BACKGROUND:Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca (Ca) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts. METHODS:Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo. RESULTS:In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca transient amplitude showed a differential response to β-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. CONCLUSIONS:Exercise disproportionately affects Ca homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular β-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable β-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy-related arrhythmias have a common mechanism.

BACKGROUND:Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methylcytosine dioxygenase TET2 acting as a master regulator of smooth muscle cell phenotype. The histone acetyl-transferases p300 and CREB-binding protein (CBP) are highly homologous and often considered to be interchangeable, and their roles in smooth muscle cell phenotypic regulation are not known. METHODS:We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (inducible knockout [iKO]; or ), and in samples of human intimal hyperplasia. RESULTS:P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in vessel remodeling after vascular injury. Medial p300 expression and activity were repressed by injury, but CBP and histone acetylation were induced in neointima. Knockdown experiments revealed opposing effects of p300 and CBP in the VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300 mice exhibited severe intimal hyperplasia after arterial injury compared with controls, whereas CBP mice were entirely protected. In normal aorta, p300 reduced, but CBP enhanced, contractile protein expression and contractility compared with controls. Mechanistically, we found that these histone acetyl-transferases oppositely regulate histone acetylation, DNA hydroxymethylation, and PolII (RNA polymerase II) binding to promoters of differentiation-specific contractile genes. Our data indicate that p300 and TET2 function together, because p300 was required for TET2-dependent hydroxymethylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 coimmunoprecipitated with p300, and this interaction was enhanced by rapamycin but repressed by platelet-derived growth factor (PDGF) treatment, with p300 promoting TET2 protein stability. CBP did not associate with TET2, but instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile protein promoters. Furthermore, CBP inhibited TET2 mRNA levels. Immunostaining of cardiac allograft vasculopathy samples revealed that p300 expression is repressed but CBP is induced in human intimal hyperplasia. CONCLUSIONS:This work reveals that p300 and CBP serve nonredundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific histone acetyl-transferases may hold therapeutic promise for cardiovascular diseases.

BACKGROUND:Dapagliflozin improved heart failure and kidney outcomes in patients with type 2 diabetes (T2DM) with or at high risk for atherosclerotic cardiovascular disease in the DECLARE-TIMI 58 trial (Dapagliflozin Effect on Cardiovascular Events - Thrombolysis in Myocardial Infarction 58). Here, the aim was to analyze the efficacy and safety of dapagliflozin stratified according to baseline systolic blood pressure (SBP). METHODS:The DECLARE-TIMI 58 trial randomly assigned patients with T2DM and either previous atherosclerotic cardiovascular disease or atherosclerotic cardiovascular disease risk factors to dapagliflozin or placebo. Patients were categorized by baseline SBP levels: <120, 120 to 129, 130 to 139, 140 to 159, and ≥160 mm Hg (normal, elevated, stage 1, stage 2, and severe hypertension, respectively). Efficacy outcomes of interest were hospitalization for heart failure and a renal-specific composite outcome (sustained decrease in estimated glomerular filtration rate by 40%, progression to end-stage renal disease, or renal death). Safety outcomes included symptoms of volume depletion, lower extremity amputations, and acute kidney injury. RESULTS:The trial comprised 17 160 patients; mean age, 64.0±6.8 years; 37.4% women; median duration of T2DM, 11 years; 40.6% with prevalent cardiovascular disease. Overall, dapagliflozin reduced SBP by 2.4 mm Hg (95% CI, 1.9-2.9; <0.0001) compared with placebo at 48 months. The beneficial effects of dapagliflozin on hospitalization for heart failure and renal outcomes were consistent across all baseline SBP categories, with no evidence of modification of treatment effect (=0.28 and 0.52, respectively). Among normotensive patients, the hazard ratios were 0.66 (95% CI, 0.42-1.05) and 0.39 (95% CI, 0.19-0.78), respectively, for hospitalization for heart failure and the renal-specific outcome. Events of volume depletion, amputation, and acute kidney injury did not differ with dapagliflozin overall or within any baseline SBP group. CONCLUSIONS:In patients with T2DM with or at high atherosclerotic cardiovascular disease risk, dapagliflozin reduced risk for hospitalization for heart failure and renal outcomes regardless of baseline SBP, with no difference in adverse events of interest at any level of baseline SBP. These results indicate that dapagliflozin provides cardiorenal benefits in patients with T2DM at high atherosclerotic cardiovascular disease risk independent of baseline blood pressure. REGISTRATION:URL: https://www. CLINICALTRIALS:gov; Unique identifier: NCT01730534.

