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.

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.

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.

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

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.

RationaleHypertrophic cardiomyopathy (HCM) is a common inherited cardiac disorder characterized by cardiac hypertrophy, fibrosis, arrhythmias, and sudden cardiac death (SCD). Although current therapies primarily target sarcomere dysfunction, the contribution of epigenetic dysregulation to HCM pathogenesis and its therapeutic potential remain poorly understood. ObjectiveTo determine whether pharmacological inhibition of histone lysine demethylases (KDMs) with JIB-04 can prevent or reverse HCM progression and to identify the underlying epigenetic mechanisms. Methods and ResultsWe evaluated the pan-KDM inhibitor JIB-04 in Myh6R403Q/+ mice carrying the murine equivalent of the pathogenic human MYH7 R403Q mutation. JIB-04 prevented disease progression, reduced cardiac hypertrophy and fibrosis, preserved cardiac function, and completely prevented SCD in cyclosporin A- accelerated HCM. JIB-04 also reversed established disease, produced sustained therapeutic benefits after drug withdrawal, and improved cardiac function in aged mice with spontaneous HCM. Bulk RNA sequencing and ATAC-seq demonstrated partial restoration of disease-associated transcriptional programs and chromatin accessibility. Proteomic analyses identified PHF2 (KDM7C) as a candidate target of JIB-04 in both mouse and human HCM hearts. PHF2 knockdown suppressed hypertrophic, inflammatory, and fibrotic gene expression in cardiomyocytes, macrophages, and fibroblasts, respectively. Human HCM hearts exhibited increased expression of multiple JIB-04-sensitive KDMs, including PHF2. In MYH7 R403Q induced pluripotent stem cell- derived cardiomyocytes, JIB-04 normalized disease-associated gene expression, restored connexin-43 membrane localization, and improved mitochondrial respiration. Although prolonged treatment induced reversible hepatomegaly with hepatic lipid accumulation, co-administration of the antioxidant N-acetylcysteine mitigated liver toxicity while preserving the therapeutic efficacy of JIB-04. ConclusionsPharmacological KDM inhibition prevents and reverses HCM through epigenetic remodeling of disease-associated transcriptional and chromatin programs. These findings identify KDM inhibition as a promising therapeutic strategy for HCM, establish PHF2 as a candidate mediator of disease pathogenesis, and support further development of KDM-targeted therapies.

Environmental temperature strongly influences muscle contractile mechanics and locomotor performance in ectotherms, yet animals routinely develop across a range of temperatures while maintaining effective movement. We tested the hypothesis that developmental temperature induces compensatory changes in the intrinsic mechanical properties of the muscles that power the fast-start escape response in larval zebrafish (Danio rerio). Larvae were reared at 25{degrees}C, 28{degrees}C, or 32{degrees}C, and contractile properties of intact tail myotomal muscles were measured across experimental temperatures. Acute changes in experimental temperature strongly affected twitch kinetics, particularly relaxation rate (Q10 = 2.1), resulting in substantial changes in twitch duration. In contrast, rearing temperature produced adaptive changes that opposed these acute thermal effects. At a common experimental temperature, muscles from cold-reared larvae exhibited faster intrinsic relaxation and greater force production during shortening at a physiologically relevant velocity, whereas warm-reared larvae showed slower relaxation and reduced shortening force. As a result, twitch kinetics were largely normalized when measurements were made at each group's rearing temperature, reducing the apparent thermal sensitivity of relaxation rate (Q10 = 1.1). To identify molecular correlates of these functional adaptations, we performed label-free quantitative LCMS proteomic analysis. Cold rearing increased the abundance of Sarco/Endoplasmic Reticulum Calcium-ATPase (SERCA) proteins, driven primarily by elevated atp2a1 expression, while warm rearing reduced the abundance of the major parvalbumin isoforms pvalb1 and pvalb2. These changes implicate remodeling of intracellular calcium handling as a mechanism underlying thermal compensation of muscle function. Together, our results demonstrate that developmental temperature modifies the intrinsic mechanical properties of larval zebrafish muscle in ways that counteract the direct effects of environmental temperature, thereby preserving the timing and power-generating capacity required for fast-start escape performance.

