with antioxidant restores AF-associated phenotypes including abnormal calcium cycling and sarcomere disassembly in Pitx2c-deficient atrial cardiomyocytes. TRANSLATIONAL PERSPECTIVEGenetic variants close to the PITX2 gene associate most strongly with atrial fibrillation. This study reveals a mechanistic link between multiple AF-associated phenotypes and mitochondrial dysfunction with subsequent accumulation of reactive oxygen species downstream of PITX2. Importantly, metabolic therapies and reducing oxidative stress may present a potential clinical strategy to reverse and prevent functional and structural remodelling related to AF.

BackgroundClinical evidence supports a greater impact of arterial stiffening in cardiovascular mortality in women versus men. Arterial stiffness increases across the menopausal transition, implicating a role of the loss of estrogens in arterial stiffening, but mediating mechanisms remain unclear. MethodsThe role of estradiol and smooth muscle cell (SMC) estrogen receptor alpha (ER) in arterial stiffening, by aortic pulse wave velocity (PWV), was assessed in 3 models: (1) the loss of estradiol in young, female mice comparing sham surgery or bilateral ovariectomy (OVEX) {+/-} estradiol, (2) the impact of sham versus OVEX surgery in young, female SMC-ER-intact and SMC-ER-knockout (KO) littermates, and (3) arterial stiffening during natural aging by comparing young and aged, female and male SMC-ER-intact and SMC-ER-KO littermates. Mechanistic pathways were assessed using histological assessment of aortic fibrosis and elastin degradation, aortic MMP expression, and atomic force microscopy. ResultsOVEX increased PWV and aortic medial fibrosis, with no impact on elastin integrity, in young female mice. Arterial stiffening and fibrosis were prevented in OVEX mice that were supplemented with estradiol. OVEX-induced arterial stiffening in SMC-ER-intact female mice was prevented in SMC-ER-KO littermates. In this model, OVEX was also associated with increased aortic medial fibrosis without changes in elastin integrity. Aging from 3 to 18 months significantly increased PWV in female and male SMC-ER-intact mice. Aging-induced stiffening was fully prevented in female and partially prevented in male SMC-ER-KO mice. SMC-ER contributes to aging-associated arterial stiffening by sex-specific mechanisms, including elastin degradation in females and phenotypic changes in SMC stiffness and probability to form cellular adhesions in males. Circulating estradiol was significantly decreased in serum from aged compared with young female mice. ConclusionsThese findings support that SMC-ER contributes to arterial stiffening in female and male mice in situations where the vasculature is exposed to low levels of estradiol.

Ventricular arrhythmias are the leading cause of sudden cardiac death. It is well-established that environmental factors contribute to the origin and penetrance of ventricular arrhythmic disorders. However, to our knowledge, no studies have considered the role of the intrauterine environment. In this study, we investigated the long-term effects of fetal hypoxia on ventricular arrhythmia susceptibility. Pregnant Wistar rats were assigned to normoxia (21% O2) or hypoxia (13% O2 between gestational days 6-20), and offspring were raised to 6 months. Hearts were isolated and loaded with the fluorescent calcium- and voltage-sensitive indicators, Rhod-2 and RH237, respectively. Optical mapping was performed while the left ventricle was burst paced (10-20hz) to induce arrhythmias. Hearts isolated from adult offspring exposed to fetal hypoxia were more susceptible to arrythmia during burst pacing, compared to controls. This phenotype was associated with prolonged Ca2+ transients and action potentials, an increased frequency of Ca2+ waves and delayed after depolarisations, as well as lower gene and protein expression of the sarcoplasmic reticulum Ca2+ ATPase. Collectively, our data shows that fetal hypoxia can programme ventricular arrhythmia sensitivity in adulthood, driven by abnormalities in excitation-contraction coupling. This is the first evidence that some ventricular arrhythmias may have a developmental origin, highlighting pregnancy as a potential window for early preventive intervention.

