Exercise training is recommended for individuals with hypertension because it has been shown to lower blood pressure and reverse left ventricular concentric remodelling and mass. However, it is unclear how hypertensive individuals respond in comparison to normotensive individuals and to what extent medical treatment affects the outcome of training. Our aim was to assess the effect of a 6 week high-intensity interval training (HIIT) intervention on cardiac adaptations in subjects with treated or untreated essential hypertension compared with normotensive control subjects. Cardiac function was evaluated by echocardiography in 11 medicated hypertensive men, who adhered to treatment during the intervention but refrained from medication for 4 days prior to and during measurements (MED-HYP), in 9 untreated hypertensive men (HYP) and in 10 age-matched normotensive men before and after HIIT. At baseline, HYP and MED-HYP had lower mitral valve E/A ratio (MED-HYP, 0.86 ± 0.2; HYP, 0.99 ± 0.1; P < 0.05) compared with normotensive men (1.37 ± 0.4). The HIIT stroke volume in normotensive men only improved maximum oxygen uptake (change, 178 ± 239 mL O2/min). The E/e' ratio (echocardiographic risk marker of cardiac events) increased (P < 0.05) with HIIT in MED-HYP, with no change in normotensive men and HYP. Men with treated and untreated essential hypertension display diminished cardiac adaptation and less improvement in cardiopulmonary fitness in response to HIIT compared with normotensive counterparts. Additionally, MED-HYP increased E/e' with HIIT, potentially raising the risk of primary cardiac events. Therefore, further research is required to assess the interactive effects of exercise training and antihypertensive treatment.

Background Cardiovascular disease is the main cause of mortality among women. However, the menstrual cycle and hormonal contraception are often overlooked in research. This study investigates whether concentrations of common cardiovascular biomarkers (natriuretic peptides, adrenomedullin, and copeptin) change during a menstrual cycle and with hormonal contraception use. Methods Concentrations of cardiovascular biomarkers in 2 prospective cohorts were measured. In The Menstrual Cycle Study, blood samples were collected throughout a menstrual cycle, and in The Contraception Study, blood samples were collected from women using different contraceptive methods. Results Blood samples from 19 women (The Menstrual Cycle study) and 638 women (The Contraception Study) were analyzed. Concentrations of mid-regional (MR)-pro-atrial natriuretic peptide (proANP) are highest during the early follicular phase (Delta %: median: 111.5%, IQR: 100.6-115.9%) and lowest during the mid-luteal phase (median: 88.0%, IQR: 80.4-103.2%) (P =.02). When compared to women with natural cycles, median concentrations of MR-proANP were reduced by 19.3% in those using combined oral contraceptives (COC)/vaginal contraceptive ring and by 18.2% in progestin-users. Additionally, copeptin concentrations were reduced by 11.9% in COC/vaginal contraceptive ring-users, while MR-proadrenomedullin (proADM) concentrations were reduced by 15.4% in COC/vaginal contraceptive ring-users and 11.5% in progestin-users compared to non-users. Conclusions MR-proANP concentrations vary across the menstrual cycle with the highest concentrations during the early follicular phase. Furthermore, concentrations of MR-proANP, copeptin, and MR-proADM are affected by hormonal contraception. Our findings underscore the necessity to consider menstrual cycle phases and use of hormonal contraception in clinical assessment of premenopausal women when using cardiovascular biomarkers.

Although CNP (C‐type natriuretic peptide) was first identified in brain tissue, its systemic expression and release is mainly located to the endothelial lining of the vasculature. As such, CNP should not be viewed as a classic blood‐borne hormone but rather as a local factor that supports the integrity of vascular function. Albeit CNP is structurally related to both ANP and BNP (A‐ and B‐type natriuretic peptide), the main signaling pathway is through a specific membrane‐bound receptor without crosstalk from endocrine ANP and BNP. CNP expression and CNP‐mediated effects can thus be uniquely targeted without interference from the related cardiac NPs. In this perspective, we present the CNP system as a largely overlooked target for potential intervention in cardiometabolic disease, hypertension, and diabetes before onset of complications and overt heart failure. To fully unleash this potential, with this perspective, we hope to fuel renewed interest with a focus on use of human in vitro and ex vivo systems as well as experimental medical approaches and human cohort data.