BACKGROUNDInterleukin-27 (IL-27) is a heterodimeric cytokine that serves as a bifunctional rheostat rather than an inherently pro- or anti-inflammatory signaling protein. However, the specific role of IL-27 in regulating systolic overload-induced cardiac inflammation and heart failure (HF) pathogenesis remains unknown. METHODSWe investigated the effects of genetic IL-27 receptor deficiency (IL-27R knockout), pharmacological IL-27 blockade, and recombinant IL-27 administration on transverse aortic constriction (TAC)-induced HF in mice. RESULTSCardiac IL-27 expression was significantly elevated in both murine and human HF tissues. The global genetic ablation of the IL-27 receptor (IL-27R) significantly suppressed TAC-induced cardiac inflammation, fibrosis, hypertrophy, HF progression, and mortality. Corroborating these protective effects, transcriptomic analysis (RNA-seq) revealed that IL-27R deficiency drastically suppressed pathways driving immune responses and antigen presentation, alongside the significant downregulation of networks governing systemic inflammation, pathogen infection, and extracellular matrix remodeling. Furthermore, pharmacological neutralization of IL-27 effectively attenuated TAC-induced left ventricular dysfunction, chamber dilation, myocardial hypertrophy, fibrosis, and leukocyte infiltration. Conversely, the administration of recombinant mouse IL-27 exacerbated the TAC-induced cardiac accumulation of multiple immune cell subsets, resulting in worsened cardiac fibrosis, cardiomyocyte hypertrophy, and overall HF progression. CONCLUSIONSOur findings demonstrate that IL-27 acts as a critical pathogenic driver of cardiac inflammation and HF development by modulating both cardiac immune cells (predominantly T cells) and non-immune cells, highlighting the IL-27 signaling axis as a promising therapeutic target.

Background: Mitral regurgitation (MR) is a major risk factor for the development of atrial fibrillation (AF), yet the molecular mechanisms linking volume overload to arrhythmogenic remodeling remain poorly understood. Although fibrosis has long been considered the primary substrate for AF, increasing evidence suggests that fibroblast heterogeneity and cell-cell interactions may play important roles in disease progression. Methods: MR was created endovascularly by chordal avulsion in 12 dogs with 6 controls. AF inducibility was assessed by transvenous burst pacing, left atrial volume by echocardiography, and collagen content by Masson trichrome and picrosirius red staining. Single-nucleus RNA sequencing (snRNA-seq) was performed on left atrial posterior wall tissue from control, 4-week, and 6-month MR animals. Fibroblast subpopulations and fibroblast-cardiomyocyte communication were analyzed and markers validated by RNA in situ hybridization in all 18 animals. Results: MR resulted in progressive left atrial dilation, but neither the change in left atrial volume from baseline nor total collagen burden correlated with the inducibility of AF (n=6 each). SnRNA-seq resolved seven major cardiac cell populations and identified four transcriptionally distinct fibroblast populations (NOX4/GRIA4, PCOLCE2, ADRB2/HCN1, PTX3/ICAM1). Fibroblast composition shifted markedly: matrix-associated PCOLCE2 fibroblasts starkly declined by 6 months, whereas inflammatory-associated PTX3/ICAM1 fibroblasts expanded stepwise over time. Cardiomyocyte-to-fibroblast signaling, dominated by PTPRM and LAMA2, was progressively redirected toward PTX3/ICAM1 fibroblasts. RNAscope confirmed a stepwise rise in ICAM1 transcripts and higher ICAM1 in AF-inducible than non-inducible animals. Conclusions: In a canine model of MR, the inducibility of AF was associated with fibroblast state remodeling rather than with atrial dilation or collagen burden. Progressive expansion of inflammatory-associated PTX3/ICAM1 fibroblasts, together with reorganized fibroblast-cardiomyocyte signaling, defines a candidate arrhythmogenic mechanism and therapeutic target in MR.

BackgroundCardiovascular disease risk is higher in men than in women. Although sex differences in aortic wall adaptation following antihypertensive treatment have been reported in acute hypertension models, the response after gradually developing hypertension, which mimics human essential hypertension, remains unclear. This study investigated sex differences in aortic wall adaptation following acute blood pressure reduction after gradually developing hypertension. MethodSeventeen-week-old spontaneously hypertensive rats (SHRs) were assigned to the Hypertensive group or the antihypertensive (Reversal) group (N = 5/sex each). The Reversal group received the antihypertensive drug captopril for 4 weeks to maintain systolic blood pressure below 130 mmHg. Age-matched Wistar Kyoto rats (N = 3/sex) served as normotensive (Normal) group. After the experimental period, arterial wall thickness, circumferential wall stress, smooth muscle cell phenotype, and histological changes were evaluated. ResultsAntihypertensive treatment significantly reduced systolic blood pressure in both sexes. Both male and female SHRs exhibited elevated circumferential wall stress during the gradual development of hypertension. In females, antihypertensive treatment significantly reduced medial thickness compared with the Hypertensive group, whereas males showed no reduction. Circumferential wall stress in female Reversal group did not differ significantly from either the Hypertensive or Normal group, whereas males exhibited a significant reduction in circumferential wall stress compared with the Hypertensive group. Furthermore, the reduced collagen area fraction in the Hypertensive group returned to the normotensive levels only in females following antihypertensive treatment. ConclusionThese findings indicate that vascular remodeling induced by gradually developing hypertension is more effectively reversed by antihypertensive treatment in females than in males.