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

BackgroundAntihypertensive and cardioprotective medications are prescribed to pregnant women and include Ca2+ channel blockers (CCBs; amlodipine, nifedipine), - (doxazosin) and {beta}-(labetalol, bisoprolol, nebivolol) adrenergic receptor antagonists, and -adrenergic receptor agonists (methyldopa). These vasoactive drugs enter the fetal circulation, with unknown effects on the fetoplacental vasculature. We aimed to investigate whether cardiovascular medications modulate human fetoplacental vascular tone, which may impair or enhance placental perfusion. MethodsChorionic plate arteries (CPAs) were obtained from the placentas of women with normotensive pregnancy (N=28), with unmedicated hypertension (N=14), and those chronically medicated (N=61) with either amlodipine, nifedipine, labetalol or bisoprolol, or a combination of CCBs and labetalol. Using wire myography, ex vivo effects of amlodipine, nifedipine, labetalol, methyldopa, doxazosin, bisoprolol and nebivolol were tested in a concentration-dependent manner (10-11-10-5M) in pre-constricted CPAs isolated from the placentas of normotensive women. Differences in CPA vascular reactivity in response to chronic exposure to hypertension and/or cardiovascular medications was assessed by vasoconstriction to high potassium physiological solution (KPSS; 120mM) and to the thromboxane A2 mimetic (U46619; 10-10-2x10-6M), and relaxation to the nitric oxide donor, sodium nitroprusside (SNP; 10-10-10-5M). ResultsIn pre-constricted CPAs isolated from normotensive women, acute exposure to amlodipine, nifedipine, doxazosin and nebivolol promoted significant vasorelaxation (P<0.05). CPAs acutely exposed to labetalol, methyldopa (P<0.05) and bisoprolol (P<0.001) exhibited increased vasoconstriction compared to their respective diluent controls. CPAs from women with chronic hypertension and from those who had chronic labetalol treatment exhibited significantly reduced vasoconstriction to KPSS (P<0.05). CPAs from women with chronic hypertension and exposure to bisoprolol also had significantly attenuated vascular responses to U46619 and SNP (P<0.01 and P<0.01, respectively), compared to normal pregnancy. ConclusionsMaternal hypertension impairs vascular responses of the placenta. Cardiovascular medications prescribed during pregnancy may dysregulate placental vascular function. Further research is warranted to evaluate the relative safety of cardiovascular medications in pregnancy, as their distinct effects on fetoplacental vascular function may have important implications for maternal and fetal outcomes. Mechanistic studies alongside clinical correlations are essential to guide evidence-based prescribing.

BackgroundEndothelial response to flow is key to vascular function in health and disease. Our earlier studies demonstrated that endothelial Kir2.1 is essential for flow-induced Akt1/eNOS signaling and for flow-induced vasodilation (FIV) but the mechanistic integration between Kir and other flow signaling pathways remained poorly understood. MethodsWe use a combination of electrophysiological recordings in real time of flow exposure, Ca2+ imaging, pressure myography of resistance arteries, and echocardiography. ResultsWe demonstrate that Kir2.1 is essential for flow-induced PI3K phosphorylation, whereas expression of myristoylated Akt1, which bypasses PI3K-dependent membrane recruitment, restores flow-induced Akt1/eNOS phosphorylation in Kir2.1-deficient endothelium. It also restores FIV in Kir2.1-deficient mesenteric arteries. We further demonstrate that Kir2.1 is essential for flow-induced Ca{superscript 2} influx mediated by Piezo1 and TRPV4 channels, whereas Ca{superscript 2} influx induced by pharmacological activation of these channels is Kir2.1 independent. Deficiency of Piezo1 does not affect endothelial Kir2.1 channels. We also discover that flow activation of endothelial Kir2.1 requires Syndecan1, thus creating a link between glycocalyx and downstream effects. Physiologically, we find that endothelial Kir2.1 is suppressed by infusion of Angiotensin-II and by advanced aging, resulting in significant impairment of FIV. In both cases, FIV is fully restored by endothelium-specific over-expression of Kir2.1. ConclusionsOur study reveals that Kir2.1 serves as a mechanistic linker between endothelial glycocalyx to Piezo1-mediated Ca2+ influx and downstream signaling suggesting a new integrated model of endothelial mechanotransduction. A functional loss of endothelial Kir2.1 is shown to play a significant role in FIV impairment in Angiotensin-induced hypertension and aging.

