Multi-chamber cardioids unravel human heart development and cardiac defects.

The number one cause of human fetal death are defects in heart development. Because the human embryonic heart is inaccessible and the impacts of mutations, drugs, and environmental factors on the specialized functions of different heart compartments are not captured by in vitro models, determining the underlying causes is difficult. Here, we established a human cardioid platform that recapitulates the development of all major embryonic heart compartments, including right and left ventricles, atria, outflow tract, and atrioventricular canal. By leveraging 2D and 3D differentiation, we efficiently generated progenitor subsets with distinct first, anterior, and posterior second heart field identities. This advance enabled the reproducible generation of cardioids with compartment-specific in vivo-like gene expression profiles, morphologies, and functions. We used this platform to unravel the ontogeny of signal and contraction propagation between interacting heart chambers and dissect how mutations, teratogens, and drugs cause compartment-specific defects in the developing human heart.

Transcription factor protein interactomes reveal genetic determinants in heart disease.

Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease.

Modeling congenital heart disease: lessons from mice, hPSC-based models, and organoids.

Congenital heart defects (CHDs) are among the most common birth defects, but their etiology has long been mysterious. In recent decades, the development of a variety of experimental models has led to a greater understanding of the molecular basis of CHDs. In this review, we contrast mouse models of CHD, which maintain the anatomical arrangement of the heart, and human cellular models of CHD, which are more likely to capture human-specific biology but lack anatomical structure. We also discuss the recent development of cardiac organoids, which are a promising step toward more anatomically informative human models of CHD.

STIGMA: Single-cell tissue-specific gene prioritization using machine learning.

Clinical exome and genome sequencing have revolutionized the understanding of human disease genetics. Yet many genes remain functionally uncharacterized, complicating the establishment of causal disease links for genetic variants. While several scoring methods have been devised to prioritize these candidate genes, these methods fall short of capturing the expression heterogeneity across cell subpopulations within tissues. Here, we introduce single-cell tissue-specific gene prioritization using machine learning (STIGMA), an approach that leverages single-cell RNA-seq (scRNA-seq) data to prioritize candidate genes associated with rare congenital diseases. STIGMA prioritizes genes by learning the temporal dynamics of gene expression across cell types during healthy organogenesis. To assess the efficacy of our framework, we applied STIGMA to mouse limb and human fetal heart scRNA-seq datasets. In a cohort of individuals with congenital limb malformation, STIGMA prioritized 469 variants in 345 genes, with UBA2 as a notable example. For congenital heart defects, we detected 34 genes harboring nonsynonymous de novo variants (nsDNVs) in two or more individuals from a set of 7,958 individuals, including the ortholog of Prdm1, which is associated with hypoplastic left ventricle and hypoplastic aortic arch. Overall, our findings demonstrate that STIGMA effectively prioritizes tissue-specific candidate genes by utilizing single-cell transcriptome data. The ability to capture the heterogeneity of gene expression across cell populations makes STIGMA a powerful tool for the discovery of disease-associated genes and facilitates the identification of causal variants underlying human genetic disorders.

Fundamental Mechanisms of Regulated Cell Death and Implications for Heart Disease.

Twelve regulated cell death programs have been described. We review in detail the basic biology of nine including death receptor-mediated apoptosis, death receptor-mediated necrosis (necroptosis), mitochondrial-mediated apoptosis, mitochondrial-mediated necrosis, autophagy-dependent cell death, ferroptosis, pyroptosis, parthanatos, and immunogenic cell death. This is followed by a dissection of the roles of these cell death programs in the major cardiac syndromes: myocardial infarction and heart failure. The most important conclusion relevant to heart disease is that regulated forms of cardiomyocyte death play important roles in both myocardial infarction with reperfusion (ischemia/reperfusion) and heart failure. While a role for apoptosis in ischemia/reperfusion cannot be excluded, regulated forms of necrosis, through both death receptor and mitochondrial pathways, are critical. Ferroptosis and parthanatos are also likely important in ischemia/reperfusion, although it is unclear if these entities are functioning as independent death programs or as amplification mechanisms for necrotic cell death. Pyroptosis may also contribute to ischemia/reperfusion injury, but potentially through effects in non-cardiomyocytes. Cardiomyocyte loss through apoptosis and necrosis is also an important component in the pathogenesis of heart failure and is mediated by both death receptor and mitochondrial signaling. Roles for immunogenic cell death in cardiac disease remain to be defined but merit study in this era of immune checkpoint cancer therapy. Biology-based approaches to inhibit cell death in the various cardiac syndromes are also discussed.