Mechanical homeostasis plays a central role in promoting and preserving optimal structure and function in the adult aorta. Although pathogenic variants can compromise homeostatic processes, it appears that intramural cells yet attempt to compensate for some genetically induced changes. In particular, lysyl oxidase is higher in the adult Marfan aorta compared with the age-matched control aorta. Here, we block lysyl oxidase in adult Fbn1C1041G/+ Marfan syndrome mice after stimulating aortic disease progression via induced hypertension. Whereas hypertension alone increases aortic dilatation, concurrent blocking of lysyl oxidase results in a dramatic increase in disease severity, driving an otherwise mild aortic phenotype in adult male Fbn1C1041G/+ Marfan mice to aneurysmal dilatations as well as dissection and rupture, with frequent premature death. Deposition and cross-linking of fibrillar collagens, among other extracellular matrix constituents, can represent a protective compensation against severe disease in the Marfan aorta. The present study emphasizes the need clinically to avoid compromising new collagen deposition and suggests that strategies to augment collagen cross-linking could be beneficial.

Background and aimsAtherosclerotic plaques form preferentially at vascular sites exposed to disturbed blood flow, yet the protein changes underlying this site-specific plaque development remain unclear. Mouse models are widely used to study atherosclerosis but yield only limited amounts of tissue, previously restricting proteomic studies. However, recent advances in mass spectrometry now enable proteomic profiling of very small tissue samples. We aimed to utilise this to uncover site-specific protein changes in aortic regions prone or resistant to plaque formation. MethodsAortic arches from apolipoprotein E-deficient (ApoE-/-) mice fed a Western diet (WD) for 16 weeks were dissected into plaques from the major branches and inner curvature and visibly healthy regions. Proteins were extracted, enzymatically digested, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). ResultsMore than 4000 proteins were identified per sample despite their small size (< 1 mg tissue). Principal component analysis showed clustering by both disease status and anatomical location within the aortic arch, indicating distinct proteomes. Proteins known to drive atherosclerosis - including vascular cell adhesion molecule 1 (Vcam1), apolipoprotein B (Apob), lipoprotein lipase (Lpl), and galectin 3 (Lgals3) - were most abundant in advanced plaques and decreased progressively across anatomical regions, reaching their lowest levels in healthy regions furthest from the plaques. Enrichment analysis highlighted pathways related to the extracellular matrix, immune system, hemostasis, and lipoprotein transport as central to disease progression. ConclusionsThis study demonstrates the feasibility of region-resolved proteomics in individual murine aortas and provide new molecular insights into the site-specific nature of atherosclerotic plaque development.

Background: Pericoronary adipose tissue (PCAT) attenuation on coronary computed tomography angiography (CTCA) has emerged as a novel non-invasive biomarker of coronary inflammation and cardiovascular risk. The degree to which PCAT reflects local or systemic inflammation remains uncertain. We hypothesized that the presence, location and extent of PCAT would be associated with transcoronary or transcardiac cytokine gradient. Methods: This prospective cohort study involved 31 adults with stable coronary artery disease who underwent clinically indicated CTCA within 90 days of invasive coronary angiography. Patients with acute coronary syndromes or unstable angina were excluded. Blood samples were obtained from peripheral vein, coronary sinus, aortic root, and right coronary artery at time of cardiac catheterization. Plasma interleukin-6 (IL-6) and interleukin-1{beta} (IL-1{beta}) concentrations from each site were used to calculate transcardiac and transcoronary cytokine gradients. PCAT attenuation was measured using semi-automatic software by readers blinded to clinical and biochemical endpoints. Results: Participants were predominantly male (76%), aged 66.6 {+/-} 9.4 years, with a high prevalence of hypercholesterolemia (76%), hypertension (73%), and diabetes (36%). Mean PCAT attenuation was -74.8 HU (RCA), -70.3 HU (LCx), and -73.6 HU (LAD). Regression analyses showed no significant associations between PCAT attenuation and IL-6 gradients across any coronary territory (all p >0.40; R2 {approx} 0), including in plaque-free subgroup analyses. IL-1{beta} was below the assay detection limit in 81% of participants; analyses using non-parametric testing and logistic no association with PCAT attenuation. RCA (OR 0.96, 95% CI 0.88-1.06, p=0.46), LCx (OR 1.00, 95% CI 0.91-1.09, p=0.94), LAD (OR 0.99, 95% CI 0.90-1.08, p=0.81). Conclusion: In a cohort with predominantly stable coronary disease, PCAT attenuation was not associated with intracardiac or intracoronary IL-6 or IL-1{beta} gradients, including in plaque-free vessels. These findings suggest that PCAT attenuation may not reflect active cytokine-mediated coronary inflammation in stable disease.