BackgroundSex differences in the epidemiology of atrial fibrillation are well-documented; however, the underlying mechanisms remain poorly understood. This gap in knowledge is compounded by limited data on sex-specific atrial electrophysiology in the absence of disease. ObjectivesThe aim of this study was to investigate sex differences in atrial electrophysiology and arrhythmia susceptibility in a translationally-relevant rabbit model. MethodsDual optical mapping of transmembrane voltage and Ca2+ was performed on intact atria of young (3.5-5 months) male and female rabbit hearts. Baseline atrial electrophysiology and arrhythmia susceptibility were investigated using rapid pacing and premature stimulation and further tested with the parasympathomimetic carbachol. Sex and regional differences in gene expression were assessed using qPCR. ResultsFemales exhibited similar action potential duration (APD), but greater APD heterogeneity across the atria at slower rates, along with longer Ca2+ transient durations compared to males. Greater APD heterogeneity in females was rate-dependent and comparable to males at faster pacing frequencies; however, it was associated with greater susceptibility to transient reentrant arrhythmias with premature stimuli. After carbachol application, males had heightened vulnerability to arrhythmia. This was associated with cholinergic-mediated APD shortening in both atria in males, but only in the right atrium in females. Sex differences in carbachol responses were linked to variations in muscarinic receptor and acetylcholine-activated potassium channel gene expression. ConclusionsThese findings demonstrate sex and regional differences in atrial electrophysiology at baseline and in response to cholinergic stimulation in the healthy heart that may contribute to sex-specific mechanisms of arrhythmia.

Background and aimsIncreased levels of -tubulin and its post-translational modifications (PTMs) are found in human heart failure and could initiate diastolic dysfunction by modulating cardiomyocyte stiffness. How these modifications occur and how they may underlie cardiac dysfunction remains unknown. Upstream kinases may play a critical role, but this has not been explored. Methods and resultsHere we address this question by, for the first time ever, determining levels of the enzymes involved in microtubule (MT) detyrosination and acetylation (TAT1, HDAC6) in a well-characterized cohort of patients with hypertrophic cardiomyopathy (HCM). In HCM patients (N=10-11), protein levels of detyrosination enzymes remain unaltered, whilst levels of TAT1 and HDAC6 were decreased and increased, respectively. Phosphoproteomics in HCM (N=24) and control (N=8) myocardium identified significant differences in over 1900 serine/threonine and 160 tyrosine phosphosites, in addition to increased EGFR/IGF1R-MAPK signaling in HCM. We subsequently showed that MT repolymerization was increased in HCM MYBPC3Arg943X hiPSC-CMs. Isoprenaline-mediated PKA activation decreased MT repolymerization in hiPSC-CMs and revealed CLASP1, MAST4 and MAP1A as potential MT modifiers in HCM. ConclusionsWe show that the altered HCM MT code cannot be attributed to levels of key MT-modifying enzymes. By combining kinome analyses in human HCM hearts with hiPSC-CM studies on MT dynamics, PTMs and contractility we unveiled a regulatory role for MTs in the cardiomyocyte response to beta-adrenergic receptor stimulation. Disease-mediated changes in the MT code thereby exert both a direct, and indirect effect on cardiac function via mediating the response to adrenergic activation. Graphical Abstract created with BioRender.com O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/706710v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1c58dc4org.highwire.dtl.DTLVardef@de502eorg.highwire.dtl.DTLVardef@1621512org.highwire.dtl.DTLVardef@557b82_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pregnant women are particularly susceptible to adverse outcomes from environmental heat, yet the physiological effects of acute heat exposure during pregnancy remain poorly understood. Some physiological changes are monitored in humans; however, investigation of underlying molecular mechanisms requires invasive methods that can only be ethically applied in mammalian models. Moreover, research with animal models has largely focused on early and lethal teratogenic effects of heat exposure and lacks longitudinal physiological monitoring, detailed parameterisation of heating regimes and in-depth investigation of underlying mechanisms. Here we used a mouse model to investigate the impact of a controlled acute heat exposure at mid-gestation (E12{middle dot}5), slowly elevating core body temperature (CBT) over 210mins to raise CBT by [~]1{degrees}C. Using high-frequency ultrasound and morphological analyses, we observed delayed alterations in placental and foetal cerebral blood flow indicative of a brain-sparing response, alongside reduced placental labyrinth zone size. Additionally, maternal cardiac function was impaired, accompanied by cardiac and renal fibrosis and elevated circulating soluble Flt-1 levels, an anti-angiogenic biomarker of gestational hypertension. These findings demonstrate that brief heat stress at mid-gestation can induce lasting effects on placental function and maternal cardiovascular health in a mammalian model, highlighting potential risks for pregnancy outcomes under increasing global temperatures. Together this data suggests that an acute exposure to heat elevating core body temperature by 1{middle dot}2{degrees}C can induce a long-term impact on both placenta and maternal health in a mouse model. It will be important to understand the molecular changes which underpin the pathophysiology and whether this is translated to humans.