Background: Preeclampsia, a pregnancy complication marked by hypertension after 20 weeks of gestation, arises from placental factors that impair maternal vascular function. C-type natriuretic peptide (CNP), known for its vasodilatory role, may help counter preeclampsia-related vascular dysfunction. This study aimed to explore the effect of CNP on preeclampsia risk using the Mendelian randomization (MR) framework. Methods: Genetic instrumental variables that mimic the effects of CNP signaling (through natriuretic peptide receptor 2 [NPR2] activation or reduced NPR3-mediated clearance) were identified in the genes encoding the two receptors. This discovery emerged from a multiancestry genome-wide association study (GWAS) involving over 5 million individuals. Female-specific genetic association estimates were obtained from individual-level data comprising 198,402 female participants in the UK Biobank. Two-sample MR analyses were conducted to investigate the effects of NPR2 activation and NPR3 function on preeclampsia, utilizing the largest publicly available GWAS on preeclampsia, which included 296,824 female participants. Results: Genetically proxied reduced NPR3 function was associated with a lower risk of preeclampsia (odds ratio (OR): 0.46, 95% confidence interval 0.30-0.69). In contrast, genetically proxied increased NPR2 activation lacked significant association, likely due to underpowered genetic instruments. Sensitivity analyses indicated robust findings with minimal pleiotropy, meaning the genetic variants used primarily influenced preeclampsia through the intended biological pathway rather than affecting multiple unrelated traits. Conclusion: This study employed the MR paradigm to provide genetic evidence supporting the protective effects of CNP (through reduced NPR3 function) on the risk of preeclampsia. However, it is important to gather additional evidence from other sources before moving forward with clinical development efforts to explore CNP as a potential treatment for preeclampsia.

C-type natriuretic peptide (CNP) has therapeutic potential in heart failure with preserved ejection fraction (HFpEF) due to its broad range of beneficial effects on cardiovascular structure and function. This study presents the design of a CNP analogue (65) for once-weekly subcutaneous administration. The design of 65 incorporated five strategic substitutions and an engineered fatty acid protractor to enhance the chemical stability, improve the pharmacokinetics, and lower the isoelectric point (pI). Low pI was found to be essential for minimizing injection site reactions and improving subcutaneous bioavailability. 65 demonstrated a promising pharmacokinetic profile for once-weekly treatment and an improved bioavailability compared to high pI CNP analogs. Furthermore, 65 was engineered for solubility at pH 6.5 to enable stability in liquid formulation. In vivo assessments supported the therapeutic potential in HFpEF of fatty acid-derivatized low pI CNP analogues. 65 is currently under clinical investigation in Phase 1.

Genome-wide association studies (GWASs) have linked numerous genetic loci active in vascular cells to coronary artery disease (CAD), implicating smooth muscle cells (SMCs) and SMC-derived mesenchymal cells as potential mediators. We combined CAD GWAS with single-cell RNA sequencing (scRNA-seq) from human atherosclerotic plaques to identify 20 risk genes with putative action in SMCs, and then performed in vitro perturbation experiments in SMCs driven toward plaque-relevant phenotypes. Although the genes encode diverse proteins, their perturbations converged on shared transcriptional programs regulating contractile machinery, cell-cycle progression, nuclear factor κB (NF-κB), and type I interferon signaling. Integrating GWAS effect-direction with cholesterol- and stretch-responsive gene modules suggest that cholesterol-induced signaling promotes pro-atherogenic SMC states and is differentially modulated by risk versus protective variants. These results delineate polygenic regulation of SMC disease mechanisms and show that GWAS effect-directionality can help prioritize cellular pathways for follow-up functional studies across genes and cell types.