Background: DNA methylation (DNAm) may capture cumulative genetic, environmental, and lifestyle influences on cardiovascular health. Composite DNAm score based on the American Heart Association Life's Essential 8 (LE8) framework have been linked to clinical events, but their association with early vascular changes and intergenerational effects is unclear. Methods: We studied up to 1432 participants from the multigenerational Young Finns Study (YFS-3G), including parents (G0) and adult offspring (G1). DNAm was measured using Illumina EPIC arrays in 2011 and/or 2018, and carotid intima--media thickness (cIMT) was assessed in 2018. The LE8 DNAm score was calculated as a weighted sum of methylation levels. Associations with cIMT were evaluated in intergenerational, prospective, and cross-sectional settings, adjusting for demographic, technical, and biological covariates and conventional cardiovascular risk factors. Results: Higher parental LE8 DNAm score was associated with lower offspring cIMT ({beta} = -0.022 mm/SD; p-value = 0.02), although the association was attenuated after adjustment for parental cardiovascular risk factors. In G1, a higher baseline DNAm score was associated with lower cIMT measured seven years later ({beta} = -0.030 mm/SD; p-value = 1.1 x 10-5). This association remained significant after adjustment for follow-up cardiovascular risk factors (p-value=0.009) but not after additional adjustment for prior cIMT. Cross-sectionally, higher DNAm score was associated with lower cIMT in both generations, with attenuation after risk factor adjustment in G1 but not G0. Associations with carotid plaque were not significant. Genes associated with the DNAm score were enriched for immune and inflammatory pathways. Conclusions: An LE8-derived DNAm score was associated with lower cIMT across the life course and, to a lesser extent, across generations. These findings suggest that blood DNAm reflects cumulative cardiovascular health and vascular burden and may complement conventional cardiovascular risk assessment.

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

Background: Electrocardiographic (ECG) markers of atrial cardiopathy (AC) are associated with stroke mortality, but whether this association is modified by blood pressure (BP) is unknown. Methods: We analyzed 7,191 adults free of cardiovascular disease from the Third National Health and Nutrition Examination Survey who underwent baseline ECG. AC was defined by three ECG markers: prolonged P-wave duration 120 ms), abnormal P-wave axis (<0{degrees} or >75{degrees}), and deep terminal negativity of the P wave in V1 (<100 V). AC burden (per additional AC marker) and AC presence (1 vs. 0 markers) were examined in relation to stroke mortality using Cox proportional hazards models. Participants were stratified by BP as normal/elevated (<130/80 mmHg), stage 1-2 hypertension (130-159/80-99 mmHg), or severe hypertension (160/100 mmHg). Interaction by BP category was assessed. Results: During a median follow-up of 13.8 years, 183 stroke deaths occurred. In multivariable adjusted model, AC burden was associated with a 41% higher risk of stroke mortality (HR (95%CI): 1.41 (1.13-1.77)). This association was significantly modified by BP (interaction P=0.003). The HRs (95% CIs) per additional AC marker were 0.88 (0.52-1.49), 1.39 (1.03-1.88), and 2.94 (1.82-4.75) for normal/elevated BP, stage 1-2 hypertension, and severe hypertension, respectively. A similar pattern of associations was observed for AC presence, although the interaction with BP was not statistically significant. Conclusions: ECG-defined AC burden was independently associated with stroke mortality, with substantially stronger associations among individuals with severe hypertension, supporting BP as an important modifier of its prognostic significance.

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