BackgroundThe cardiac isoform of phosphofructokinase-2/fructose 2,6-bisphosphatase (PFKFB2) is the hearts strongest glycolytic regulator but is degraded in the absence of insulin signaling. This makes PFKFB2 loss critical to understand in metabolic heart disease, of which impaired insulin signaling is a hallmark. Prolongation of the QT interval, risk of arrhythmia, and sudden cardiac death are also augmented in metabolic heart disease, raising a question as to whether potential crosstalk between glycolytic dysregulation and electrophysiological dysfunction exists. MethodsWe therefore assessed the impact of PFKFB2 loss on cardiac electrophysiology using a cardiomyocyte-specific PFKFB2 knockout mouse model (cKO) and litter-matched controls (CON). To do so, we employed electrocardiography in the fed state and following 12 hours of fasting, examining physiology both at baseline and in the presence of an acute stimulant stress. To further investigate the arrhythmia mechanism, we used patch-clamp electrophysiology and IonOptix Ca2+ transient measurements in ventricular cardiomyocytes isolated from CON and cKO hearts. ResultsThe hearts of cKO mice exhibited prolonged repolarization, marked by QT and action potential duration prolongations. This occurred with impaired Ca2+ reuptake and increased spontaneous Ca2+ release events in ventricular cardiomyocytes. Ultimately, these changes culminated in ventricular tachyarrhythmia in cKO mice, which was enhanced in the fed relative to the fasted state. ConclusionThese data suggest that in the presence of sufficient glucose availability, cardiac glycolytic dysregulation at the phosphofructokinase nexus is sufficient to promote cardiac electrophysiological instability. Clinical PerspectiveO_ST_ABSWhat is KnownC_ST_ABSO_LIMetabolic heart diseases, such as heart failure with preserved ejection fraction and diabetic cardiomyopathy, are associated with heightened risks of arrhythmogenesis and sudden cardiac death. C_LI What the Study AddsO_LIHere, we show for the first time that PFKFB2 is decreased in human hearts with heart failure with preserved ejection fraction. C_LIO_LIFurthermore, we show that loss of cardiac PFKFB2 is sufficient to promote impaired ventricular repolarization at baseline and ventricular tachyarrhythmia upon stress test. C_LIO_LIThis identifies PFKFB2 stabilization and activation as key potential targets in conferring electrophysiological stability in metabolic heart disease. C_LI

BackgroundCoronary autoregulation is the ability of the normal heart to maintain constant coronary blood flow (CBF) over a wide range of coronary driving pressures (CDP). Despite being vital for survival, the mechanism of coronary autoregulation is unknown. We hypothesized that GPR39, present in vascular smooth muscle cells, together with its endogenous agonist 15-hydroxyeicosatetraenoic acid (15-HETE) orchestrate coronary autoregulation. MethodsWe created coronary stenoses of varying degrees in open-chest, anesthetized dogs where we measured CBF and CDP. In a subset of animals, coronary venous blood was sampled for eicosanoid, adenosine, endothelin-1, polyunsaturated fatty acids, and prostaglandins levels. Stenoses were recreated during intravenous administration of VC108, a specific GPR39 antagonist and systemic, pulmonary, and coronary hemodynamics measured. ResultsGPR39 was identified in coronary arterioles by immunohistochemistry and in heart tissue by western blot. In-vivo, 15-HETE correlated linearly with CDP over the autoregulatory range (r2=0.47, p=0.0024). Apart from 6-keto PGF1 no other metabolite had any relation with CDP. Prior to administration of VC108, CBF did not change within the autoregulatory range. VC108 had no effect of systemic and pulmonary hemodynamics but increased CBF (p=0.02 versus vehicle) by decreasing coronary microvascular resistance (p=0.01 versus vehicle), indicating that GPR39 participates in control of normal coronary vascular tone. With VC108, coronary autoregulation was abolished and CBF became CDP dependent (r2=0.96, p=0.004). ConclusionGPR39 and its endogenous agonist 15-HETE together orchestrate coronary autoregulation when CDP is reduced. These novel findings provide a mechanism for coronary autoregulation and could direct pharmacological treatment of various coronary syndromes in humans.

