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Background: Hundreds of candidate genes have been associated with coronary artery disease (CAD) through genome-wide association studies. However, a systematic way to understand the causal mechanism(s) of these genes, and a means to prioritize them for further study, has been lacking. This represents a major roadblock for developing novel disease- and gene-specific therapies for patients with CAD. Recently, powerful integrative genomics analyses pipelines have emerged to identify and prioritize candidate causal genes by integrating tissue/cell-specific gene expression data with genome-wide association study data sets. Methods: We aimed to develop a comprehensive integrative genomics analyses pipeline for CAD and to provide a prioritized list of causal CAD genes. To this end, we leveraged several complimentary informatics approaches to integrate summary statistics from CAD genome-wide association studies (from UK Biobank and CARDIoGRAMplusC4D) with transcriptomic and expression quantitative trait loci data from 9 cardiometabolic tissue/cell types in the STARNET study (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task). Results: We identified 162 unique candidate causal CAD genes, which exerted their effect from between one and up to 7 disease-relevant tissues/cell types, including the arterial wall, blood, liver, skeletal muscle, adipose, foam cells, and macrophages. When their causal effect was ranked, the top candidate causal CAD genes were CDKN2B (associated with the 9p21.3 risk locus) and PHACTR1 ; both exerting their causal effect in the arterial wall. A majority of candidate causal genes were represented in cross-tissue gene regulatory co-expression networks that are involved with CAD, with 22/162 being key drivers in those networks. Conclusions: We identified and prioritized candidate causal CAD genes, also localizing their tissue(s) of causal effect. These results should serve as a resource and facilitate targeted studies to identify the functional impact of top causal CAD genes.

Background and aims The atherosclerotic plaque microenvironment is highly complex, and selective agents that modulate plaque stability are not yet available. We sought to develop a scRNA-seq analysis workflow to investigate this environment and uncover potential therapeutic approaches. We designed a user-friendly, reproducible workflow that will be applicable to other disease-specific scRNA-seq datasets. Methods Here we incorporated automated cell labeling, pseudotemporal ordering, ligand-receptor evaluation, and drug-gene interaction analysis into a ready-to-deploy workflow. We applied this pipeline to further investigate a previously published human coronary single-cell dataset by Wirka et al. Notably, we developed an interactive web application to enable further exploration and analysis of this and other cardiovascular single-cell datasets. Results We revealed distinct derivations of fibroblast-like cells from smooth muscle cells (SMCs), and showed the key changes in gene expression along their de-differentiation path. We highlighted several key ligand-receptor interactions within the atherosclerotic environment through functional expression profiling and revealed several avenues for future pharmacological development for precision medicine. Further, our interactive web application, PlaqView ( www.plaqview.com ), allows lay scientists to explore this and other datasets and compare scRNA-seq tools without prior coding knowledge. Conclusions This publicly available workflow and application will allow for more systematic and user-friendly analysis of scRNA datasets in other disease and developmental systems. Our analysis pipeline provides many hypothesis-generating tools to unravel the etiology of coronary artery disease. We also highlight potential mechanisms for several drugs in the atherosclerotic cellular environment. Future releases of PlaqView will feature more scRNA-seq and scATAC-seq atherosclerosis-related datasets to provide a critical resource for the field, and to promote data harmonization and biological interpretation.

Significance Here, we present unequivocal evidence of physical association of AMPK holoenzymes with mitochondrial reticulum (mitoAMPK) across multiple mouse tissues with evidence of conservation in human skeletal muscle and heart. We demonstrate that mitoAMPK is activated heterogeneously across the mitochondrial reticulum by mitochondrial energetic stress. Finally, we present evidence that suggests activation of mitoAMPK in skeletal muscle is required for mitophagy. We propose that mitoAMPK responds to mitochondrial microenvironment cues to maintain energetic homeostasis through mitochondrial quality control., Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5′ AMP-activated protein kinase (AMPKα1/α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

Coronary artery disease (CAD) is a complex inflammatory disease involving genetic influences across cell types. Genome-wide association studies have identified over 200 loci associated with CAD, where the majority of risk variants reside in noncoding DNA sequences impacting cis-regulatory elements. Here, we applied single-nucleus assay for transposase-accessible chromatin with sequencing to profile 28,316 nuclei across coronary artery segments from 41 patients with varying stages of CAD, which revealed 14 distinct cellular clusters. We mapped ~320,000 accessible sites across all cells, identified cell-type-specific elements and transcription factors, and prioritized functional CAD risk variants. We identified elements in smooth muscle cell transition states (for example, fibromyocytes) and functional variants predicted to alter smooth muscle cell- and macrophage-specific regulation of MRAS (3q22) and LIPA (10q23), respectively. We further nominated key driver transcription factors such as PRDM16 and TBX2. Together, this single-nucleus atlas provides a critical step towards interpreting regulatory mechanisms across the continuum of CAD risk.

Aims De-differentiation and activation of pro-inflammatory pathways are key transitions vascular smooth muscle cells (SMCs) make during atherogenesis. Here, we explored the upstream regulators of this 'atherogenic transition'. Methods and results Genome-wide sequencing studies, including ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and RNA-seq, were performed on cells isolated from both murine SMC-lineage tracing models of atherosclerosis and human atherosclerotic lesions. At the bulk level, alterations in chromatin accessibility were associated with the atherogenic transitioning of lesional SMCs, especially in relation to genes that govern differentiation status and complement-dependent inflammation. Using computational biology, we observed that a transcription factor previously related to coronary artery disease, ATF3 (Activating transcription factor 3), was predicted to be an upstream regulator of genes altered during the transition. At the single-cell level, our results indicated that ATF3 is a key repressor of SMC transitioning towards the subset of cells that promote vascular inflammation by activating the complement cascade. The expression of ATF3 and complement component C3 were negatively correlated in SMCs from human atherosclerotic lesions, suggesting translational relevance. Phenome-wide association studies indicated that genetic variation that results in reduced expression of ATF3 is correlated with an increased risk for atherosclerosis, and the expression of ATF3 was significantly downregulated in humans with advanced vascular disease. Conclusion Our study indicates that the plasticity of atherosclerotic SMCs may in part be explained by dynamic changes in their chromatin architecture, which in turn may contribute to their maladaptive response to inflammation-induced stress. Translational perspective The recent CANTOS and COLCOT trials have shown that targeting inflammatory pathways lowers the risk of major adverse cardiovascular events. However, more specific targets are needed to avoid immunosuppressive side effects. Our data identify an upstream regulator of pro-inflammatory SMCs, ATF3, which is involved in the initial atherogenic transitioning of lesional SMCs. Restoring ATF3 activity may prevent the de-differentiation of SMCs and offer a novel translational approach for the suppression of complement-dependent inflammation in atherosclerotic lesions.

