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ObjectivesDetermine the role of apolipoprotein M (ApoM) in anthracycline (Dox) cardiotoxicity.BackgroundApoM binds the cardioprotective sphingolipid sphingosine-1-phosphate (S1P). Circulating ApoM is inversely associated with mortality in human heart failure (HF).MethodsIn the Penn HF Study (PHFS), we tested the relationship between ApoM and mortality in a subset with anthracycline-induced cardiomyopathy. We measured ApoM in humans and mice treated with Dox and utilized hepatic ApoM transgenic (ApomTG), ApoM knockout (ApomKO), ApoM knock-in mice with impaired S1P binding, and S1P receptor 3 (S1PR3) knockout mice in Dox cardiotoxicity. We assayed autophagy in left ventricular tissue from anthracycline-induced HF patients versus donor controls.ResultsApoM was inversely associated with mortality in PHFS, and Dox reduced circulating ApoM in mice and breast cancer patients.ApomTGmice were protected from Dox-induced cardiac dysfunction and loss of left ventricular mass.ApomTGattenuated Dox-induced impairment in autophagic flux in vivo and accumulation of insoluble p62, which was also observed in the myocardium of patients with anthracycline-induced HF. In vehicle-treated mice, ApoM negatively regulated transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis. The effect of ApoM on TFEB required both S1P binding and S1PR3. In the presence of Dox, ApoM preserved TFEB and cardiomyocyte lysosomal abundance assessed as lysosomal associated membrane protein 1 positive structures in vivo, while S1P mimetic pretreatment of cardiomyocytes prevented Dox-induced changes in lysosomal pH.ConclusionsApoM attenuates Dox cardiotoxicity via the autophagy-lysosome pathway. The association between ApoM and reduced mortality may be explained by its role in sustaining autophagy.HighlightsCirculating ApoM is inversely associated with survival in human anthracycline-induced cardiomyopathyAnthracycline treatment reduces circulating ApoM in humans and miceIncreasing ApoM attenuates doxorubicin cardiotoxicity, lysosomal injury and preserves myocardial autophagic flux, but does not impact doxorubicin anti-neoplastic efficacyAutophagic impairment is characteristic of human anthracycline cardiomyopathy

BackgroundHigh-dimensional electronic health records (EHR) data can be used to phenotype complex diseases. The aim of this study is to apply unsupervised clustering to EHR-based traits derived in a cohort of patients with heart failure (HF) from a large integrated health system.MethodsUsing the institutional EHR, we identified 8569 patients with HF and extracted 1263 EHR-based input features, including clinical, echocardiographic, and comorbidity data, prior to the time of HF diagnosis. Principal component analysis, Uniform Manifold Approximation and Projection, and spectral clustering were applied to the input features after sex stratification of the cohort. The optimal number of clusters for each sex-stratified group was selected by highest Silhouette score and by within-cluster and between-cluster sums of squares. Determinants of cluster assignment were evaluated.ResultsWe identified four clusters in each of the female-only (44%) and male-only (56%) cohorts. Sex-specific cohorts differed significantly by age of HF diagnosis, left ventricular chamber size, markers of renal and hepatic function, and comorbidity burden (all pConclusionReadily available EHR data collected in the course of routine care can be leveraged to accurately classify patients into major phenotypic HF subtypes using data driven approaches.

Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the regulatory landscape in heart disease is unclear. Here, we use RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from up to 26 healthy controls, 18 individuals with idiopathic dilated cardiomyopathy (DCM), and five fetal hearts. Healthy individuals have a highly reproducible epigenomic landscape, consisting of more than 33,000 predicted heart enhancers. In contrast, we observe reproducible disease-associated changes in activity at 6,850 predicted heart enhancers. Combined analysis of adult and fetal samples reveals that the heart disease epigenome and transcriptome both acquire fetal-like characteristics, with 3,400 individual enhancers sharing fetal regulatory properties. We also provide a comprehensive data resource (http://heart.lbl.gov) for the mechanistic exploration of DCM etiology.