Heart failure encompasses a diverse group of cardiomyopathies, including myocardial infarction, hypertrophic, dilated, and arrhythmogenic forms, each defined by distinct etiologies. By integrating single-nuclei transcriptomic data from human heart tissue across these conditions, we constructed a unified atlas containing 1.8 million nuclei from 195 individuals. The atlas reveals disease-specific cellular clusters, transcriptional changes and altered ligand-receptor interactions. We found cell states specific to the ischemic zone of myocardial infarction, explored the influence of cytokines on fibroblast cell-states, and identified etiological pathways in different cell types. The integration of summary statistics of 52 genome-wide association studies with atlas-wide gene expression highlighted genetically associated pathways involving metabolic dysregulation and ion channel dysfunction. Implementation of an AI agent led to the identification of ZLN005, a small molecule that boosts mitochondrial biogenesis via PGC-1α, whose cardioprotective effect we validated experimentally, underscoring the utility of the atlas in early therapeutic target discovery for heart failure.

Intro: Vascular smooth muscle cell (VSMC) proliferation following endovascular interventions leads to restenosis, treated with drug-eluting stents. However, current drugs completely abolish vascular healing, cause thrombosis and clinical events. PCSK6 is a key protease in vascular remodeling and VSMCs activation upon vessel injury, via regulation of MMP2/14 activity. Here, we investigate whether local PCSK6 inhibition reduces VSMC proliferation and intimal hyperplasia. Methods: PCSK6 peptide inhibitor was tested in vitro on VSMCs migration, proliferation, apoptosis in comparison to rapamycin and paclitaxel. Internalization of a FITC labelled inhibitor was studied by flow cytometry and immunofluorescence. In vivo , PCSK6 inhibitor was administered at 3mg/kg IP using cationic microbubbles, followed by ultrasound-mediated local delivery in mice developing intimal hyperplasia after carotid ligation (n=8 vs 11). Carotid ligation in Tagln Cre+ /Pcsk6 fl/fl mice investigated the effect of PCSK6 specific ablation in VSMCs on intimal hyperplasia (n=7 vs 9). Carotids were collected from these and constitutive Pcsk6 -/- mice vs. controls for transcriptomic and histological analyses. Results: In vitro , PCSK6 inhibitor was internalized and inhibited both VSMC migration and proliferation (p<0.0001), reducing the expression of PCSK6, MMP2 and GDF15 . In comparison, paclitaxel showed severe VSMC toxicity, whereas rapamycin inhibited more strongly VSMC migration and proliferation than the PCSK6 inhibitor. In vivo, the inhibitor reduced intimal hyperplasia (p=0.026), again, by repressing PCSK6 and MMP2 expression in treated vs. control mice, with a favorable safety profile. Likewise, conditional VSMC Pcsk6 knockouts showed less intimal hyperplasia (p=0.053). In carotids from Pcsk6 -/- mice, cytoskeletal (Synm) and peptidase (Pgpep1l, Klk1) genes were strongly downregulated, showing its importance for vascular matrix composition. Conclusion: Our proof-of-concept translational study shows that local application of a PCSK6 inhibitor is a viable method for specifically modulating VSMC activation in intimal hyperplasia following vascular injury

The significant morbidity and premature mortality of type 2 diabetes mellitus (T2DM) is largely associated with its cardiovascular consequences. Focus has long been on the arterial atheromatosis of DM giving rise to early stroke and myocardial infarctions, whereas less attention has been given to its non-ischemic cardiovascular consequences. Irrespective of ischemic changes, T2DM is associated with heart failure (HF) most commonly with preserved ejection fraction (HFpEF). Largely due to increasing population ages, hypertension, obesity and T2DM, HFpEF is becoming the most prevalent form of heart failure. Unfortunately, randomized controlled trials of HFpEF have largely been futile, and it now seems logical to address the important different phenotypes of HFpEF to understand their underlying pathophysiology. In the early phases, HFpEF is associated with a significantly impaired ability to increase cardiac output with exercise. The lowered cardiac output with exercise results from both cardiac and peripheral causes. T2DM is associated with left ventricular (LV) diastolic dysfunction based on LV hypertrophy with myocardial disperse fibrosis and significantly impaired ability for myocardial blood flow increments with exercise. T2DM is also associated with impaired ability for skeletal muscle vasodilation during exercise, and as is the case in the myocardium, such changes may be related to vascular rarefaction. The present review discusses the underlying phenotypical changes of the heart and peripheral vascular system and their importance for an adequate increase in cardiac output. Since many of the described cardiovascular changes with T2DM must be considered difficult to change if fully developed, it is suggested that patients with T2DM are early evaluated with respect to their cardiovascular compromise.