RationaleRegular aerobic exercise protects against vascular aging and reshapes the circulating molecular milieu, but the relation between vascular function, circulating molecules, and exercise dose at extreme volumes remains poorly defined. The vascular and molecular consequences of chronic, multi-stage ultra-endurance running are particularly unclear. ObjectiveTo define circulating molecular signatures associated with vascular dysfunction following the 64-stage, 4,486-km Trans Europe Foot Race (TEFR). Methods and ResultsIntegrated multiomics analysis (proteomics, lipidomics, metabolomics) of plasma from 27 finishers revealed a coordinated systemic shift driving an oxidative phenotype. Specifically, we identified altered arginine metabolism and a universal upregulation of lipotoxic ceramides consistent with incomplete fatty acid oxidation. In conjunction, we identified upregulation of innate immune system pathways including the acute phase response and the complement system. Central pulse wave velocity (cPWV) increased significantly after the race, consistent with arterial stiffening. To test whether the post-race circulating milieu could directly influence vascular mechanics, naive murine aortic rings were incubated with participant plasma. Post-race plasma acutely increased aortic elastic modulus, and this effect was attenuated by the superoxide dismutase mimetic TEMPOL, supporting a ROS-dependent component. In human aortic endothelial cells (HAECs), post-race plasma increased reactive oxygen species generation without detectable changes in eNOS phosphorylation, total eNOS abundance, or stimulated nitric oxide production. Endothelial ROS responses were associated with components of the terminal complement pathway. ConclusionsExtreme multi-stage ultra-endurance exercise induces a distinct systemic milieu associated with arterial stiffening through ROS-sensitive mechanisms. This response is characterized by remodeling of arginine-related metabolism, ceramide accumulation, innate immune activation, and oxidative stress, without evidence of reduced measured eNOS abundance or stimulated NO production. These findings identify candidate molecular pathways linking prolonged metabolic stress to vascular dysfunction.

Lysosomal function is essential for cardiac proteostasis and cellular health, yet its regulation during ageing remains poorly defined. We hypothesised that ageing alters both the abundance of acidic organelles and the machinery supporting their acidification. Using fluorescence-based In Vivo Imaging Systems (IVIS) with Lysotracker Red in young (2-4 months) and aged (18 months) mouse hearts, we quantified whole-heart acidic-vesicle signals and assessed expression of lysosomal and autophagy-related genes (Lamp2, Atp6v1a, Sqstm1, Cd63, Atg12, Nfe2l2, M6pr) by RT-qPCR. Whole-heart labelled Lysotracker fluorescence did not differ significantly between age groups, indicating preservation of the total acidic-vesicle pool. No changes in Atp6v1a and Lamp2 expression suggest acidification capacity and structural stability are maintained, whereas the minor, upregulation of Sqstm1 might indicate increased autophagic demand and altered vesicle trafficking, which warrants further investigation. No statistical significant changes in M6pr, Atg12, or Nfe2l2 were detected, suggesting transcriptional stability in enzyme trafficking, core autophagy, and oxidative stress pathways. Regionally, atria showed higher Lysotracker signal than ventricles, consistent with known enrichment of acidic vesicular stores in atrial physiology. These findings highlight the utility of IVIS imaging of Lysotracker-labelled hearts, providing rapid whole-organ assessment of acidic vesicle distribution, albeit with limited depth resolution. Complementary techniques such as RT-qPCR analysis is essential to interpret IVIS findings, enabling insight into underlying molecular changes in lysosomal and autophagy pathways during cardiac ageing.

Background: For decades, cardiovascular physiology has been built on the assumption that arterial baroreceptors adjust heart rate (HR) to maintain a defined blood pressure set point. We challenge this paradigm fundamentally. Blood pressure and heart rate both change substantially in response to physiological stress and neither returns reliably to a fixed baseline value. This raises the question of whether a higher-order variable, one that remains stable while blood pressure and heart rate reset freely might better represent a truly defended, set-point quantity. Hypothesis: We hypothesized that the coefficient of variation of the instantaneous baroreceptor gain (IBS CV), expressed as the change in R-R interval per unit change in systolic blood pressure (SBP), is invariant across different physiological challenges. If IBS CV is fixed, then HR and SBP must vary proportionally, maintaining a stable gain relationship even as each changes in magnitude. Methods: To test this hypothesis, we had healthy adult volunteers undergo either the cold pressor test or passive orthostatic challenge. HR, SBP, IBS, and the coefficients of variation (CV, i.e. standard deviation / mean value) of each were measured at baseline and during each stress perturbation. Results: During orthostatic challenge, HR rose significantly while SBP fell significantly. Classically, this HR rise is attributed to baroreflex compensation for falling pressure. However, the critical observation is that SBP was not restored to baseline. Instead, it remained substantially reduced while HR stayed persistently elevated and HR CV increased significantly. A system primarily defending a blood pressure set point should augment baroreflex gain and suppress pressure variability; instead mean IBS showed no significant change, SBP CV amplified more than threefold, and IBS CV was unchanged. During the cold pressor test, both HR and SBP rose simultaneously, which is inconsistent with a pressure-defending system that would have suppressed HR in response to the large rise in SBP. IBS CV was also stable across this perturbation while SBP CV amplified dramatically. Conclusion: These findings challenge the classical baroreceptor set-point model and suggest that IBS CV, not blood pressure, is the primary regulated cardiovascular variable. Furthermore, IBS CV is likely to prove to be a more sensitive marker than blood pressure or heart rate variability for risk stratification in patients with hypertension, heart failure, or autonomic insufficiency.