In response to various stimuli, vascular smooth muscle cells (SMCs) can de-differentiate, proliferate and migrate in a process known as phenotypic modulation. However, the phenotype of modulated SMCs in vivo during atherosclerosis and the influence of this process on coronary artery disease (CAD) risk have not been clearly established. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomic phenotype of modulated SMCs in vivo in atherosclerotic lesions of both mouse and human arteries and found that these cells transform into unique fibroblast-like cells, termed 'fibromyocytes', rather than into a classical macrophage phenotype. SMC-specific knockout of TCF21-a causal CAD gene-markedly inhibited SMC phenotypic modulation in mice, leading to the presence of fewer fibromyocytes within lesions as well as within the protective fibrous cap of the lesions. Moreover, TCF21 expression was strongly associated with SMC phenotypic modulation in diseased human coronary arteries, and higher levels of TCF21 expression were associated with decreased CAD risk in human CAD-relevant tissues. These results establish a protective role for both TCF21 and SMC phenotypic modulation in this disease.

Aims While most patients with myocardial infarction (MI) have underlying coronary atherosclerosis, not all patients with coronary artery disease (CAD) develop MI. We sought to address the hypothesis that some of the genetic factors which establish atherosclerosis may be distinct from those that predispose to vulnerable plaques and thrombus formation. Methods and results We carried out a genome-wide association study for MI in the UK Biobank (n∼472 000), followed by a meta-analysis with summary statistics from the CARDIoGRAMplusC4D Consortium (n∼167 000). Multiple independent replication analyses and functional approaches were used to prioritize loci and evaluate positional candidate genes. Eight novel regions were identified for MI at the genome wide significance level, of which effect sizes at six loci were more robust for MI than for CAD without the presence of MI. Confirmatory evidence for association of a locus on chromosome 1p21.3 harbouring choline-like transporter 3 (SLC44A3) with MI in the context of CAD, but not with coronary atherosclerosis itself, was obtained in Biobank Japan (n∼165 000) and 16 independent angiography-based cohorts (n∼27 000). Follow-up analyses did not reveal association of the SLC44A3 locus with CAD risk factors, biomarkers of coagulation, other thrombotic diseases, or plasma levels of a broad array of metabolites, including choline, trimethylamine N-oxide, and betaine. However, aortic expression of SLC44A3 was increased in carriers of the MI risk allele at chromosome 1p21.3, increased in ischaemic (vs. non-diseased) coronary arteries, up-regulated in human aortic endothelial cells treated with interleukin-1β (vs. vehicle), and associated with smooth muscle cell migration in vitro. Conclusions A large-scale analysis comprising ∼831 000 subjects revealed novel genetic determinants of MI and implicated SLC44A3 in the pathophysiology of vulnerable plaques.

In this second of a 5-part Focus Seminar series, we focus on precision medicine in the context of vascular disease. The most common vascular disease worldwide is atherosclerosis, which is the primary cause of coronary artery disease, peripheral vascular disease, and a large proportion of strokes and other disorders. Atherosclerosis is a complex genetic disease that likely involves many hundreds to thousands of single nucleotide polymorphisms, each with a relatively modest effect for causing disease. Conversely, although less prevalent, there are many vascular disorders that typically involve only a single genetic change, but these changes can often have a profound effect that is sufficient to cause disease. These are termed “Mendelian vascular diseases,” which include Marfan and Loeys-Dietz syndromes. Given the very different genetic basis of atherosclerosis versus Mendelian vascular diseases, this article was divided into 2 parts to cover the most promising precision medicine approaches for these disease types.

Rationale: Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. Objective: To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs. Methods and Results: We quantified 12 atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multiethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed genome-wide association studies for 12 atherosclerosis-relevant phenotypes and identified 4 genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD loci with our data set and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3 . The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 (melanoma inhibitory activity protein 3) in VSMCs resulted in lower proliferation, consistent with human genetics findings. Furthermore, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans. Conclusions: Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Furthermore, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of “dedifferentiated” vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don’t eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

Summary Identification of functional transcription factors that regulate a given gene set is an important problem in gene regulation studies. Conventional approaches for identifying transcription factors, such as DNA sequence motif analysis, are unable to predict functional binding of specific factors and not sensitive enough to detect factors binding at distal enhancers. Here, we present binding analysis for regulation of transcription (BART), a novel computational method and software package for predicting functional transcription factors that regulate a query gene set or associate with a query genomic profile, based on more than 6000 existing ChIP-seq datasets for over 400 factors in human or mouse. This method demonstrates the advantage of utilizing publicly available data for functional genomics research. Availability and implementation BART is implemented in Python and available at http://faculty.virginia.edu/zanglab/bart. Supplementary information Supplementary data are available at Bioinformatics online.

Coronary artery disease is a complex cardiovascular disease involving an interplay of genetic and environmental influences over a lifetime. Although considerable progress has been made in understanding lifestyle risk factors, genetic factors identified from genome-wide association studies may capture additional hidden risk undetected by traditional clinical tests. These genetic discoveries have highlighted many candidate genes and pathways dysregulated in the vessel wall, including those involving smooth muscle cell phenotypic modulation and injury responses. Here, we summarize experimental evidence for a few genome-wide significant loci supporting their roles in smooth muscle cell biology and disease. We also discuss molecular quantitative trait locus mapping as a powerful discovery and fine-mapping approach applied to smooth muscle cell and coronary artery disease-relevant tissues. We emphasize the critical need for alternative genetic strategies, including cis/trans-regulatory network analysis, genome editing, and perturbations, as well as single-cell sequencing in smooth muscle cell tissues and model organisms, under both normal and disease states. By integrating multiple experimental and analytical modalities, these multidimensional datasets should improve the interpretation of coronary artery disease genome-wide association studies and molecular quantitative trait locus signals and inform candidate targets for therapeutic intervention or risk prediction.