Background: The TOPCAT trial (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial) suggested clinical benefits of spironolactone treatment among patients with heart failure with preserved ejection fraction enrolled in the Americas. However, a comprehensive assessment of biologic pathways impacted by spironolactone therapy in heart failure with preserved ejection fraction has not been performed. Methods: We conducted aptamer-based proteomic analysis utilizing 5284 modified aptamers to 4928 unique proteins on plasma samples from TOPCAT participants from the Americas (n=164 subjects with paired samples at baseline and 1 year) to identify proteins and pathways impacted by spironolactone therapy in heart failure with preserved ejection fraction. Mean percentage change from baseline was calculated for each protein. Additionally, we conducted pathway analysis of proteins altered by spironolactone. Results: Spironolactone therapy was associated with proteome-wide significant changes in 7 proteins. Among these, CARD18 (caspase recruitment domain-containing protein 18), PKD2 (polycystin 2), and PSG2 (pregnancy-specific glycoprotein 2) were upregulated, whereas HGF (hepatic growth factor), PLTP (phospholipid transfer protein), IGF2R (insulin growth factor 2 receptor), and SWP70 (switch-associated protein 70) were downregulated. CARD18, a caspase-1 inhibitor, was the most upregulated protein by spironolactone (−0.5% with placebo versus +66.5% with spironolactone, P Conclusions: Proteomic analysis in the TOPCAT trial revealed proteins and pathways altered by spironolactone, including the caspase inhibitor CARD18 and multiple pathways that involved collagens. In addition to effects on fibrosis, our studies suggest potential antiapoptotic effects of spironolactone in heart failure with preserved ejection fraction, a hypothesis that merits further exploration.

Objectives This study sought to assess if clinical phenogroups differ in comprehensive biomarker profiles, cardiac and arterial structure/function, and responses to spironolactone therapy. Background Previous studies identified distinct subgroups (phenogroups) of patients with heart failure with preserved ejection fraction (HFpEF). Methods Among TOPCAT (Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist Trial) participants, we performed latent-class analysis to identify HFpEF phenogroups based on standard clinical features and assessed differences in multiple biomarkers measured from frozen plasma; cardiac and arterial structure/function measured with echocardiography and arterial tonometry; prognosis; and response to spironolactone. Results Three HFpEF phenogroups were identified. Phenogroup 1 (n = 1,214) exhibited younger age, higher prevalence of smoking, preserved functional class, and the least evidence of left ventricular (LV) hypertrophy and arterial stiffness. Phenogroup 2 (n = 1,329) was older, with normotrophic concentric LV remodeling, atrial fibrillation, left atrial enlargement, large-artery stiffening, and biomarkers of innate immunity and vascular calcification. Phenogroup 3 (n = 899) demonstrated more functional impairment, obesity, diabetes, chronic kidney disease, concentric LV hypertrophy, high renin, and biomarkers of tumor necrosis factor-alpha–mediated inflammation, liver fibrosis, and tissue remodeling. Compared with phenogroup 1, phenogroup 3 exhibited the highest risk of the primary endpoint of cardiovascular death, heart failure hospitalization, or aborted cardiac arrest (hazard ratio [HR]: 3.44; 95% confidence interval [CI]: 2.79 to 4.24); phenogroups 2 and 3 demonstrated similar all-cause mortality (phenotype 2 HR: 2.36; 95% CI: 1.89 to 2.95; phenotype 3 HR: 2.26, 95% CI: 1.77 to 2.87). Spironolactone randomized therapy was associated with a more pronounced reduction in the risk of the primary endpoint in phenogroup 3 (HR: 0.75; 95% CI: 0.59 to 0.95; p for interaction = 0.016). Results were similar after excluding participants from Eastern Europe. Conclusions We identified important differences in circulating biomarkers, cardiac/arterial characteristics, prognosis, and response to spironolactone across clinical HFpEF phenogroups. These findings suggest distinct underlying mechanisms across clinically identifiable phenogroups of HFpEF that may benefit from different targeted interventions.