Abstract Background: Poor sleep quality is associated with increased cardiovascular risk, although its relationship with left ventricle (LV) structure is poorly understood and ethnic differences in the relationship between sleep and LV structure have not been studied. We investigated the association between poor sleep quality and LV structure in a tri-ethnic cohort. Methods: A total of 1284 participants were analysed from the Southall and Brent Revisited (SABRE) study (age=49.9{+/-} 6.2y; male 75.9%, Europeans (EU)=615, South Asians (SA)=457, African/African-Caribbean (AC)=212). A composite sleep quality score was calculated, and LV structure was measured using echocardiography. Associations between sleep quality and LV mass indexed to height1.7 (LVMi), relative wall thickness (RWT) and LV end-diastolic volume indexed to height1.7 (LVEDVi) were estimated using multivariable linear regression with adjustment for demographic and lifestyle factors across three models. Analyses were performed in the whole cohort and stratified by ethnicity. Results: Compared with those who reported very good sleep quality, participants with poorer sleep quality had higher LVMi (4.8 (95% CI 1.4; 8.2)g/(m1.7*unit sleep score); p=0.006). When stratifying by ethnicity, the association between sleep quality and LVMi was unconvincing in EU (1.9(-3.5, 7.3)g/(m1.7*unit sleep score); p=0.493), whereas poor sleep was associated with higher LVMi in AC and SA participants (9.1(1.3;16.8)g/(m1.7*unit sleep score); p=0.023 and 5.8(0.5;11.0)g/(m1.7*unit sleep score); p=0.031 respectively). Conclusions: Poor sleep quality is associated with higher LVMi in older African/African-Caribbeans and South Asians, but not in Europeans. This may contribute to cardiovascular risk. Keywords: sleep, left ventricle, hypertrophy, remodelling

BackgroundInterindividual variation in systolic blood pressure (SBP)-heart rate (HR) coupling reflects differences in autonomic and vascular regulation. The primary objective of this study was to determine whether SBP-HR synchronization identifies distinct hemodynamic phenotypes associated with demographic characteristics and blood pressure (BP) profiles in hypertensive and normotensive individuals. MethodsWe conducted a cross-sectional cohort analysis of 1,122 adults who underwent continuous 24-hour ambulatory monitoring of SBP, diastolic blood pressure (DBP), and HR in an outpatient clinical setting. Participants were classified as hypertensive or normotensive using guideline-based criteria. The primary exposure variable was SBP-HR synchronization, quantified using zero-lag cross-correlation coefficients. The primary outcomes were demographic characteristics (age, sex, stature) and BP phenotypes, including isolated systolic hypertension (ISH). Unsupervised partitioning around medoids clustering was used to identify synchronization-based phenotypes. Group comparisons were performed using Welchs ANOVA and {chi}{superscript 2} testing, with multivariable adjustment for age and sex where applicable. ResultsAll 1,122 participants were included in the final analysis (mean age 60.7 years; 52% female). Three synchronization phenotypes were identified: low, moderate, and high. Compared with the low-synchronization phenotype, the high-synchronization phenotype was younger (55.2 {+/-} 15.0 vs. 66.6 {+/-} 13.9 years), taller (170.4 {+/-} 9.4 vs. 167.4 {+/-} 10.5 cm), more frequently male (56% vs. 38%), and had lower baseline SBP (131.8 {+/-} 17.5 vs. 136.7 {+/-} 24.2 mmHg). Overall hypertension prevalence did not differ across phenotypes; however, ISH was less frequent in the high-synchronization phenotype (15.7% vs. 23.4%). Cluster assignments were robust across sensitivity analyses. ConclusionsSBP-HR synchronization identifies distinct hemodynamic phenotypes associated with age, sex, stature, and BP characteristics. Stronger synchronization reflects a physiological profile consistent with preserved autonomic-vascular integration and lower prevalence of isolated systolic hypertension, supporting its potential role in refined cardiovascular phenotyping.