Background Genome-wide association studies have identified over 160 loci that are associated with coronary artery disease. As with other complex human diseases, risk in coronary disease loci is determined primarily by altered expression of the causal gene, due to variation in binding of transcription factors and chromatin-modifying proteins that directly regulate the transcriptional apparatus. We have previously identified a coronary disease network downstream of the disease-associated transcription factor TCF21, and in work reported here extends these studies to investigate the mechanisms by which it interacts with the AP-1 transcription complex to regulate local epigenetic effects in these downstream coronary disease loci. Methods Genomic studies, including chromatin immunoprecipitation sequencing, RNA sequencing, and protein-protein interaction studies, were performed in human coronary artery smooth muscle cells. Results We show here that TCF21 and JUN regulate expression of two presumptive causal coronary disease genes, SMAD3 and CDKN2B-AS1, in part by interactions with histone deacetylases and acetyltransferases. Genome-wide TCF21 and JUN binding is jointly localized and particularly enriched in coronary disease loci where they broadly modulate H3K27Ac and chromatin state changes linked to disease-related processes in vascular cells. Heterozygosity at coronary disease causal variation, or genome editing of these variants, is associated with decreased binding of both JUN and TCF21 and loss of expression in cis, supporting a transcriptional mechanism for disease risk. Conclusions These data show that the known chromatin remodeling and pioneer functions of AP-1 are a pervasive aspect of epigenetic control of transcription, and thus, the risk in coronary disease-associated loci, and that interaction of AP-1 with TCF21 to control epigenetic features, contributes to the genetic risk in loci where they co-localize. Electronic supplementary material The online version of this article (10.1186/s13073-019-0635-9) contains supplementary material, which is available to authorized users.

Recent genome-wide association studies (GWAS) have identified multiple new loci which appear to alter coronary artery disease (CAD) risk via arterial wall-specific mechanisms. One of the annotated genes encodes LMOD1 (Leiomodin 1), a member of the actin filament nucleator family that is highly enriched in smooth muscle-containing tissues such as the artery wall. However, it is still unknown whether LMOD1 is the causal gene at this locus and also how the associated variants alter LMOD1 expression/function and CAD risk. Using epigenomic profiling we recently identified a non-coding regulatory variant, rs34091558, which is in tight linkage disequilibrium (LD) with the lead CAD GWAS variant, rs2820315. Herein we demonstrate through expression quantitative trait loci (eQTL) and statistical fine-mapping in GTEx, STARNET, and human coronary artery smooth muscle cell (HCASMC) datasets, rs34091558 is the top regulatory variant for LMOD1 in vascular tissues. Position weight matrix (PWM) analyses identify the protective allele rs34091558-TA to form a conserved Forkhead box O3 (FOXO3) binding motif, which is disrupted by the risk allele rs34091558-A. FOXO3 chromatin immunoprecipitation and reporter assays show reduced FOXO3 binding and LMOD1 transcriptional activity by the risk allele, consistent with effects of FOXO3 downregulation on LMOD1. LMOD1 knockdown results in increased proliferation and migration and decreased cell contraction in HCASMC, and immunostaining in atherosclerotic lesions in the SMC lineage tracing reporter mouse support a key role for LMOD1 in maintaining the differentiated SMC phenotype. These results provide compelling functional evidence that genetic variation is associated with dysregulated LMOD1 expression/function in SMCs, together contributing to the heritable risk for CAD., Author summary It is now estimated that nearly ~50% of one’s lifetime risk for coronary artery disease is genetically predetermined. While genetic studies have led to the identification of several novel disease risk factors, the mechanisms driving many of these associations are yet to be fully identified. Here, we take advantage of a recent finding identifying genetic variation in a smooth muscle cell restricted gene called Leiomodin1, and investigate the causal gene and mechanism by which this risk factor predisposes individuals to coronary artery disease. By implementing several complementary approaches, we extend previous findings demonstrating that this genetic variant is associated with attenuated expression of Leiomodin 1. Interestingly, we demonstrate that this association is attributed to altered binding of a transcription factor Forkhead Box 3. More importantly, we show that reduced Leiomodin 1 results in phenotypic modulation of smooth muscle cells, a process related to diseases such as atherosclerosis. Interestingly, Leiomodin 1 levels were found to be reduced in smooth muscle cells within atherosclerotic lesions relative to those around the fibrous cap. These studies provide new insights into the heritable mechanisms of coronary artery disease with Leiomodin 1 as a potential therapeutic target.

Although numerous genetic loci have been associated with coronary artery disease (CAD) with genome wide association studies, efforts are needed to identify the causal genes in these loci and link them into fundamental signaling pathways. Recent studies have investigated the disease mechanism of CAD associated gene SMAD3, a central transcription factor (TF) in the TGFβ pathway, investigating its role in smooth muscle biology. In vitro studies in human coronary artery smooth muscle cells (HCASMC) revealed that SMAD3 modulates cellular phenotype, promoting expression of differentiation marker genes while inhibiting proliferation. RNA sequencing and chromatin immunoprecipitation sequencing studies in HCASMC identified downstream genes that reside in pathways which mediate vascular development and atherosclerosis processes in this cell type. HCASMC phenotype, and gene expression patterns promoted by SMAD3 were noted to have opposing direction of effect compared to another CAD associated TF, TCF21. At sites of SMAD3 and TCF21 colocalization on DNA, SMAD3 binding was inversely correlated with TCF21 binding, due in part to TCF21 locally blocking chromatin accessibility at the SMAD3 binding site. Further, TCF21 was able to directly inhibit SMAD3 activation of gene expression in transfection reporter gene studies. In contrast to TCF21 which is protective toward CAD, SMAD3 expression in HCASMC was shown to be directly correlated with disease risk. We propose that the pro-differentiation action of SMAD3 inhibits dedifferentiation that is required for HCASMC to expand and stabilize disease plaque as they respond to vascular stresses, counteracting the protective dedifferentiating activity of TCF21 and promoting disease risk., Author summary Coronary artery disease (CAD) is the worldwide leading cause of death. The majority of risk for CAD is genetic in nature, i.e., a feature of the genetic information that is transmitted to each individual from both parents, and primarily affects the disease processes in the blood vessel wall that regulate the disease molecular pathways. Modern genetic approaches have allowed mapping of the regions of the human genome that encode information that mediates this risk. The SMAD3 gene has been identified through these studies, a known master regulatory of other genes and molecular pathways, and we have investigated the functions of this gene that are important for disease risk. SMAD3 affects basic functions of a cellular component of the vessel wall, the smooth muscle cell (SMC), that is responsible for responding to vascular stresses to heal the lesions that are produced in conjunction with elevated lipids and other classic risk factors. Studies reported here show that SMAD3 actually inhibits the cellular processes that allow SMC to repair the vascular lesions, and its expression is promoted by the disease related variable sequences in the disease associated regions of the genome. SMAD3 is opposed by another CAD gene, TCF21, that functions to block the effects of SMAD3 expression, and these studies identify genetic mechanisms by which this is done. Thus, these studies identify an interactive pathway that directly contributes to disease risk, and the ability to block SMAD3 or promote TCF21 function could be exploited to inhibit vascular events such as myocardial infarction.