BACKGROUND:Timely diagnosis of structural heart disease improves patient outcomes, yet many remain underdiagnosed. While population screening with echocardiography is impractical, ECG-based prediction models can help target high-risk patients. We developed a novel ECG-based machine learning approach to predict multiple structural heart conditions, hypothesizing that a composite model would yield higher prevalence and positive predictive values to facilitate meaningful recommendations for echocardiography. METHODS:Using 2 232 130 ECGs linked to electronic health records and echocardiography reports from 484 765 adults between 1984 to 2021, we trained machine learning models to predict the presence or absence of any of 7 echocardiography-confirmed diseases within 1 year. This composite label included the following: moderate or severe valvular disease (aortic/mitral stenosis or regurgitation, tricuspid regurgitation), reduced ejection fraction <50%, or interventricular septal thickness >15 mm. We tested various combinations of input features (demographics, laboratory values, structured ECG data, ECG traces) and evaluated model performance using 5-fold cross-validation, multisite validation trained on 1 site and tested on 10 independent sites, and simulated retrospective deployment trained on pre-2010 data and deployed in 2010. RESULTS:Our composite rECHOmmend model used age, sex, and ECG traces and had a 0.91 area under the receiver operating characteristic curve and a 42% positive predictive value at 90% sensitivity, with a composite label prevalence of 17.9%. Individual disease models had area under the receiver operating characteristic curves from 0.86 to 0.93 and lower positive predictive values from 1% to 31%. Area under the receiver operating characteristic curves for models using different input features ranged from 0.80 to 0.93, increasing with additional features. Multisite validation showed similar results to cross-validation, with an aggregate area under the receiver operating characteristic curve of 0.91 across our independent test set of 10 clinical sites after training on a separate site. Our simulated retrospective deployment showed that for ECGs acquired in patients without preexisting structural heart disease in the year 2010, 11% were classified as high risk and 41% (4.5% of total patients) developed true echocardiography-confirmed disease within 1 year. CONCLUSIONS:An ECG-based machine learning model using a composite end point can identify a high-risk population for having undiagnosed, clinically significant structural heart disease while outperforming single-disease models and improving practical utility with higher positive predictive values. This approach can facilitate targeted screening with echocardiography to improve underdiagnosis of structural heart disease.

BACKGROUND:There are good data to support using a single high-sensitivity cardiac troponin T (hs-cTnT) below the limit of detection of 5 ng/L to exclude acute myocardial infarction. Per the US Food and Drug Administration, hs-cTnT can only report to the limit of quantitation of 6 ng/L, a threshold for which there are limited data. Our goal was to determine whether a single hs-cTnT below the limit of quantitation of 6 ng/L is a safe strategy to identify patients at low risk for acute myocardial injury and infarction. METHODS:The efficacy (proportion identified as low risk based on baseline hs-cTnT<6 ng/L) of identifying low-risk patients was examined in a multicenter (n=22 sites) US cohort study of emergency department patients undergoing at least 1 hs-cTnT (CV Data Mart Biomarker cohort). We then determined the performance of a single hs-cTnT<6 ng/L (biomarker alone) to exclude acute myocardial injury (subsequent hs-cTnT >99th percentile in those with an initial hs-cTnT<6 ng/L). The clinically intended rule-out strategy combining a nonischemic ECG with a baseline hs-cTnT<6 ng/L was subsequently tested in an adjudicated cohort in which the diagnostic performance for ruling out acute myocardial infarction and safety (myocardial infarction or death at 30 days) were evaluated. RESULTS:A total of 85 610 patients were evaluated in the CV Data Mart Biomarker cohort, among which 24 646 (29%) had a baseline hs-cTnT<6 ng/L. Women were more likely than men to have hs-cTnT<6 ng/L (38% versus 20%, <0.0001). Among 11 962 patients with baseline hs-cTnT<6 ng/L and serial measurements, only 1.2% developed acute myocardial injury, resulting in a negative predictive value of 98.8% (95% CI, 98.6-99.0) and sensitivity of 99.6% (95% CI, 99.5-99.6). In the adjudicated cohort, a nonischemic ECG with hs-cTnT<6 ng/L identified 33% of patients (610/1849) as low risk and resulted in a negative predictive value and sensitivity of 100% and a 30-day rate of 0.2% for myocardial infarction or death. CONCLUSIONS:A single hs-cTnT below the limit of quantitation of 6 ng/L is a safe and rapid method to identify a substantial number of patients at very low risk for acute myocardial injury and infarction.