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

Background: AI-enabled electrocardiographic age (AI-ECG age) is a digital biomarker of electrophysiological cardiac health. Although cardiovascular physiology exhibits circadian organization, the circadian behavior of AI-ECG age and its structural correlates have not been defined in AF-naive individuals. Objectives: To determine whether AI-ECG age exhibits reproducible circadian patterns and whether disruption of these patterns is associated with left atrial (LA) remodeling, a marker of atrial myopathy. Methods: Continuous single-lead wearable ECGs were analyzed from two independent prospective cohorts (S-Patch [ClinicalTrials.gov: NCT05119725, registered November 2021]; Memo Patch [ClinicalTrials.gov: NCT05355948, registered May 2022]). In AF-naive participants with 48 hours of data, AI-ECG age was estimated every 10 minutes. Unsupervised clustering was used to identify intrinsic circadian trajectories. For clinical interpretability, participants were classified using a day-night difference cutoff (Age 0.6 years) as Restorative (Age >0.6) or Disrupted (Age 0.6). We assessed phenotype reproducibility and examined associations with left atrial volume index (LAVI) using multivariable regression and meta-analysis. Results: Unsupervised learning consistently identified three circadian trajectory patterns across cohorts. Under the simplified binary classification, the Restorative phenotype was observed in approximately half of the participants (47.6-50.2%). Phenotype reproducibility was moderate (Cohen's 0.518; ICC=0.51-0.54) and was not fully explained by conventional heart rate variability measures. Among participants with echocardiography (n=122), the Disrupted phenotype was associated with higher LAVI (adjusted mean difference 6.09 mL/m2; 95% CI 1.46-10.72; p=0.010) and higher odds of severe LA enlargement (adjusted OR 4.17; 95% CI 1.58?10.99; p=0.004), with negligible heterogeneity (I2=0%). Conclusions: Wearable-derived AI-ECG age exhibits circadian patterns in AF-naive individuals, with unsupervised learning identifying distinct trajectories. Attenuation of a nocturnal decline the Disrupted phenotype is associated with left atrial enlargement, independent of conventional comorbidities and static AI-ECG age metrics. These findings suggest that circadian electrophysiological aging phenotyping may capture a dimension of atrial structural vulnerability not reflected by point-in-time assessments, and support prospective studies to evaluate its clinical utility.

Background and ObjectivesLeft ventricular pressure-strain loop (LV-PSL) analysis provides noninvasive myocardial work indices that may reflect ventricular-arterial (VA) coupling, but their behavior under controlled physiologic stressors is incompletely defined. We aimed to characterize directional changes in LV-PSL, derived indices during standardized interventions predominantly affecting preload, afterload, or contractility in healthy adults. MethodsIn this prospective, within-subject repeated-measures study, 61 healthy volunteers underwent interventions designed to elicit domain-specific hemodynamic perturbations. Group 1 (n=31) performed isotonic exercise (contractility-dominant). Group 2 (n=30) performed isometric handgrip (afterload-increasing) and passive leg raising (PLR; preload augmentation with concurrent afterload change). LV-PSL indices were assessed at baseline and during each intervention. Six co-primary endpoints were prespecified: Global Work Index (GWI), peak systolic strain, strain range, systolic strain rate (SSR), arterial elastance (Ea), and end-systolic pressure (ESP). Within-subject changes were analyzed using paired tests with multiplicity adjustment and determined effect sizes. Reproducibility was evaluated using intraclass correlation coefficients (ICC). ResultsLV-PSL responses were directionally consistent with established pressure-volume physiology. Isotonic exercise produced large increases in contractility-sensitive indices, including GWI (dz=1.03), peak systolic strain (dz=0.88), strain range (dz=1.10), SSR (dz=1.29), and ESP (r=1.26), all adjusted p<0.001, while Ea remained unchanged. In contrast, isometric handgrip and PLR elicited afterload-dominant responses, with significant increases in ESP (dz=1.11 and 1.21, respectively; adjusted p<0.001) and Ea (dz=0.79 and 0.77; adjusted p[&le;]0.001), without significant changes in GWI or strain-derived indices after adjustment. Intraobserver reproducibility was good-to-excellent (ICC 0.86-0.90), and interobserver reproducibility was moderate-to-good (ICC 0.72-0.87). ConclusionsIn healthy adults, LV-PSL indices demonstrate good reproducibility and appropriate sensitivity to hemodynamic perturbations. Exercise elicited contractility-dominant responses, whereas handgrip and PLR induced afterload-dominant changes. These physiologically coherent response patterns support LV-PSL as a practical non-invasive surrogate for invasive pressure-volume assessment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/26347879v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@113c652org.highwire.dtl.DTLVardef@1413e5aorg.highwire.dtl.DTLVardef@64b898org.highwire.dtl.DTLVardef@930462_HPS_FORMAT_FIGEXP M_FIG Central Illustration - Validation of Non-Invasive Pressure-Strain Loops for Assessing Ventriculo-Arterial Coupling The study evaluated left ventricle pressure-strain loop (LV-PSL) derived indices during three hemodynamic interventions in healthy volunteers: Group 1 - exercise (contractility-dominant), Group 2 - isometric handgrip (afterload-dominant), and passive leg raising (preload/afterload modulation). Center heatmap shows effect sizes (Cohens dz or rank-biserial r) for six co-primary PSL endpoints. Color intensity indicates effect magnitude (red = positive, blue = negative); asterisks denote significance after Holm-Bonferroni correction (**p[&le;]0.001). Exercise produced robust responses in 5/6 parameters, while handgrip and passive leg raising showed intervention-specific patterns, particularly for afterload indices. PSL methodology demonstrates high reproducibility and physiological sensitivity for non-invasive ventriculo-arterial coupling assessment. Abbreviations: LV-PSL, Left ventricle pressure-strain loop; PLR, passive leg raising; VA, ventriculo-arterial C_FIG