Objective— Key augmented processes in atherosclerosis have been identified, whereas less is known about downregulated pathways. Here, we applied a systems biology approach to examine suppressed molecular signatures, with the hypothesis that they may provide insight into mechanisms contributing to plaque stability. Approach and Results— Muscle contraction, muscle development, and actin cytoskeleton were the most downregulated pathways (false discovery rate=6.99e-21, 1.66e-6, 2.54e-10, respectively) in microarrays from human carotid plaques (n=177) versus healthy arteries (n=15). In addition to typical smooth muscle cell (SMC) markers, these pathways also encompassed cytoskeleton-related genes previously not associated with atherosclerosis. SYNPO2, SYNM, LMOD1, PDLIM7 , and PLN expression positively correlated to typical SMC markers in plaques (Pearson r >0.6, P r >0.8, P PDLIM7, PLN , and SYNPO2 loci associated with progression of carotid intima-media thickness in high-risk subjects without symptoms of cardiovascular disease (n=3378). By eQTL (expression quantitative trait locus), rs11746443 also associated with PDLIM7 expression in plaques. Mechanistically, silencing of PDLIM7 in vitro led to downregulation of SMC markers and disruption of the actin cytoskeleton, decreased cell spreading, and increased proliferation. Conclusions— We identified a panel of genes that reflect the altered phenotype of SMCs in vascular disease and could be early sensitive markers of SMC dedifferentiation.

Background The genetic determinants of heart failure (HF) and response to medical therapy remain unknown. We hypothesized that identifying genetic variants of HF that associate with response to medical therapy would elucidate the genetic basis of cardiac function. Objectives This study sought to identify genetic variations associated with response to HF therapy. Methods This study compared extremes of response to medical therapy in 866 HF patients using a genome-wide approach that informed the systems-based design of a customized single nucleotide variant array. The effect of genotype on gene expression was measured using allele-specific luciferase reporter assays. Candidate gene transcription-deficient mice underwent echocardiography and treadmill exercise. The ability of the target gene agonist to rescue mice from chemically-induced HF was assessed with echocardiography. Results Of 866 HF patients, 136 had an ejection fraction improvement of 20% attributed to resynchronization (n = 83), revascularization (n = 7), tachycardia resolution (n = 2), alcohol cessation (n = 1), or medications (n = 43). Those with the minor allele for rs7767652, upstream of hypocretin (orexin) receptor-2 (HCRTR2), were less likely to have improved left ventricular function (odds ratio: 0.40 per minor allele; p = 3.29 × 10−5). In a replication cohort of 798 patients, those with a minor allele for rs7767652 had a lower prevalence of ejection fraction >35% (odds ratio: 0.769 per minor allele; p = 0.021). In an HF model, HCRTR2-deficient mice exhibited poorer cardiac function, worse treadmill exercise capacity, and greater myocardial scarring. Orexin, an HCRTR2 agonist, rescued function in this HF mouse model. Conclusions A systems approach identified a novel genetic contribution to human HF and a promising therapeutic agent efficacious in an HF model.

The clinical importance of microbiomes to the chronicity of wounds is widely appreciated, yet little is understood about patient-specific processes shaping wound microbiome composition. Here, a two-cohort microbiome-genome wide association study is presented through which patient genomic loci associated with chronic wound microbiome diversity were identified. Further investigation revealed that alternative TLN2 and ZNF521 genotypes explained significant inter-patient variation in relative abundance of two key pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis. Wound diversity was lowest in Pseudomonas aeruginosa infected wounds, and decreasing wound diversity had a significant negative linear relationship with healing rate. In addition to microbiome characteristics, age, diabetic status, and genetic ancestry all significantly influenced healing. Using structural equation modeling to identify common variance among SNPs, six loci were sufficient to explain 53% of variation in wound microbiome diversity, which was a 10% increase over traditional multiple regression. Focusing on TLN2, genotype at rs8031916 explained expression differences of alternative transcripts that differ in inclusion of important focal adhesion binding domains. Such differences are hypothesized to relate to wound microbiomes and healing through effects on bacterial exploitation of focal adhesions and/or cellular migration. Related, other associated loci were functionally enriched, often with roles in cytoskeletal dynamics. This study, being the first to identify patient genetic determinants for wound microbiomes and healing, implicates genetic variation determining cellular adhesion phenotypes as important drivers of infection type. The identification of predictive biomarkers for chronic wound microbiomes may serve as risk factors and guide treatment by informing patient-specific tendencies of infection., Author summary Chronic, or non-healing, wounds represent a costly burden to patients, and bacterial infection of wounds is an important driver of chronicity. A variety of bacterial species often occur in chronic wounds, but it is unknown why certain species are observed in some wound infections and not others. In this study, genetic variation of wound clinic patients was compared to the bacteria observed in their infected wounds. Through these comparisons, genetic variation in the TLN2 and ZNF521 genes was found to be associated with both the number of bacteria observed in wounds and the abundance of common pathogens (primarily Pseudomonas aeruginosa and Staphylococcus epidermidis). Moreover, Pseudomonas infected wounds were found to have fewer species present and wounds with fewer species were slower to heal. Furthermore, patient genes associated with microbiomes commonly encode proteins known to be important for cellular structures important to healing and to which bacteria directly interact. Experimental investigation of one such gene, TLN2, identified genotype-dependent differences in the expression of functionally different versions of TLN2 that is hypothesized to shape differences in cellular adhesion structures. Finally, a new statistical approach is presented in which patient biomarkers are used to predict the number of species observed during infection. Overall, our results describe how patient genetic variation influence the types of bacteria likely to infect an individual as well as influence healing.