Background Better risk stratification strategies are needed to enhance clinical care and trial design in heart failure with preserved ejection fraction (HFpEF). Objectives The purpose of this study was to assess the value of a targeted plasma multi-marker approach to enhance our phenotypic characterization and risk prediction in HFpEF. Methods In this study, the authors measured 49 plasma biomarkers from TOPCAT (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist) trial participants (n = 379) using a Multiplex assay. The relationship between biomarkers and the risk of all-cause death or heart failure-related hospital admission (DHFA) was assessed. A tree-based pipeline optimizer platform was used to generate a multimarker predictive model for DHFA. We validated the model in an independent cohort of HFpEF patients enrolled in the PHFS (Penn Heart Failure Study) (n = 156). Results Two large, tightly related dominant biomarker clusters were found, which included biomarkers of fibrosis/tissue remodeling, inflammation, renal injury/dysfunction, and liver fibrosis. Other clusters were composed of neurohormonal regulators of mineral metabolism, intermediary metabolism, and biomarkers of myocardial injury. Multiple biomarkers predicted incident DHFA, including 2 biomarkers related to mineral metabolism/calcification (fibroblast growth factor-23 and OPG [osteoprotegerin]), 3 inflammatory biomarkers (tumor necrosis factor-alpha, sTNFRI [soluble tumor necrosis factor-receptor I], and interleukin-6), YKL-40 (related to liver injury and inflammation), 2 biomarkers related to intermediary metabolism and adipocyte biology (fatty acid binding protein-4 and growth differentiation factor-15), angiopoietin-2 (related to angiogenesis), matrix metalloproteinase-7 (related to extracellular matrix turnover), ST-2, and N-terminal pro–B-type natriuretic peptide. A machine-learning–derived model using a combination of biomarkers was strongly predictive of the risk of DHFA (standardized hazard ratio: 2.85; 95% confidence interval: 2.03 to 4.02; p Conclusions Various novel circulating biomarkers in key pathophysiological domains are predictive of outcomes in HFpEF, and a multimarker approach coupled with machine-learning represents a promising strategy for enhancing risk stratification in HFpEF.

In heart failure with reduced ejection fraction (HFrEF), elevated soluble neprilysin (sNEP) levels are associated with an increased risk of cardiovascular death, and its inhibition with sacubitril/valsartan has improved survival.This study sought to determine the relevance of sNEP as a biomarker in heart failure with preserved ejection fraction (HFpEF) and to compare circulating sNEP levels in patients with HFpEF with normal controls.A case-control study was performed in 242 symptomatic patients with HFpEF previously enrolled in the Phosphodiesterase-5 Inhibition to Improve Clinical Status and Exercise Capacity in Heart Failure with Preserved Ejection Fraction (RELAX) and Nitrates's Effect on Activity Tolerance in Heart Failure With Preserved Ejection (NEAT-HFpEF) clinical trials and 891 asymptomatic subjects without HF or diastolic dysfunction (confirmed by NT-proBNP levels 200 pg/ml and echocardiography) who were enrolled in the Prevalence of Asymptomatic Left Ventricular Dysfunction study. sNEP was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) in all subjects.Overall, sNEP levels were lower in HFpEF compared with controls (3.5 ng/ml; confidence interval [CI]: 2.5 to 4.8 vs. 8.5 ng/ml; CI: 7.2 to 10.0; p 0.001). After adjusting for age, gender, body mass index (BMI), and smoking history, mean sNEP levels were also lower in HFpEF compared with controls (4.0 ng/ml [CI: 2.7 to 5.4] vs. 8.2 ng/ml [CI: 6.8 to 9.7]; p = 0.002). The cohorts were propensity matched based on age, BMI, diabetes, hypertension, smoking history, and renal function, and sNEP levels remained lower in HFpEF compared with controls (median 2.4 ng/ml [interquartile range: 0.6 to 27.7] vs. 4.9 ng/ml [interquartile range: 1.2 to 42.2]; p = 0.02).Patients with HFpEF on average have significantly lower circulating sNEP levels compared with controls. These findings challenge our current understanding of the complex biology of circulating sNEP in HFpEF.