Variants of the promoter of MYH6 gene in congenital isolated and sporadic patent ductus arteriosus: case-control study and cellular functional analyses.

Patent ductus arteriosus (PDA) is a common form of congenital heart disease. The MYH6 gene has important effects on cardiovascular growth and development, but the effect of variants in the MYH6 gene promoter on ductus arteriosus is unknown. DNA was extracted from blood samples of 721 subjects (428 patients with isolated and sporadic PDA and 293 healthy controls) and analyzed by sequencing for MYH6 gene promoter region variants. Cellular function experiments with three cell lines (HEK-293, HL-1, and H9C2 cells) and bioinformatics analyses were performed to verify their effects on gene expression. In the MYH6 gene promoter, 11 variants were identified. Four variants were found only in patients with PDA and 2 of them (g.3434G>C and g.4524C>T) were novel. Electrophoretic mobility shift assay showed that the transcription factors bound by the promoter variants were significantly altered in comparison to the wild-type in all three cell lines. Dual luciferase reporter showed that all the 4 variants reduced the transcriptional activity of the MYH6 gene promoter (P < 0.05). Prediction of transcription factors bound by the variants indicated that these variants alter the transcription factor binding sites. These pathological alterations most likely affect the contraction of the smooth muscle of ductus arteriosus, leading to PDA. This study is the first to focus on variants at the promoter region of the MYH6 gene in PDA patients with cellular function tests. Therefore, this study provides new insights to understand the genetic basis and facilitates further studies on the mechanism of PDA formation.

Pros and Cons of Long-Chain Omega-3 Polyunsaturated Fatty Acids in Cardiovascular Health.

The long-chain omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are found in seafood, supplements, and concentrated pharmaceutical preparations. Prospective cohort studies demonstrate an association between higher intakes of EPA+DHA or higher levels of EPA and DHA in the body and lower risk of developing cardiovascular disease (CVD), especially coronary heart disease and myocardial infarction, and of cardiovascular mortality in the general population. The cardioprotective effect of EPA and DHA is due to the beneficial modulation of a number of risk factors for CVD. Some large trials support the use of EPA+DHA (or EPA alone) in high-risk patients, although the evidence is inconsistent. This review presents key studies of EPA and DHA in the primary and secondary prevention of CVD, briefly describes potential mechanisms of action, and discusses recently published RCTs and meta-analyses. Potential adverse aspects of long-chain omega-3 fatty acids in relation to CVD are discussed.

Complex Congenital Heart Disease in the Adult.

Congenital heart disease (CHD), a heterogeneous group of structural abnormalities of the cardiovascular system, is the most frequent cause of severe birth defects. Related to improved pediatric outcomes, there are now more adults living with CHD, including complex lesions, than children. Adults with CHD are at high risk for complications related to their underlying anatomy and past surgical palliative interventions. Adults with CHD require close monitoring and proactive management strategies to improve outcomes.

The H2Bub1-deposition complex is required for human and mouse cardiogenesis.