Aims Recent genome-wide association studies (GWAS) have identified that the JCAD locus is associated with risk of coronary artery disease (CAD) and myocardial infarction (MI). However, the mechanisms whereby candidate gene JCAD confers disease risk remain unclear. We addressed whether and how JCAD affects the development of atherosclerosis, the common cause of CAD. Methods and results By mining data in the Genotype-Tissue Expression (GTEx) database, we found that CAD-associated risk variants at the JCAD locus are linked to increased JCAD gene expression in human arteries, implicating JCAD as a candidate causal CAD gene. We therefore generated global and endothelial cell (EC) specific-JCAD knockout mice, and observed that JCAD deficiency attenuated high fat diet-induced atherosclerosis in ApoE-deficient mice. JCAD-deficiency in mice also improved endothelium-dependent relaxation. Genome-wide transcriptional profiling of JCAD-depleted human coronary artery ECs showed that JCAD depletion inhibited the activation of YAP/TAZ pathway, and the expression of downstream pro-atherogenic genes, including CTGF and Cyr61. As a result, JCAD-deficient ECs attracted fewer monocytes in response to lipopolysaccharide (LPS) stimulation. Moreover, JCAD expression in ECs was decreased under unidirectional laminar flow in vitro and in vivo. Proteomics studies suggest that JCAD regulates YAP/TAZ activation by interacting with actin-binding protein TRIOBP, thereby stabilizing stress fiber formation. Finally, we observed that endothelial JCAD expression was increased in mouse and human atherosclerotic plaques. Conclusion The present study demonstrates that the GWAS-identified CAD risk gene JCAD promotes endothelial dysfunction and atherosclerosis, thus highlighting the possibility of new therapeutic strategies for CAD by targeting JCAD.

Coronary artery disease (CAD) is the leading cause of death globally. Genome-wide association studies (GWAS) have identified more than 95 independent loci that influence CAD risk, most of which reside in non-coding regions of the genome. To interpret these loci, we generated transcriptome and whole-genome datasets using human coronary artery smooth muscle cells (HCASMC) from 52 unrelated donors, as well as epigenomic datasets using ATAC-seq on a subset of 8 donors. Through systematic comparison with publicly available datasets from GTEx and ENCODE projects, we identified transcriptomic, epigenetic, and genetic regulatory mechanisms specific to HCASMC. We assessed the relevance of HCASMC to CAD risk using transcriptomic and epigenomic level analyses. By jointly modeling eQTL and GWAS datasets, we identified five genes (SIPA1, TCF21, SMAD3, FES, and PDGFRA) that modulate CAD risk through HCASMC, all of which have relevant functional roles in vascular remodeling. Comparison with GTEx data suggests that SIPA1 and PDGFRA influence CAD risk predominantly through HCASMC, while other annotated genes may have multiple cell and tissue targets. Together, these results provide new tissue-specific and mechanistic insights into the regulation of a critical vascular cell type associated with CAD in human populations.

Both environmental factors and genetic loci have been associated with coronary artery disease (CAD), however gene-gene and gene-environment interactions that might identify molecular mechanisms of risk are not easily studied by human genetic approaches. We have previously identified the transcription factor TCF21 as the causal CAD gene at 6q23.2 and characterized its downstream transcriptional network that is enriched for CAD GWAS genes. Here we investigate the hypothesis that TCF21 interacts with a downstream target gene, the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (e.g., tobacco smoke). Perturbation of TCF21 expression in human coronary artery smooth muscle cells (HCASMC) revealed that TCF21 promotes expression of AHR, its heterodimerization partner ARNT, and cooperates with these factors to upregulate a number of inflammatory downstream disease related genes including IL1A, MMP1, and CYP1A1. TCF21 was shown to bind in AHR, ARNT and downstream target gene loci, and co-localization was noted for AHR-ARNT and TCF21 binding sites genome-wide in regions of HCASMC open chromatin. These regions of co-localization were found to be enriched for GWAS signals associated with cardio-metabolic as well as chronic inflammatory disease phenotypes. Finally, we show that similar to TCF21, AHR gene expression is increased in atherosclerotic lesions in mice in vivo using laser capture microdissection, and AHR protein is localized in human carotid atherosclerotic lesions where it is associated with protein kinases with a critical role in innate immune response. These data suggest that TCF21 can cooperate with AHR to activate an inflammatory gene expression program that is exacerbated by environmental stimuli, and may contribute to the overall risk for CAD., Author summary Coronary heart disease is the leading cause of death in the world. Both genes and the environment are important risk factors for the progression of disease, however, how genes may modulate the harmful response to the disease promoting environment is unknown and difficult to study. Here, we show that a common heritable variation in the gene TCF21 may regulate coronary heart disease risk by regulating the response of downstream gene activation by the disease environment. We find that a well-known environmental sensor, aryl-hydrocarbon receptor (AHR), is regulated by TCF21 and also interacts with TCF21, resulting in regulation of pro-inflammatory gene expression in coronary artery smooth muscle cells. We further show that oxidized LDL, a well-known driver of atherosclerosis in the plaque can activate the AHR pathway. This work describes a heritable form of gene-environment interaction identified through genome wide association studies in coronary artery disease, and presents an opportunity to define causal gene-gene and gene-environment interactions.