Abstract Background Drug target Mendelian randomization describes the use of genetic variants as instrumental variables for studying the effects of pharmacological agents. The paradigm can be used to inform on all aspects of drug development and has become increasingly popular over the last decade, particularly given the time- and cost-efficiency with which it can be performed even before commencing clinical studies. Main body In this review, we describe the recent emergence of drug target Mendelian randomization, its common pitfalls, how best to address them, as well as potential future directions. Throughout, we offer advice based on our experiences on how to approach these types of studies, which we hope will be useful for both practitioners and those translating the findings from such work. Conclusions Drug target Mendelian randomization is nuanced and requires a combination of biological, statistical, genetic, epidemiological, clinical, and pharmaceutical expertise to be utilized to its full potential. Unfortunately, these skillsets are relatively infrequently combined in any given study.

Atherosclerosis is a pervasive contributor to ischemic heart disease and stroke. Despite the advance of lipid-lowering therapies and anti-hypertensive agents, the residual risk of an atherosclerotic event remains high, and developing therapeutic strategies has proven challenging. This is due to the complexity of atherosclerosis with a spatial interplay of multiple cell types within the vascular wall. In this study, we generated an integrative high-resolution map of human atherosclerotic plaques combining single-cell RNA sequencing from multiple studies and spatial transcriptomics data from 12 human specimens with different stages of atherosclerosis. Here we show cell-type-specific and atherosclerosis-specific expression changes and spatially constrained alterations in cell–cell communication. We highlight the possible recruitment of lymphocytes via ACKR1 endothelial cells of the vasa vasorum, the migration of vascular smooth muscle cells toward the lumen by transforming into fibromyocytes and cell–cell communication in the plaque region, indicating an intricate cellular interplay within the adventitia and the subendothelial space in human atherosclerosis. Bleckwehl et al. present a spatial transcriptomic map of atherosclerotic plaques across disease stages, revealing cellular recruitment and migration patterns and intercellular communication dynamics as a valuable resource for future research.

Measurement of natriuretic peptides (NPs) has proven its clinical value as biomarker, especially in the context of heart failure (HF). In contrast, a state of partial NP deficiency appears integral to several conditions in which lower NP concentrations in plasma presage overt cardiometabolic disease. Here, obesity and type 2 diabetes have attracted considerable attention. Other factors-including age, sex, race, genetics, and diurnal regulation-affect the NP "armory" and may leave some individuals more prone to development of cardiovascular disease. The molecular maturation of NPs has also proven complex, with highly variable O-glycosylation within the biosynthetic precursors. The relevance of this regulatory step in post-translational propeptide maturation has recently become recognized in biomarker measurement/interpretation and cardiovascular pathophysiology. An important proportion of people appear to have reduced effective net NP bioactivity in terms of receptor activation and physiological effects. The state of NP deficiency both entails a potential for further biomarker development and could also offer novel pharmacological possibilities. Alleviating the state of NP deficiency before development of overt cardiometabolic disease in selected patients could be a future path for improving precision medicine.