Nutritionally inadequate dietary intake is a leading contributor to chronic cardiometabolic diseases. Differences in dietary quality contribute to socioeconomic and racial and ethnic health disparities. Food insecurity, a household-level social or economic condition of limited access to sufficient food, is a common cause of inadequate dietary intake. Although US food assistance policies and programs are designed to improve food security, there is growing consensus that they should have a broader focus on nutrition security. In this policy statement, we define nutrition security as an individual or household condition of having equitable and stable availability, access, affordability, and utilization of foods and beverages that promote well-being and prevent and treat disease. Despite existing policies and programs, significant gaps remain for achieving equity in nutrition security across the life span. We provide recommendations for expanding and improving current food assistance policies and programs to achieve nutrition security. These recommendations are guided by several overarching principles: emphasizing nutritional quality, improving reach, ensuring optimal utilization, improving coordination across programs, ensuring stability of access to programs across the life course, and ensuring equity and dignity for access and utilization. We suggest a critical next step will be to develop and implement national measures of nutrition security that can be added to the current US food security measures. Achieving equity in nutrition security will require coordinated and sustained efforts at the federal, state, and local levels. Future advocacy, innovation, and research will be needed to expand existing food assistance policies and programs and to develop and implement new policies and programs that will improve cardiovascular health and reduce disparities in chronic disease.

BACKGROUND:IDO1 (indoleamine 2,3-dioxygenase 1) is the rate-limiting enzyme for tryptophan metabolism. IDO1 malfunction is involved in the pathogenesis of atherosclerosis. Vascular smooth muscle cells (VSMCs) with an osteogenic phenotype promote calcification and features of plaque instability. However, it remains unclear whether aberrant IDO1-regulated tryptophan metabolism causes VSMCs osteogenic reprogramming and calcification. METHODS:We generated global () and double knockout mice, and knockout mice with specific deletion of IDO1 in VSMCs or macrophages. Arterial intimal calcification was evaluated by a Western diet-induced atherosclerotic calcification model. RESULTS:Global deficiency of IDO1 boosted calcific lesion formation without sex bias in vivo. Conditional IDO1 loss of function in VSMCs rather than macrophages promoted calcific lesion development and the abundance of RUNX2 (runt-related transcription factor 2). In contrast, administration of kynurenine via intraperitoneal injection markedly delayed the progression of intimal calcification in parallel with decreased RUNX2 expression in both and mice. We found that IDO1 deletion restrained RUNX2 from proteasomal degradation, which resulted in enhanced osteogenic reprogramming of VSMCs. Kynurenine administration downregulated RUNX2 in an aryl hydrocarbon receptor-dependent manner. Kynurenine acted as the endogenous ligand of aryl hydrocarbon receptor, controlled resultant interactions between cullin 4B and aryl hydrocarbon receptor to form an E3 ubiquitin ligase that bound with RUNX2, and subsequently promoted ubiquitin-mediated instability of RUNX2 in VSMCs. Serum samples from patients with coronary artery calcification had impaired IDO1 activity and decreased kynurenine catabolites compared with those without calcification. CONCLUSIONS:Kynurenine, an IDO1-mediated tryptophan metabolism main product, promotes RUNX2 ubiquitination and subsequently leads to its proteasomal degradation via an aryl hydrocarbon receptor-dependent nongenomic pathway. Insufficient kynurenine exerts the deleterious role of IDO1 ablation in promoting RUNX2-mediated VSMCs osteogenic reprogramming and calcification in vivo.