Genetic variation at the chromosome 9p21 risk locus promotes cardiovascular disease; however, it is unclear how or which proteins encoded at this locus contribute to disease. We have previously demonstrated that loss of one candidate gene at this locus, cyclin-dependent kinase inhibitor 2B (Cdkn2b), in mice promotes vascular SMC apoptosis and aneurysm progression. Here, we investigated the role of Cdnk2b in atherogenesis and found that in a mouse model of atherosclerosis, deletion of Cdnk2b promoted advanced development of atherosclerotic plaques composed of large necrotic cores. Furthermore, human carriers of the 9p21 risk allele had reduced expression of CDKN2B in atherosclerotic plaques, which was associated with impaired expression of calreticulin, a ligand required for activation of engulfment receptors on phagocytic cells. As a result of decreased calreticulin, CDKN2B-deficient apoptotic bodies were resistant to efferocytosis and not efficiently cleared by neighboring macrophages. These uncleared SMCs elicited a series of proatherogenic juxtacrine responses associated with increased foam cell formation and inflammatory cytokine elaboration. The addition of exogenous calreticulin reversed defects associated with loss of Cdkn2b and normalized engulfment of Cdkn2b-deficient cells. Together, these data suggest that loss of CDKN2B promotes atherosclerosis by increasing the size and complexity of the lipid-laden necrotic core through impaired efferocytosis.

Coronary artery disease (or coronary heart disease), is the leading cause of mortality in many of the developing as well as the developed countries of the world. Cholesterol-enriched plaques in the heart's blood vessels combined with inflammation lead to the lesion expansion, narrowing of blood vessels, reduced blood flow, and may subsequently cause lesion rupture and a heart attack. Even though several environmental risk factors have been established, such as high LDL-cholesterol, diabetes, and high blood pressure, the underlying genetic composition may substantially modify the disease risk; hence, genome composition and gene-environment interactions may be critical for disease progression. Ongoing scientific efforts have seen substantial advancements related to the fields of genetics and genomics, with the major breakthroughs yet to come. As genomics is the most rapidly advancing field in the life sciences, it is important to present a comprehensive overview of current efforts. Here, we present a summary of various genetic and genomics assays and approaches applied to coronary artery disease research.

Cardiac fibroblasts become activated and differentiate to smooth muscle-like myofibroblasts in response to hypertension and myocardial infarction (MI), resulting in extracellular matrix (ECM) remodeling, scar formation and impaired cardiac function. cAMP and cGMP-dependent signaling have been implicated in cardiac fibroblast activation and ECM synthesis. Dysregulation of cyclic nucleotide phosphodiesterase (PDE) activity/expression is also associated with various diseases and several PDE inhibitors are currently available or in development for treating these pathological conditions. The objective of this study is to define and characterize the specific PDE isoform that is altered during cardiac fibroblast activation and functionally important for regulating myofibroblast activation and ECM synthesis. We have found that Ca(2+)/calmodulin-stimulated PDE1A isoform is specifically induced in activated cardiac myofibroblasts stimulated by Ang II and TGF-β in vitro as well as in vivo within fibrotic regions of mouse, rat, and human diseased hearts. Inhibition of PDE1A function via PDE1-selective inhibitor or PDE1A shRNA significantly reduced Ang II or TGF-β-induced myofibroblast activation, ECM synthesis, and pro-fibrotic gene expression in rat cardiac fibroblasts. Moreover, the PDE1 inhibitor attenuated isoproterenol-induced interstitial fibrosis in mice. Mechanistic studies revealed that PDE1A modulates unique pools of cAMP and cGMP, predominantly in perinuclear and nuclear regions of cardiac fibroblasts. Further, both cAMP-Epac-Rap1 and cGMP-PKG signaling was involved in PDE1A-mediated regulation of collagen synthesis. These results suggest that induction of PDE1A plays a critical role in cardiac fibroblast activation and cardiac fibrosis, and targeting PDE1A may lead to regression of the adverse cardiac remodeling associated with various cardiac diseases.

Coronary artery disease (CAD) is the leading cause of mortality and morbidity, driven by both genetic and environmental risk factors. Meta-analyses of genome-wide association studies have identified >150 loci associated with CAD and myocardial infarction susceptibility in humans. A majority of these variants reside in non-coding regions and are co-inherited with hundreds of candidate regulatory variants, presenting a challenge to elucidate their functions. Herein, we use integrative genomic, epigenomic and transcriptomic profiling of perturbed human coronary artery smooth muscle cells and tissues to begin to identify causal regulatory variation and mechanisms responsible for CAD associations. Using these genome-wide maps, we prioritize 64 candidate variants and perform allele-specific binding and expression analyses at seven top candidate loci: 9p21.3, SMAD3, PDGFD, IL6R, BMP1, CCDC97/TGFB1 and LMOD1. We validate our findings in expression quantitative trait loci cohorts, which together reveal new links between CAD associations and regulatory function in the appropriate disease context., Coronary heart disease is the leading cause of death worldwide with multiple environmental and genetic risk factors. Here the authors integrate genomic, epigenomic and transcriptomic mapping to elucidate causal variation and mechanisms of known genetic associations.

Growing evidence suggests that multiple spatially, temporally, and functionally distinct pools of cyclic nucleotides exist and regulate cardiac performance, from acute myocardial contractility to chronic gene expression and cardiac structural remodeling. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP and cyclic GMP, regulate the amplitude, duration, and compartmentation of cyclic nucleotide-mediated signaling. In particular, PDE3 enzymes play a major role in regulating cAMP metabolism in the cardiovascular system. PDE3 inhibitors, by raising cAMP content, have acute inotropic and vasodilatory effects in treating congestive heart failure but have increased mortality in long-term therapy. PDE3A expression is downregulated in human and animal failing hearts. In vitro, inhibition of PDE3A function is associated with myocyte apoptosis through sustained induction of a transcriptional repressor ICER (inducible cAMP early repressor) and thereby inhibition of antiapoptotic molecule Bcl-2 expression. Sustained induction of ICER may also cause the change of other protein expression implicated in human and animal failing hearts. These data suggest that the downregulation of PDE3A observed in failing hearts may play a causative role in the progression of heart failure, in part, by inducing ICER and promoting cardiac myocyte dysfunction. Hence, strategies that maintain PDE3A function may represent an attractive approach to circumvent myocyte apoptosis and cardiac dysfunction.

Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including “vascular disease,” “disorder of artery,” and “occlusion of artery,” as well as disease-related cellular functions including “cellular movement” and “cellular growth and proliferation.” In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall., Author Summary Coronary artery disease (CAD) is responsible for the majority of deaths in the Western world, and is due in part to environmental and metabolic factors. However, half of the risk for developing heart disease is genetically predetermined. Genome-wide association studies in human populations have identified over 100 sites in the genome that appear to be associated with CAD, however, the mechanisms by which variation in these regions are responsible for predisposition to CAD remain largely unknown. We have begun to study a gene that contributes to CAD risk, the TCF21 gene. Through genomic studies we show that this gene is involved in processes related to alterations in vascular gene expression, and in particular those related to the smooth muscle cell biology. With cell culture models, we show that TCF21 regulates the differentiation state of this cell type, which is believed critical for vascular disease. Using mouse genetic models of atherosclerotic vascular disease we provide evidence that this gene is expressed in precursor cells that migrate into the disease lesions and contribute to the formation of the fibrous cap that is believed to stabilize these lesions and prevent heart attacks.

To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including “growth factor binding,” “matrix interaction,” and “smooth muscle contraction.” We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology., Author Summary While coronary artery disease (CAD) is due in part to environmental and metabolic factors, about half of the risk is genetically predetermined. Genome-wide association studies in human populations have identified approximately 150 sites in the genome that appear to be associated with CAD. The mechanisms by which mutations in these regions are responsible for predisposition to CAD remain largely unknown. To begin to explore how disease-specific gene sequences and disease gene function promotes pathology, we have mapped the loci and genes that are downstream of the transcription factor TCF21, which is strongly associated with CAD. By identifying genes that are regulated by TCF21 we have been able to link together multiple other CAD associated genes and begin to identify the critical molecular processes that mediate atherosclerosis in the blood vessel wall and contribute to the genesis of ischemic cardiovascular events.

Large-scale genome-wide association studies (GWAS) have identified 46 loci that are associated with coronary heart disease (CHD). Additionally, 104 independent candidate variants (false discovery rate of 5 %) have been identified (Schunkert H, Konig IR, Kathiresan S, Reilly MP, Assimes TL, Holm H et al. Nat Genet 43:333-8, 2011; Deloukas P, Kanoni S, Willenborg C, Farrall M, Assimes TL, Thompson JR et al. Nat Genet 45:25-33, 2012; C4D Genetics Consortium. Nat Genet 43:339-44, 2011). The majority of the causal genes in these loci function independently of conventional risk factors. It is postulated that a number of the CHD-associated genes regulate basic processes in the vascular cells involved in atherosclerosis, and that study of the signaling pathways that are modulated in this cell type by causal regulatory variation will provide critical new insights for targeting the initiation and progression of disease. In this review, we will discuss the types of experimental approaches and data that are critical to understanding the molecular processes that underlie the disease risk at 9p21.3, TCF21, SORT1, and other CHD-associated loci.

Coronary heart disease (CHD) is the leading cause of mortality in both developed and developing countries worldwide. Genome-wide association studies (GWAS) have now identified 46 independent susceptibility loci for CHD, however, the biological and disease-relevant mechanisms for these associations remain elusive. The large-scale meta-analysis of GWAS recently identified in Caucasians a CHD-associated locus at chromosome 6q23.2, a region containing the transcription factor TCF21 gene. TCF21 (Capsulin/Pod1/Epicardin) is a member of the basic-helix-loop-helix (bHLH) transcription factor family, and regulates cell fate decisions and differentiation in the developing coronary vasculature. Herein, we characterize a cis-regulatory mechanism by which the lead polymorphism rs12190287 disrupts an atypical activator protein 1 (AP-1) element, as demonstrated by allele-specific transcriptional regulation, transcription factor binding, and chromatin organization, leading to altered TCF21 expression. Further, this element is shown to mediate signaling through platelet-derived growth factor receptor beta (PDGFR-β) and Wilms tumor 1 (WT1) pathways. A second disease allele identified in East Asians also appears to disrupt an AP-1-like element. Thus, both disease-related growth factor and embryonic signaling pathways may regulate CHD risk through two independent alleles at TCF21., Author Summary As much as half of the risk of developing coronary heart disease is genetically predetermined. Genome-wide association studies in human populations have now uncovered multiple sites of common genetic variation associated with heart disease. However, the biological mechanisms responsible for linking the disease associations with changes in gene expression are still underexplored. One of these variants occurs within the vascular developmental factor, TCF21, leading to dysregulated gene expression. Using various in silico and molecular approaches, we identify an intricate allele-specific regulatory mechanism underlying altered expression of TCF21. Notably, we observe that two apparently independent risk alleles identified in distinct populations function through a similar regulatory mechanism. Together these data suggest that conserved upstream pathways may organize the complex genetic etiology of coronary heart disease and potentially lead to new treatment opportunities.

Objective— Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear. Methods and Results— Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow–derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model. Conclusion— These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.

High throughput sequencing has facilitated a precipitous drop in the cost of genomic sequencing, prompting predictions of a revolution in medicine via genetic personalization of diagnostic and therapeutic strategies. There are significant barriers to realizing this goal that are related to the difficult task of interpreting personal genetic variation. A comprehensive, widely accessible application for interpretation of whole genome sequence data is needed. Here, we present a series of methods for identification of genetic variants and genotypes with clinical associations, phasing genetic data and using Mendelian inheritance for quality control, and providing predictive genetic information about risk for rare disease phenotypes and response to pharmacological therapy in single individuals and father-mother-child trios. We demonstrate application of these methods for disease and drug response prognostication in whole genome sequence data from twelve unrelated adults, and for disease gene discovery in one father-mother-child trio with apparently simplex congenital ventricular arrhythmia. In doing so we identify clinically actionable inherited disease risk and drug response genotypes in pre-symptomatic individuals. We also nominate a new candidate gene in congenital arrhythmia, ATP2B4, and provide experimental evidence of a regulatory role for variants discovered using this framework., Author Summary Technological advances have dramatically reduced the cost of sequencing the human genome. Tools for analyzing such data across families including annotation of clinically important variants and aggregation of variants for personalizing drug prescriptions have been developed but few are publically available. Here we describe such tools then demonstrate their application in several distinct data sets. In particular, we use the tools to define the genetic basis of a new congenital arrhythmia syndrome.