De novo variants affecting monoubiquitylation of histone H2B (H2Bub1) are enriched in human congenital heart disease. H2Bub1 is required in stem cell differentiation, cilia function, post-natal cardiomyocyte maturation and transcriptional elongation. However, how H2Bub1 affects cardiogenesis is unknown. We show that the H2Bub1-deposition complex (RNF20-RNF40-UBE2B) is required for mouse cardiogenesis and for differentiation of human iPSCs into cardiomyocytes. Mice with cardiac-specific Rnf20 deletion are embryonic lethal and have abnormal myocardium. We then analyzed H2Bub1 marks during differentiation of human iPSCs into cardiomyocytes. H2Bub1 is erased from most genes at the transition from cardiac mesoderm to cardiac progenitor cells but is preserved on a subset of long cardiac-specific genes. When H2Bub1 is reduced in iPSC-derived cardiomyocytes, long cardiac-specific genes have fewer full-length transcripts. This correlates with H2Bub1 accumulation near the center of these genes. H2Bub1 accumulation near the center of tissue-specific genes was also observed in embryonic fibroblasts and fetal osteoblasts. In summary, we show that normal H2Bub1 distribution is required for cardiogenesis and cardiomyocyte differentiation, and suggest that H2Bub1 regulates tissue-specific gene expression by increasing the amount of full-length transcripts.

Circadian and Diurnal Regulation of Cerebral Blood Flow.

Circadian and diurnal variation in cerebral blood flow directly contributes to the diurnal variation in the risk of stroke, either through factors that trigger stroke or due to impaired compensatory mechanisms. Cerebral blood flow results from the integration of systemic hemodynamics, including heart rate, cardiac output, and blood pressure, with cerebrovascular regulatory mechanisms, including cerebrovascular reactivity, autoregulation, and neurovascular coupling. We review the evidence for the circadian and diurnal variation in each of these mechanisms and their integration, from the detailed evidence for mechanisms underlying the nocturnal nadir and morning surge in blood pressure to identifying limited available evidence for circadian and diurnal variation in cerebrovascular compensatory mechanisms. We, thus, identify key systemic hemodynamic factors related to the diurnal variation in the risk of stroke but particularly identify the need for further research focused on cerebrovascular regulatory mechanisms.

Mutations in Bcl9 and Pygo genes cause congenital heart defects by tissue-specific perturbation of Wnt/ß-catenin signaling.

Bcl9 and Pygopus (Pygo) are obligate Wnt/ß-catenin cofactors in Drosophila, yet their contribution to Wnt signaling during vertebrate development remains unresolved. Combining zebrafish and mouse genetics, we document a conserved, ß-catenin-associated function for BCL9 and Pygo proteins during vertebrate heart development. Disrupting the ß-catenin-BCL9-Pygo complex results in a broadly maintained canonical Wnt response yet perturbs heart development and proper expression of key cardiac regulators. Our work highlights BCL9 and Pygo as selective ß-catenin cofactors in a subset of canonical Wnt responses during vertebrate development. Moreover, our results implicate alterations in BCL9 and BCL9L in human congenital heart defects.

Forecasting the Burden of Cardiovascular Disease and Stroke in the United States Through 2050-Prevalence of Risk Factors and Disease: A Presidential Advisory From the American Heart Association.