Introduction: The contribution of common genetic variation to heart failure (HF) risk has not been fully elucidated. Here, we applied multi-ancestry and multivariate methods to summary genetic data from >1 million individuals to improve power to identify common genetic variants, genes, cells, tissues, and circulating proteins/metabolites associated with HF and related cardiac imaging traits. Methods: Trans-ancestry meta-analysis of HF (56,722 cases and 1,133,054 controls) was performed using METAL. Multivariate analysis including GWAS of HF and imaging traits (MRI and echocardiogram) was performed using N-GWAMA. Downstream transcriptome-wide association studies (TWAS; S-PrediXcan), tissue/cell enrichment (LDSC-SEG using RNAseq and snRNAseq of human left ventricle samples from the MAGnet consortium), and Mendelian randomization (MR) analyses were performed. Results: The multi-ancestry HF GWAS identified 15 loci associated with all-cause HF (p < 5 x 10 -8 ). Multivariate analysis identified 48 (16 novel) loci (p < 5 x 10 -8 ), with enrichment for loci near Mendelian cardiomyopathy genes (p < 1 x 10 -4 ). Genetic associations were enriched (FDR < 0.05) for cardiac and musculoskeletal gene expression and chromatin marks. Gene expression (p = 0.007) and splicing events (p = 0.01) were enriched for established cardiomyopathy genes. Branch chain amino acid dehydrogenase ( BCKDHA ) expression was prioritized in TWAS, and MR identified causal associations between circulating branch chain amino acids and cardiac imaging traits: LVEDV MRI (leucine β = -0.137, 95% CI -0.25 to -0.022, p = 0.02; isoleucine β = -0.276, 95% CI -0.38 to -0.17, p = 3 x 10 -7 ) and LVSEV MRI (leucine β = -.131, 95% CI -0.24 to -0.026, p = 0.01; isoleucine β = -0.217, 95% CI -0.33 to -0.11, p = 1 x 10 -4 ). Unbiased proteome-wide MR of 725 circulating proteins identified 18 significant (FDR < 0.05) causal protein-trait associations, including between lipoprotein(a) and HF (OR 1.09 per 1-SD increase in lipoprotein(a), 95% CI 1.06 to 1.11, p = 1.6 x 10 -11 ). Conclusion: These analyses implicate novel common genetic variation in the pathogenesis of HF, highlight Mendelian cardiomyopathy genes in common HF, and identify circulating metabolites and proteins that may represent new treatment targets.

Introduction: MTSS1 is an I-BAR protein that interacts with the actin cytoskeleton and the cell membrane and is speculated to play a role in cell motility, tumor metastasis, and cardiogenesis. Enhancer variants that reduce the expression of MTSS1 in the human left ventricle have been associated with cardioprotective traits in genome-wide association studies. However, the effect of MTSS1 downregulation on the development of heart disease has not been studied in experimental models. Here we tested the hypothesis that reduction of MTSS1 protects against dilated cardiomyopathy (DCM) in vitro and in vivo . Methods: We used CRISPR-Cas9 to knock out MTSS1 in induced pluripotent stem cells (iPSCs) harboring pathogenic variants from patients with severe DCM. DCM iPSC lines with and without MTSS1 were then differentiated into cardiomyocytes and phenotyped in vitro . A DCM mouse model containing a TPM1 D230N transgene was crossed with a mouse model heterozygous for Mtss1 knockout. Progeny from this F1 cross were assessed for differences in cardiac structure and function via echocardiography. Results: MTSS1 deletion restored the beta-adrenergic response in DCM iPSC cardiomyocytes. In parallel, Mtss1 deletion partially rescued DCM-associated phenotypes including left ventricular dilation and reduced ejection fraction in mice. Conclusions: Our initial results suggest a causal link between MTSS1 reduction and cardioprotection in DCM. Inhibiting cardiac MTSS1 may hold therapeutic potential for DCM, though the cellular mechanisms through which MTSS1 affects cardiac phenotypes remain under investigation.

Truncating variants in TTN (TTNtvs) are the most common known cause of nonischemic dilated cardiomyopathy (DCM), but how TTNtvs cause disease has remained controversial. Efforts to detect truncated titin proteins in affected human DCM hearts have failed, suggesting that disease is caused by haploinsufficiency, but reduced amounts of titin protein have not yet been demonstrated. Here, we leveraged a collection of 184 explanted posttransplant DCM hearts to show, using specialized electrophoretic gels, Western blotting, allelic phasing, and unbiased proteomics, that truncated titin proteins can quantitatively be detected in human DCM hearts. The sizes of truncated proteins corresponded to that predicted by their respective TTNtvs; the truncated proteins were encoded by the TTNtv-bearing allele; and no degradation fragments from protein encoded by either allele were detectable. In parallel, full-length titin was less abundant in TTNtv(+) than in TTNtv(−) DCM hearts. Disease severity or need for transplantation did not correlate with TTNtv location. Transcriptomic profiling revealed few differences in splicing or allelic imbalance of the TTN transcript between TTNtv(+) and TTNtv(−) DCM hearts. Studies with isolated human adult cardiomyocytes revealed no defects in contractility in cells from TTNtv(+) compared to TTNtv(−) DCM hearts. Together, these data demonstrate the presence of truncated titin protein in human TTNtv(+) DCM, show reduced amounts of full-length titin protein in TTNtv(+) DCM hearts, and support combined dominant-negative and haploinsufficiency contributions to disease.