Objective— The phenotypic modulation of vascular smooth muscle cells (VSMCs) to a synthetic phenotype is vital during pathological vascular remodeling and the development of various vascular diseases. An increase in type I collagen (collagen I) has been implicated in synthetic VSMCs, and cyclic nucleotide signaling is critical in collagen I regulation. Herein, we investigate the role and underlying mechanism of cyclic nucleotide phosphodiesterase 1 (PDE1) in regulating collagen I in synthetic VSMCs. Methods and Results— The PDE1 inhibitor IC86340 significantly reduced collagen I in human saphenous vein explants undergoing spontaneous remodeling via ex vivo culture. In synthetic VSMCs, high basal levels of intracellular and extracellular collagen I protein were markedly decreased by IC86340. This attenuation was due to diminished protein but not mRNA. Inhibition of lysosome function abolished the effect of IC86340 on collagen I protein expression. PDE1C but not PDE1A is the major isoform responsible for mediating the effects of IC86340. Bicarbonate-sensitive soluble adenylyl cyclase/cAMP signaling was modulated by PDE1C, which is critical in collagen I degradation in VSMCs. Conclusion— These data demonstrate that PDE1C regulates soluble adenylyl cyclase/cAMP signaling and lysosome-mediated collagen I protein degradation, and they suggest that PDE1C plays a critical role in regulating collagen homeostasis during pathological vascular remodeling.

The second messengers, cAMP and cGMP, regulate a number of physiological processes in the myocardium, from acute contraction/relaxation to chronic gene expression and cardiac structural remodeling. Emerging evidence suggests that multiple spatiotemporally distinct pools of cyclic nucleotides can discriminate specific cellular functions from a given cyclic nucleotide-mediated signal. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing intracellular cyclic AMP and/or cyclic GMP, control the amplitude, duration, and compartmentation of cyclic nucleotide signaling. To date, more than 60 different isoforms have been described and grouped into 11 broad families (PDE1-PDE11) based on differences in their structure, kinetic and regulatory properties, as well as sensitivity to chemical inhibitors. In the heart, PDE isozymes from at least six families have been investigated. Studies using selective PDE inhibitors and/or genetically manipulated animals have demonstrated that individual PDE isozymes play distinct roles in the heart by regulating unique cyclic nucleotide signaling microdomains. Alterations of PDE activity and/or expression have also been observed in various cardiac disease models, which may contribute to disease progression. Several family-selective PDE inhibitors have been used clinically or pre-clinically for the treatment of cardiac or vascular-related diseases. In this review, we will highlight both recent advances and discrepancies relevant to cardiovascular PDE expression, pathophysiological function, and regulation. In particular, we will emphasize how these properties influence current and future development of PDE inhibitors for the treatment of pathological cardiac remodeling and dysfunction.

Rationale: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca 2+ /calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca 2+ /CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. Objective: Herein, we investigate the role of Ca 2+ /CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. Methods and Results: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. Conclusions: These results elucidate a novel role for Ca 2+ /CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca 2+ and cGMP signaling cross-talk during cardiac hypertrophy.

Genome-wide association studies (GWAS) have identified chromosomal loci that affect risk of coronary heart disease (CHD) independent of classical risk factors. One such association signal has been identified at 6q23.2 in both Caucasians and East Asians. The lead CHD-associated polymorphism in this region, rs12190287, resides in the 3′ untranslated region (3′-UTR) of TCF21, a basic-helix-loop-helix transcription factor, and is predicted to alter the seed binding sequence for miR-224. Allelic imbalance studies in circulating leukocytes and human coronary artery smooth muscle cells (HCASMC) showed significant imbalance of the TCF21 transcript that correlated with genotype at rs12190287, consistent with this variant contributing to allele-specific expression differences. 3′ UTR reporter gene transfection studies in HCASMC showed that the disease-associated C allele has reduced expression compared to the protective G allele. Kinetic analyses in vitro revealed faster RNA-RNA complex formation and greater binding of miR-224 with the TCF21 C allelic transcript. In addition, in vitro probing with Pb2+ and RNase T1 revealed structural differences between the TCF21 variants in proximity of the rs12190287 variant, which are predicted to provide greater access to the C allele for miR-224 binding. miR-224 and TCF21 expression levels were anti-correlated in HCASMC, and miR-224 modulates the transcriptional response of TCF21 to transforming growth factor-β (TGF-β) and platelet derived growth factor (PDGF) signaling in an allele-specific manner. Lastly, miR-224 and TCF21 were localized in human coronary artery lesions and anti-correlated during atherosclerosis. Together, these data suggest that miR-224 interaction with the TCF21 transcript contributes to allelic imbalance of this gene, thus partly explaining the genetic risk for coronary heart disease associated at 6q23.2. These studies implicating rs12190287 in the miRNA-dependent regulation of TCF21, in conjunction with previous studies showing that this variant modulates transcriptional regulation through activator protein 1 (AP-1), suggests a unique bimodal level of complexity previously unreported for disease-associated variants., Author Summary Both genetic and environmental factors cumulatively contribute to coronary heart disease risk in human populations. Large-scale meta-analyses of genome-wide association studies have now leveraged common genetic variation to identify multiple sites of disease susceptibility; however, the causal mechanisms for these associations largely remain elusive. One of these disease-associated variants, rs12190287, resides in the 3′untranslated region of the vascular developmental transcription factor, TCF21. Intriguingly, this variant is shown to disrupt the seed binding sequence for microRNA-224, and through altered RNA secondary structure and binding kinetics, leads to dysregulated TCF21 gene expression in response to disease-relevant stimuli. Importantly TCF21 and miR-224 expression levels were perturbed in human atherosclerotic lesions. Along with our previous reports on the transcriptional regulatory mechanisms altered by this variant, these studies shed new light on the complex heritable mechanisms of coronary heart disease risk that are amenable to therapeutic intervention.