BACKGROUND:Sodium-glucose cotransporter-2 inhibitors are foundational therapy in patients with heart failure with reduced ejection fraction (HFrEF), but underlying mechanisms of benefit are not well defined. We sought to investigate the relationships between sodium-glucose cotransporter-2 inhibitor treatment, changes in metabolic pathways, and outcomes using targeted metabolomics. METHODS:DEFINE-HF (Dapagliflozin Effects on Biomarkers, Symptoms and Functional Status in Patients With HF With Reduced Ejection Fraction) was a placebo-controlled trial of dapagliflozin in HFrEF. We performed targeted mass spectrometry profiling of 63 metabolites (45 acylcarnitines [markers of fatty acid oxidation], 15 amino acids, and 3 conventional metabolites) in plasma samples at randomization and 12 weeks. Using mixed models, we identified principal components analysis-defined metabolite clusters that changed differentially with treatment and examined the relationship between change in metabolite clusters and change in Kansas City Cardiomyopathy Questionnaire scores and NT-proBNP (N-terminal probrain natriuretic peptide). Models were adjusted for relevant clinical covariates and nominal <0.05 with false discovery rate-adjusted <0.10 was used to determine statistical significance. RESULTS:Among the 234 DEFINE-HF participants with targeted metabolomic data, the mean age was 62.0±11.1 years, 25% were women, 38% were Black, and mean ejection fraction was 27±8%. Dapagliflozin increased ketone-related and short-chain acylcarnitine as well as medium-chain acylcarnitine principal components analysis-defined metabolite clusters compared with placebo (nominal =0.01, false discovery rate-adjusted =0.08 for both clusters). However, ketosis (β-hydroxybutyrate levels >500 μmol/L) was achieved infrequently (3 [2.5%] in dapagliflozin arm versus 1 [0.9%] in placebo arm) and supraphysiologic levels were not observed. Increases in long-chain acylcarnitine, long-chain dicarboxylacylcarnitine, and aromatic amino acid metabolite clusters were associated with decreases in Kansas City Cardiomyopathy Questionnaire scores (ie, worse quality of life) and increases in NT-proBNP levels, without interaction by treatment group. CONCLUSIONS:In this study of targeted metabolomics in a placebo-controlled trial of sodium-glucose cotransporter-2 inhibitors in HFrEF, we observed effects of dapagliflozin on key metabolic pathways, supporting a role for altered ketone and fatty acid biology with sodium-glucose cotransporter-2 inhibitors in patients with HFrEF. Only physiologic levels of ketosis were observed. In addition, we identified several metabolic biomarkers associated with adverse HFrEF outcomes. REGISTRATION:URL: https://www. CLINICALTRIALS:gov; Unique identifier: NCT02653482.

BACKGROUND:Early neonates of both large and small mammals are able to regenerate the myocardium through cardiomyocyte proliferation for only a short period after birth. This myocardial regenerative capacity declines in parallel with withdrawal of cardiomyocytes from the cell cycle in the first few postnatal days. No mammalian species examined to date has been found capable of a meaningful regenerative response to myocardial injury later than 1 week after birth. METHODS:We examined cardiomyocyte proliferation in neonates of the marsupial opossum () by immunostaining at various times after birth. The regenerative capacity of the postnatal opossum myocardium was assessed after either apex resection or induction of myocardial infarction at postnatal day 14 or 29, whereas that of the postnatal mouse myocardium was assessed after myocardial infarction at postnatal day 7. Bioinformatics data analysis, immunofluorescence staining, and pharmacological and genetic intervention were applied to determine the role of AMPK (5'-AMP-activated protein kinase) signaling in regulation of the mammalian cardiomyocyte cell cycle. RESULTS:Opossum neonates were found to manifest cardiomyocyte proliferation for at least 2 weeks after birth at a frequency similar to that apparent in early neonatal mice. Moreover, the opossum heart at postnatal day 14 showed substantial regenerative capacity both after apex resection and after myocardial infarction injury, whereas this capacity had diminished by postnatal day 29. Transcriptomic and immunofluorescence analyses indicated that AMPK signaling is activated in postnatal cardiomyocytes of both opossum and mouse. Pharmacological or genetic inhibition of AMPK signaling was sufficient to extend the postnatal window of cardiomyocyte proliferation in both mouse and opossum neonates as well as of cardiac regeneration in neonatal mice. CONCLUSIONS:The marsupial opossum maintains cardiomyocyte proliferation and a capacity for myocardial regeneration for at least 2 weeks after birth. As far as we are aware, this is the longest postnatal duration of such a capacity among mammals examined to date. AMPK signaling was implicated as an evolutionarily conserved regulator of mammalian postnatal cardiomyocyte proliferation.