BACKGROUND: Cardiovascular disease and stroke are common and costly, and their prevalence is rising. Forecasts on the prevalence of risk factors and clinical events are crucial. METHODS: Using the 2015 to March 2020 National Health and Nutrition Examination Survey and 2015 to 2019 Medical Expenditure Panel Survey, we estimated trends in prevalence for cardiovascular risk factors based on adverse levels of Life's Essential 8 and clinical cardiovascular disease and stroke. We projected through 2050, overall and by age and race and ethnicity, accounting for changes in disease prevalence and demographics. RESULTS: We estimate that among adults, prevalence of hypertension will increase from 51.2% in 2020 to 61.0% in 2050. Diabetes (16.3% to 26.8%) and obesity (43.1% to 60.6%) will increase, whereas hypercholesterolemia will decline (45.8% to 24.0%). The prevalences of poor diet, inadequate physical activity, and smoking are estimated to improve over time, whereas inadequate sleep will worsen. Prevalences of coronary disease (7.8% to 9.2%), heart failure (2.7% to 3.8%), stroke (3.9% to 6.4%), atrial fibrillation (1.7% to 2.4%), and total cardiovascular disease (11.3% to 15.0%) will rise. Clinical CVD will affect 45 million adults, and CVD including hypertension will affect more than 184 million adults by 2050 (>61%). Similar trends are projected in children. Most adverse trends are projected to be worse among people identifying as American Indian/Alaska Native or multiracial, Black, or Hispanic. CONCLUSIONS: The prevalence of many cardiovascular risk factors and most established diseases will increase over the next 30 years. Clinical and public health interventions are needed to effectively manage, stem, and even reverse these adverse trends.

Evaluation and Management of Chronic Heart Failure in Children and Adolescents With Congenital Heart Disease: A Scientific Statement From the American Heart Association.

With continued medical and surgical advancements, most children and adolescents with congenital heart disease are expected to survive to adulthood. Chronic heart failure is increasingly being recognized as a major contributor to ongoing morbidity and mortality in this population as it ages, and treatment strategies to prevent and treat heart failure in the pediatric population are needed. In addition to primary myocardial dysfunction, anatomical and pathophysiological abnormalities specific to various congenital heart disease lesions contribute to the development of heart failure and affect potential strategies commonly used to treat adult patients with heart failure. This scientific statement highlights the significant knowledge gaps in understanding the epidemiology, pathophysiology, staging, and outcomes of chronic heart failure in children and adolescents with congenital heart disease not amenable to catheter-based or surgical interventions. Efforts to harmonize the definitions, staging, follow-up, and approach to heart failure in children with congenital heart disease are critical to enable the conduct of rigorous scientific studies to advance our understanding of the actual burden of heart failure in this population and to allow the development of evidence-based heart failure therapies that can improve outcomes for this high-risk cohort.

Environmental Exposures and Pediatric Cardiology: A Scientific Statement From the American Heart Association.

Environmental toxicants and pollutants are causes of adverse health consequences, including well-established associations between environmental exposures and cardiovascular diseases. Environmental degradation is widely prevalent and has a long latency period between exposure and health outcome, potentially placing a large number of individuals at risk of these health consequences. Emerging evidence suggests that environmental exposures in early life may be key risk factors for cardiovascular conditions across the life span. Children are a particularly sensitive population for the detrimental effects of environmental toxicants and pollutants given the long-term cumulative effects of early-life exposures on health outcomes, including congenital heart disease, acquired cardiac diseases, and accumulation of cardiovascular disease risk factors. This scientific statement highlights representative examples for each of these cardiovascular disease subtypes and their determinants, focusing specifically on the associations between climate change and congenital heart disease, airborne particulate matter and Kawasaki disease, blood lead levels and blood pressure, and endocrine-disrupting chemicals with cardiometabolic risk factors. Because children are particularly dependent on their caregivers to address their health concerns, this scientific statement highlights the need for clinicians, research scientists, and policymakers to focus more on the linkages of environmental exposures with cardiovascular conditions in children and adolescents.

Unraveling the Mechanisms of Valvular Heart Disease to Identify Medical Therapy Targets: A Scientific Statement From the American Heart Association.

Valvular heart disease is a common cause of morbidity and mortality worldwide and has no effective medical therapy. Severe disease is managed with valve replacement procedures, which entail high health care-related costs and postprocedural morbidity and mortality. Robust ongoing research programs have elucidated many important molecular pathways contributing to primary valvular heart disease. However, there remain several key challenges inherent in translating research on valvular heart disease to viable molecular targets that can progress through the clinical trials pathway and effectively prevent or modify the course of these common conditions. In this scientific statement, we review the basic cellular structures of the human heart valves and discuss how these structures change in primary valvular heart disease. We focus on the most common primary valvular heart diseases, including calcific aortic stenosis, bicuspid aortic valves, mitral valve prolapse, and rheumatic heart disease, and outline the fundamental molecular discoveries contributing to each. We further outline potential therapeutic molecular targets for primary valvular heart disease and discuss key knowledge gaps that might serve as future research priorities.