BackgroundWingless/Int-1 (Wnts) proteins are canonical Frizzled receptor ligands. Recent evidence indicates that some Wnts, including Wnt9b and Wnt5a, bind to polycystin 1 (PKD1), a transmembrane protein which can couple to polycystin 2 (PKD2) to form a non-selective cation channel. The functional significance of Wnts binding to PKD1 is unclear. Here, we tested the hypothesis that Wnts act through PKD1/PKD2 channels on endothelial cells (ECs) to regulate arterial contractility and blood pressure and investigated the cellular source and secretory regulation of vasoactive Wnt proteins. MethodsA wide variety of approaches, including inducible EC-specific PKD1 and PKD2 knockout mice, reverse-transcription polymerase chain reaction, Western blotting, immunofluorescence, pressurized artery myography, blood pressure measurements, patch-clamp electrophysiology, in vivo and in vitro Wnt and nitric oxide assays, and Wnt secretion assays. ResultsIntravascular Wnt9b or Wnt5a administration stimulates an EC PKD1/PKD2-dependent dilation in pressurized resistance-size arteries. Wnt9b and Wnt5a are present in serum and plasma and intravenous infusion rapidly stimulates a blood pressure reduction which requires EC PKD1. Wnts stimulate a PKD1-dependent non-selective cation current in ECs which through Ca2+ signaling activates endothelial nitric oxide synthase (eNOS) and small conductance Ca2+-activated K+ channels to induce vasodilation. Wnt9b acts solely via PKD1/PKD2 channels, whereas Wnt5a stimulates signaling through PKD1/PKD2, Frizzled-7 (Fzd-7), Dishevelled and c-Jun N-terminal kinase (JNK). Intravascular flow stimulates angiotensin II type 1 (AT1) receptors, which through Gq/11 and Porcupine activate Wnt9b and Wnt5a secretion in ECs. Wnts secreted in response to flow activate PKD1/PKD2 signaling in ECs and contribute to flow-mediated vasodilation. ConclusionsIntravascular flow activates AT1 receptors, which through Gq/11 and Porcupine stimulate Wnt9b and Wnt5a secretion in ECs. Wnt9b activates PKD1/PKD2 channels whereas Wnt5a stimulates both PKD1/PKD2 and Fzd-7 in ECs to induce vasodilation. Wnts contribute to flow-mediated autocrine/paracrine dilation and reduce blood pressure. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/712518v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@158bad1org.highwire.dtl.DTLVardef@5113eforg.highwire.dtl.DTLVardef@f3b94eorg.highwire.dtl.DTLVardef@10ab479_HPS_FORMAT_FIGEXP M_FIG C_FIG