There is a paucity of data regarding sex differences in the profiles and outcomes of ambulatory patients on left ventricular assist device (LVAD) support who present to the emergency department (ED).We performed a retrospective analysis of 57,200 LVAD-related ED patient encounters from the 2010 to 2018 Nationwide Emergency Department Sample. International Classification of Diseases Clinical Modification, Ninth Revision and Tenth Revision, codes identified patients aged 18 years or older with LVADs and associated primary and comorbidity diagnoses. Clinical characteristics and outcomes were stratified by sex and compared. Multivariable logistic regression was used to evaluate predictors of hospital admission and death. Female patient encounters comprised 27.2% of ED visits and occurred at younger ages and more frequently with obesity and depression (all P.01). There were no sex differences in presentation for device complication, stroke, infection, or heart failure (all P.05); however, female patient encounters were more often respiratory- and genitourinary or gynecological related (both P.01). After adjustment for age group, diabetes, depression, and hypertension, male patient encounters had a 38% increased odds of hospital admission (95% confidence interval 1.20-1.58), but there was no sex difference in the adjusted odds of death (odds ratio 1.11, 95% confidence interval 0.86-1.45).Patient encounters of females on LVAD support have significantly different comorbidities and outcomes compared with males. Further inquiry into these sex differences is imperative to improve long-term outcomes.

Heart failure affects millions of people worldwide with mortality near 50% within five years. This disease is characterized by widespread cardiac and systemic metabolic changes, but a comprehensive evaluation of metabolism in failing human hearts is lacking. Here, we provide a comprehensive depiction of cardiac and systemic metabolic changes in 89 explanted failing and non-failing human hearts through integration of plasma and cardiac tissue metabolomics, genome-wide RNAseq, and proteomic data. The data confirm a profound bioenergetic defect in end-stage human heart failure and demonstrate extensive changes in metabolic homeostasis. The data indicate a substantial defect in fatty acid (FA) use in failing hearts, in particular unsaturated FAs. Reduction of FAs and acyl-carnitines in failing tissue in contrast to concomitant elevations in plasma suggest a defect in import of FAs into the cell, rather than a defect in FA oxidation. Intermediates of glycolysis, the pentose phosphate pathway, and glycogen synthesis are all similarly reduced, as is expression of GLUT1, indicating diminished glucose uptake. However, there was no significant change in tissue pyruvate content, suggesting an increase in lactate utilization. The data suggest increased flux of pyruvate into mitochondria, likely promoting pyruvate oxidation but not pyruvate carboxylation. Blunted anabolic pyruvate flux, in turn, likely leads to insufficient TCA cycle intermediates. Ketone levels were increased in both failing tissue and plasma, as previously reported. The phospholipid content of failing human hearts is greatly increased in both failing tissue and plasma. Nucleotide synthesis pathways also appear to be reprogrammed, with a notable decrease in adenosine metabolism, specifically. Together, these data indicate widespread change in the local cardiac and greater systemic metabolic landscape in severe human heart failure.