2023 ACC/AHA/ACCP/HRS Guideline for the Diagnosis and Management of Atrial Fibrillation: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines.

AIM: The "2023 ACC/AHA/ACCP/HRS Guideline for the Diagnosis and Management of Atrial Fibrillation" provides recommendations to guide clinicians in the treatment of patients with atrial fibrillation. METHODS: A comprehensive literature search was conducted from May 12, 2022, to November 3, 2022, encompassing studies, reviews, and other evidence conducted on human subjects that were published in English from PubMed, EMBASE, the Cochrane Library, the Agency for Healthcare Research and Quality, and other selected databases relevant to this guideline. Additional relevant studies, published through November 2022, during the guideline writing process, were also considered by the writing committee and added to the evidence tables, where appropriate. STRUCTURE: Atrial fibrillation is the most sustained common arrhythmia, and its incidence and prevalence are increasing in the United States and globally. Recommendations from the "2014 AHA/ACC/HRS Guideline for the Management of Patients With Atrial Fibrillation" and the "2019 AHA/ACC/HRS Focused Update of the 2014 AHA/ACC/HRS Guideline for the Management of Patients With Atrial Fibrillation" have been updated with new evidence to guide clinicians. In addition, new recommendations addressing atrial fibrillation and thromboembolic risk assessment, anticoagulation, left atrial appendage occlusion, atrial fibrillation catheter or surgical ablation, and risk factor modification and atrial fibrillation prevention have been developed.

Valvular Heart Disease: New Concepts in Pathophysiology and Therapeutic Approaches.

This review discusses recent advancements in the field of valvular heart disease. Topics covered include recognition of the impact of atrial fibrillation on development and assessment of valvular disease, strategies for global prevention of rheumatic heart disease, understanding and management of secondary mitral regurgitation, the updated classification of bicuspid aortic valve disease, recognition of heightened cardiovascular risk associated with moderate aortic stenosis, and a growing armamentarium of transcatheter therapies.

Neither cardiac mitochondrial DNA variation nor copy number contribute to congenital heart disease risk.

The well-established manifestation of mitochondrial mutations in functional cardiac disease (e.g., mitochondrial cardiomyopathy) prompted the hypothesis that mitochondrial DNA (mtDNA) sequence and/or copy number (mtDNAcn) variation contribute to cardiac defects in congenital heart disease (CHD). MtDNAcns were calculated and rare, non-synonymous mtDNA mutations were identified in 1,837 CHD-affected proband-parent trios, 116 CHD-affected singletons, and 114 paired cardiovascular tissue/blood samples. The variant allele fraction (VAF) of heteroplasmic variants in mitochondrial RNA from 257 CHD cardiovascular tissue samples was also calculated. On average, mtDNA from blood had 0.14 rare variants and 52.9 mtDNA copies per nuclear genome per proband. No variation with parental age at proband birth or CHD-affected proband age was seen. mtDNAcns in valve/vessel tissue (320 ± 70) were lower than in atrial tissue (1,080 ± 320, p = 6.8E-21), which were lower than in ventricle tissue (1,340 ± 280, p = 1.4E-4). The frequency of rare variants in CHD-affected individual DNA was indistinguishable from the frequency in an unaffected cohort, and proband mtDNAcns did not vary from those of CHD cohort parents. In both the CHD and the comparison cohorts, mtDNAcns were significantly correlated between mother-child, father-child, and mother-father. mtDNAcns among people with European (mean = 52.0), African (53.0), and Asian haplogroups (53.5) were calculated and were significantly different for European and Asian haplogroups (p = 2.6E-3). Variant heteroplasmic fraction (HF) in blood correlated well with paired cardiovascular tissue HF (r = 0.975) and RNA VAF (r = 0.953), which suggests blood HF is a reasonable proxy for HF in heart tissue. We conclude that mtDNA mutations and mtDNAcns are unlikely to contribute significantly to CHD risk.