BACKGROUND:Supervised aerobic exercise training (ET) is recommended for stable outpatients with heart failure (HF) with reduced ejection fraction (HFrEF). Frailty, a syndrome characterized by increased vulnerability and decreased physiologic reserve, is common in patients with HFrEF and associated with a higher risk of adverse outcomes. The effect modification of baseline frailty on the efficacy of aerobic ET in HFrEF is not known. METHODS:Stable outpatients with HFrEF randomized to aerobic ET versus usual care in the HF-ACTION (Heart Failure: A Controlled Trial Investigating Outcomes of Exercise Training) trial were included. Baseline frailty was estimated using the Rockwood frailty index (FI), a deficit accumulation-based model of frailty assessment; participants with FI scores >0.21 were identified as frail. Multivariable Cox proportional hazard models with multiplicative interaction terms (frailty × treatment arm) were constructed to evaluate whether frailty modified the treatment effect of aerobic ET on the primary composite end point (all-cause hospitalization or mortality), secondary end points (composite of cardiovascular death or cardiovascular hospitalization, and cardiovascular death or HF hospitalization), and Kansas City Cardiomyopathy Questionnaire score. Separate models were constructed for continuous (FI) and categorical (frail versus not frail) measures of frailty. RESULTS:Among 2130 study participants (age, 59±13 years; 28% women), 1266 (59%) were characterized as frail (FI>0.21). Baseline frailty burden significantly modified the treatment effect of aerobic ET ( interaction: FI × treatment arm=0.02; frail status [frail versus nonfrail] × treatment arm=0.04) with a lower risk of primary end point in frail (hazard ratio [HR], 0.83 [95% CI, 0.72-0.95]) but not nonfrail (HR, 1.04 [95% CI, 0.87-1.25]) participants. The favorable effect of aerobic ET among frail participants was driven by a significant reduction in the risk of all-cause hospitalization (HR, 0.84 [95% CI, 0.72-0.99]). The treatment effect of aerobic ET on all-cause mortality and other secondary endpoints was not different between frail and nonfrail patients ( interaction>0.1 for each). Aerobic ET was associated with a nominally greater improvement in Kansas City Cardiomyopathy Questionnaire scores at 3 months among frail versus nonfrail participants without a significant treatment interaction by frailty status ( interaction>0.2). CONCLUSIONS:Among patients with chronic stable HFrEF, baseline frailty modified the treatment effect of aerobic ET with a greater reduction in the risk of all-cause hospitalization but not mortality.

BACKGROUND:The insulin-like growth factor 1 (IGF1) pathway is a key regulator of cellular metabolism and aging. Although its inhibition promotes longevity across species, the effect of attenuated IGF1 signaling on cardiac aging remains controversial. METHODS:We performed a lifelong study to assess cardiac health and lifespan in 2 cardiomyocyte-specific transgenic mouse models with enhanced versus reduced IGF1 receptor (IGF1R) signaling. Male mice with human IGF1R overexpression or dominant negative phosphoinositide 3-kinase mutation were examined at different life stages by echocardiography, invasive hemodynamics, and treadmill coupled to indirect calorimetry. In vitro assays included cardiac histology, mitochondrial respiration, ATP synthesis, autophagic flux, and targeted metabolome profiling, and immunoblots of key IGF1R downstream targets in mouse and human explanted failing and nonfailing hearts, as well. RESULTS:Young mice with increased IGF1R signaling exhibited superior cardiac function that progressively declined with aging in an accelerated fashion compared with wild-type animals, resulting in heart failure and a reduced lifespan. In contrast, mice with low cardiac IGF1R signaling exhibited inferior cardiac function early in life, but superior cardiac performance during aging, and increased maximum lifespan, as well. Mechanistically, the late-life detrimental effects of IGF1R activation correlated with suppressed autophagic flux and impaired oxidative phosphorylation in the heart. Low IGF1R activity consistently improved myocardial bioenergetics and function of the aging heart in an autophagy-dependent manner. In humans, failing hearts, but not those with compensated hypertrophy, displayed exaggerated IGF1R expression and signaling activity. CONCLUSIONS:Our findings indicate that the relationship between IGF1R signaling and cardiac health is not linear, but rather biphasic. Hence, pharmacological inhibitors of the IGF1 pathway, albeit unsuitable for young individuals, might be worth considering in older adults.