The role of C‐type natriuretic peptide (CNP) in the regulation of cardiac function in humans remains to be established as previous investigations have been confined to animal model systems. Here, we used well‐characterized engineered cardiac tissues (ECTs) generated from human stem cell‐derived cardiomyocytes and fibroblasts to study the acute effects of CNP on contractility. Application of CNP elicited a positive inotropic response as evidenced by increases in maximum twitch amplitude, maximum contraction slope and maximum calcium amplitude. This inotropic response was accompanied by a positive lusitropic response as demonstrated by reductions in time from peak contraction to 90% of relaxation and time from peak calcium transient to 90% of decay that paralleled increases in maximum contraction decay slope and maximum calcium decay slope. To establish translatability, CNP‐induced changes in contractility were also assessed in rat ex vivo (isolated heart) and in vivo models. Here, the effects on force kinetics observed in ECTs mirrored those observed in both the ex vivo and in vivo model systems, whereas the increase in maximal force generation with CNP application was only detected in ECTs. In conclusion, CNP induces a positive inotropic and lusitropic response in ECTs, thus supporting an important role for CNP in the regulation of human cardiac function. The high degree of translatability between ECTs, ex vivo and in vivo models further supports a regulatory role for CNP and expands the current understanding of the translational value of human ECTs.

BACKGROUND:C-type natriuretic peptide (CNP) is a known target for promoting growth and has been implicated as a therapeutic opportunity for the prevention and treatment of cardiovascular disease (CVD). This study aimed to explore the effect of CNP on CVD risk using the Mendelian randomization (MR) framework. METHODS:Instrumental variables mimicking the effects of pharmacological intervention on CNP were identified as uncorrelated genetic variants located in the genes coding for its primary receptors, natriuretic peptide receptors-2 and 3 (NPR2 and NPR3), that associated with height. We performed MR and colocalization analyses to investigate the effects of NPR2 signalling and NPR3 function on CVD outcomes and risk factors. MR estimates were compared to those obtained when considering height variants from throughout the genome. RESULTS:Genetically-proxied reduced NPR3 function was associated with a lower risk of CVD, with odds ratio (OR) 0.74 per standard deviation (SD) higher NPR3-predicted height, and 95% confidence interval (95% CI) 0.64-0.86. This effect was greater in magnitude than observed when considering height variants from throughout the genome. For CVD subtypes, similar MR associations for NPR3-predicted height were observed when considering the outcomes of coronary artery disease (0.75, 95% CI 0.60-0.92), stroke (0.69, 95% CI 0.50-0.95) and heart failure (0.77, 95% CI 0.58-1.02). Consideration of CVD risk factors identified systolic blood pressure (SBP) as a potential mediator of the NPR3-related CVD risk lowering. For stroke, we found that the MR estimate for NPR3 was greater in magnitude than could be explained by a genetically predicted SBP effect alone. Colocalization results largely supported the MR findings, with no evidence of results being driven by effects due to variants in linkage disequilibrium. There was no MR evidence supporting effects of NPR2 on CVD risk, although this null finding could be attributable to fewer genetic variants being identified to instrument this target. CONCLUSIONS:This genetic analysis supports the cardioprotective effects of pharmacologically inhibiting NPR3 receptor function, which is only partly mediated by an effect on blood pressure. There was unlikely sufficient statistical power to investigate the cardioprotective effects of NPR2 signalling.