Sex-dependent expression of galectin-1, a cardioprotective ß-galactoside-binding lectin, in human calcific aortic stenosis.

We aimed to analyze sex-related differences in galectin-1 (Gal-1), a ß-galactoside-binding lectin, in aortic stenosis (AS) and its association with the inflammatory and fibrocalcific progression of AS. Gal-1 was determined in serum and aortic valves (AVs) from control and AS donors by western blot and immunohistochemistry. Differences were validated by ELISA and qPCR in AS samples. In vitro experiments were conducted in primary cultured valve interstitial cells (VICs). Serum Gal-1 was not different neither between control and AS nor between men and women. There was no association between circulating and valvular Gal-1 levels. The expression of Gal-1 in stenotic AVs was higher in men than women, even after adjusting for confounding factors, and was associated with inflammation, oxidative stress, extracellular matrix remodeling, fibrosis, and osteogenesis. Gal-1 (LGALS1) mRNA was enhanced within fibrocalcific areas of stenotic AVs, especially in men. Secretion of Gal-1 was up-regulated over a time course of 2, 4, and 8 days in men's calcifying VICs, only peaking at day 4 in women's VICs. In vitro, Gal-1 was associated with similar mechanisms to those in our clinical cohort. ß-estradiol significantly up-regulated the activity of an LGALS1 promoter vector and the secretion of Gal-1, only in women's VICs. Supplementation with rGal-1 prevented the effects elicited by calcific challenge including the metabolic shift to glycolysis. In conclusion, Gal-1 is up-regulated in stenotic AVs and VICs from men in association with inflammation, oxidative stress, matrix remodeling, and osteogenesis. Estrogens can regulate Gal-1 expression with potential implications in post-menopause women. Exogenous rGal-1 can diminish calcific phenotypes in both women and men.

Metabolic profiling of aortic stenosis and hypertrophic cardiomyopathy identifies mechanistic contrasts in substrate utilization.

Aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM) are distinct disorders leading to left ventricular hypertrophy (LVH), but whether cardiac metabolism substantially differs between these in humans remains to be elucidated. We undertook an invasive (aortic root, coronary sinus) metabolic profiling in patients with severe AS and HCM in comparison with non-LVH controls to investigate cardiac fuel selection and metabolic remodeling. These patients were assessed under different physiological states (at rest, during stress induced by pacing). The identified changes in the metabolome were further validated by metabolomic and orthogonal transcriptomic analysis, in separately recruited patient cohorts. We identified a highly discriminant metabolomic signature in severe AS in all samples, regardless of sampling site, characterized by striking accumulation of long-chain acylcarnitines, intermediates of fatty acid transport across the inner mitochondrial membrane, and validated this in a separate cohort. Mechanistically, we identify a downregulation in the PPAR-α transcriptional network, including expression of genes regulating fatty acid oxidation (FAO). In silico modeling of ß-oxidation demonstrated that flux could be inhibited by both the accumulation of fatty acids as a substrate for mitochondria and the accumulation of medium-chain carnitines which induce competitive inhibition of the acyl-CoA dehydrogenases. We present a comprehensive analysis of changes in the metabolic pathways (transcriptome to metabolome) in severe AS, and its comparison to HCM. Our results demonstrate a progressive impairment of ß-oxidation from HCM to AS, particularly for FAO of long-chain fatty acids, and that the PPAR-α signaling network may be a specific metabolic therapeutic target in AS.