BACKGROUND:Cardiovascular health (CVH) from young adulthood is strongly associated with an individual's future risk of cardiovascular disease (CVD) and total mortality. Defining epigenomic biomarkers of lifelong CVH exposure and understanding their roles in CVD development may help develop preventive and therapeutic strategies for CVD. METHODS:In 1085 CARDIA study (Coronary Artery Risk Development in Young Adults) participants, we defined a clinical cumulative CVH score that combines body mass index, blood pressure, total cholesterol, and fasting glucose measured longitudinally from young adulthood through middle age over 20 years (mean age, 25-45). Blood DNA methylation at >840 000 methylation markers was measured twice over 5 years (mean age, 40 and 45). Epigenome-wide association analyses on the cumulative CVH score were performed in CARDIA and compared in the FHS (Framingham Heart Study). We used penalized regression to build a methylation-based risk score to evaluate the risk of incident coronary artery calcification and clinical CVD events. RESULTS:We identified 45 methylation markers associated with cumulative CVH at false discovery rate <0.01 (=4.7E-7-5.8E-17) in CARDIA and replicated in FHS. These associations were more pronounced with methylation measured at an older age. , , and appeared as the most prominent genes. The 45 methylation markers were mostly located in transcriptionally active chromatin and involved lipid metabolism, insulin secretion, and cytokine production pathways. Three methylation markers located in genes , , and statistically mediated 20.4% of the total effect between CVH and risk of incident coronary artery calcification. The methylation risk score added information and significantly (=0.004) improved the discrimination capacity of coronary artery calcification status versus CVH score alone and showed association with risk of incident coronary artery calcification 5 to 10 years later independent of cumulative CVH score (odds ratio, 1.87; =9.66E-09). The methylation risk score was also associated with incident clinical CVD in FHS (hazard ratio, 1.28; =1.22E-05). CONCLUSIONS:Cumulative CVH from young adulthood contributes to midlife epigenetic programming over time. Our findings demonstrate the role of epigenetic markers in response to CVH changes and highlight the potential of epigenomic markers for precision CVD prevention, and earlier detection of subclinical CVD, as well.

BACKGROUND:Certain nonmammalian species such as zebrafish have an elevated capacity for innate heart regeneration. Understanding how heart regeneration occurs in these contexts can help illuminate cellular and molecular events that can be targets for heart failure prevention or treatment. The epicardium, a mesothelial tissue layer that encompasses the heart, is a dynamic structure that is essential for cardiac regeneration in zebrafish. The extent to which different cell subpopulations or states facilitate heart regeneration requires research attention. METHODS:To dissect epicardial cell states and associated proregenerative functions, we performed single-cell RNA sequencing and identified 7 epicardial cell clusters in adult zebrafish, 3 of which displayed enhanced cell numbers during regeneration. We identified paralogs of as factors associated with the extracellular matrix and largely expressed in cluster 1. We assessed expression in published single-cell RNA sequencing data sets from different stages and injury states of murine and human hearts, and we performed molecular genetics to determine the requirements for -expressing cells and functions of each paralog. RESULTS:A particular cluster of epicardial cells had the strongest association with regeneration and was marked by expression of and . The paralogs are expressed in epicardial cells that enclose dedifferentiated and proliferating cardiomyocytes during regeneration. Induced genetic depletion of -expressing cells or genetic inactivation of altered deposition of the key extracellular matrix component hyaluronic acid, disrupted cardiomyocyte proliferation, and inhibited heart regeneration. We also found that -expressing epicardial cells first emerge at the juvenile stage, when they associate with and are required for focused cardiomyocyte expansion events that direct maturation of the ventricular wall. CONCLUSIONS:Our findings identify a subset of epicardial cells that emerge in postembryonic zebrafish and sponsor regions of active cardiomyogenesis during cardiac growth and regeneration. We provide evidence that, as the heart achieves its mature structure, these cells facilitate hyaluronic acid deposition to support formation of the compact muscle layer of the ventricle. They are also required, along with the function of paralog, in the production and organization of hyaluronic acid-containing matrix in cardiac injury sites, enabling normal cardiomyocyte proliferation and muscle regeneration.