Introduction: Genome-wide association studies identified 100s of loci that are associated with the risk of common cardio-metabolic diseases (T2D and CVD). Here we functionally characterise 99 candidate genes for a role in relevant traits using zebrafish larvae. Methods: Fifty-six T2D and 46 CVD candidate genes (three overlap) were functionally characterised by targeting their zebrafish orthologs using CRISPR/Cas9. On day 9-11 post-fertilisation, we acquired optical sections of the pancreatic islet, liver and vasculature using semi-automated fluorescence microscopy, in 16,141 CRISPR/Cas9-edited zebrafish larvae, followed by image analysis using deep learning. Results: Perturbing all nine T2D genes with at least moderate prior evidence affects ≥1 T2D trait (i.e., beta cell mass, beta cell insulin expression, liver fat, and/or glucose content), while no consequence is observed for 22 genes without prior evidence of a role in T2D. For 12 genes with at most modest prior evidence, perturbation also affects at least 1 T2D trait. Perturbation of 23 genes prioritised for a role in CVD affects at least one vascular trait, while no consequence is observed for 15 non-prioritised genes. Effects of mutations in mice and/or humans are published for 14 of 23 genes, 78% of which show directionally consistent effects across zebrafish larvae and mammals. Of the 15 genes influencing liver fat upon CRISPR/Cas9 editing, mutations in 12 previously showed an effect on liver fat in mice and/or humans, with 75% congruence in direction of effect across species. Conclusion: Systematically characterising candidate genes for a role in image-based T2D, liver fat and CVD traits in CRISPR/Cas9-edited zebrafish larvae shows highly congruent results with mice and humans, and prioritises promising genes for further in-depth characterisation. Disclosure E. Mujica: None. H. Sun: None. M. Nyberg: Employee; Novo Nordisk A/S. D. Djordjevic: Employee; Novo Nordisk. S. Vienberg: Employee; Novo Nordisk A/S. A. L. Gloyn: Other Relationship; Genentech, Inc., Roche Pharmaceuticals. A. Larsson: None. A. Allalou: None. M. Den hoed: None. H. Zhang: None. A. Emmanouilidou: None. E. Mazzaferro: None. N. Cook: None. C. Metzendorf: None. G. Alavioon: None. M. Bandaru: None. A. Rottner: Employee; AstraZeneca. Funding Swedish Research Council (201901417); Swedish Heart and Lung Foundation (20200602, 20200781)

Introduction: Genome-wide association studies identified 100s of loci that are associated with the risk of common cardio-metabolic diseases (T2D and CVD). Here we functionally characterise 99 candidate genes for a role in relevant traits using zebrafish larvae. Methods: Fifty-six T2D and 46 CVD candidate genes (three overlap) were functionally characterised by targeting their zebrafish orthologs using CRISPR/Cas9. On day 9-11 post-fertilisation, we acquired optical sections of the pancreatic islet, liver and vasculature using semi-automated fluorescence microscopy, in 16,141 CRISPR/Cas9-edited zebrafish larvae, followed by image analysis using deep learning. Results: Perturbing all nine T2D genes with at least moderate prior evidence affects ≥1 T2D trait (i.e., beta cell mass, beta cell insulin expression, liver fat, and/or glucose content), while no consequence is observed for 22 genes without prior evidence of a role in T2D. For 12 genes with at most modest prior evidence, perturbation also affects at least 1 T2D trait. Perturbation of 23 genes prioritised for a role in CVD affects at least one vascular trait, while no consequence is observed for 15 non-prioritised genes. Effects of mutations in mice and/or humans are published for 14 of 23 genes, 78% of which show directionally consistent effects across zebrafish larvae and mammals. Of the 15 genes influencing liver fat upon CRISPR/Cas9 editing, mutations in 12 previously showed an effect on liver fat in mice and/or humans, with 75% congruence in direction of effect across species. Conclusion: Systematically characterising candidate genes for a role in image-based T2D, liver fat and CVD traits in CRISPR/Cas9-edited zebrafish larvae shows highly congruent results with mice and humans, and prioritises promising genes for further in-depth characterisation. Disclosure E. Mujica: None. H. Sun: None. M. Nyberg: Employee; Novo Nordisk A/S. D. Djordjevic: Employee; Novo Nordisk. S. Vienberg: Employee; Novo Nordisk A/S. A. L. Gloyn: Other Relationship; Genentech, Inc., Roche Pharmaceuticals. A. Larsson: None. A. Allalou: None. M. Den hoed: None. H. Zhang: None. A. Emmanouilidou: None. E. Mazzaferro: None. N. Cook: None. C. Metzendorf: None. G. Alavioon: None. M. Bandaru: None. A. Rottner: Employee; AstraZeneca. Funding Swedish Research Council (201901417); Swedish Heart and Lung Foundation (20200602, 20200781)