Aims The HERMES (HEart failure Molecular Epidemiology for Therapeutic targets) consortium aims to identify the genomic and molecular basis of heart failure.Methods and results The consortium currently includes 51 studies from 11 countries, including 68 157 heart failure cases and 949 888 controls, with data on heart failure events and prognosis. All studies collected biological samples and performed genome-wide genotyping of common genetic variants. The enrolment of subjects into participating studies ranged from 1948 to the present day, and the median follow-up following heart failure diagnosis ranged from 2 to 116 months. Forty-nine of 51 individual studies enrolled participants of both sexes; in these studies, participants with heart failure were predominantly male (34-90%). The mean age at diagnosis or ascertainment across all studies ranged from 54 to 84 years. Based on the aggregate sample, we estimated 80% power to genetic variant associations with risk of heart failure with an odds ratio of >1.10 for common variants (allele frequency > 0.05) and >1.20 for low-frequency variants (allele frequency 0.01-0.05) at P < 5 x 10(-8) under an additive genetic model.Conclusions HERMES is a global collaboration aiming to (i) identify the genetic determinants of heart failure; (ii) generate insights into the causal pathways leading to heart failure and enable genetic approaches to target prioritization; and (iii) develop genomic tools for disease stratification and risk prediction.

Heart failure (HF) is a leading cause of cardiovascular morbidity and mortality, yet the contribution of common genetic variation to HF risk has not been fully elucidated, particularly in comparison to other common cardiometabolic traits. We conducted a multi-ancestry genome-wide association study (GWAS) meta-analysis of all-cause HF including up to 56,722 HF cases and 1,133,054 controls, identifying 4 novel loci. We then performed a multi-ancestry multivariate association study of HF and related cardiac imaging endophenotypes, identifying 71 conditionally-independent variants, including 16 novel loci. Secondary colocalization and transcriptome-wide association analyses identified known and novel candidate cardiomyopathy genes, which were validated in gene-expression profiling of failing and healthy human hearts. Colocalization, gene expression profiling, and Mendelian randomization provided convergent evidence for the roles ofBCKDHAand circulating branch-chain amino acids in heart failure and cardiac structure. Finally, proteome-wide Mendelian randomization revealed 11 circulating proteins associated with HF or quantitative imaging traits. These analyses highlight similarities and differences among heart failure and associated cardiovascular imaging endophenotypes, implicate novel common genetic variation in the pathogenesis of HF, and identify circulating proteins that may represent novel cardiomyopathy treatment targets.

Aims Our objective was to better understand the genetic bases of dilated cardiomyopathy (DCM), a leading cause of systolic heart failure. Methods and results We conducted the largest genome-wide association study performed so far in DCM, with 2719 cases and 4440 controls in the discovery population. We identified and replicated two new DCM-associated loci on chromosome 3p25.1 [lead single-nucleotide polymorphism (SNP) rs62232870, P = 8.7 × 10−11 and 7.7 × 10−4 in the discovery and replication steps, respectively] and chromosome 22q11.23 (lead SNP rs7284877, P = 3.3 × 10−8 and 1.4 × 10−3 in the discovery and replication steps, respectively), while confirming two previously identified DCM loci on chromosomes 10 and 1, BAG3 and HSPB7. A genetic risk score constructed from the number of risk alleles at these four DCM loci revealed a 3-fold increased risk of DCM for individuals with 8 risk alleles compared to individuals with 5 risk alleles (median of the referral population). In silico annotation and functional 4C-sequencing analyses on iPSC-derived cardiomyocytes identify SLC6A6 as the most likely DCM gene at the 3p25.1 locus. This gene encodes a taurine transporter whose involvement in myocardial dysfunction and DCM is supported by numerous observations in humans and animals. At the 22q11.23 locus, in silico and data mining annotations, and to a lesser extent functional analysis, strongly suggest SMARCB1 as the candidate culprit gene. Conclusion This study provides a better understanding of the genetic architecture of DCM and sheds light on novel biological pathways underlying heart failure.

Background: Peripartum cardiomyopathy (PPCM) occurs in ≈1:2000 deliveries in the United States and worldwide. The genetic underpinnings of PPCM remain poorly defined. Approximately 10% of women with PPCM harbor truncating variants in TTN (TTNtvs). Whether mutations in other genes can predispose to PPCM is not known. It is also not known if the presence of TTNtvs predicts clinical presentation or outcomes. Nor is it known if the prevalence of TTNtvs differs in women with PPCM and preeclampsia, the strongest risk factor for PPCM. Methods: Women with PPCM were retrospectively identified from several US and international academic centers, and clinical information and DNA samples were acquired. Next-generation sequencing was performed on 67 genes, including TTN , and evaluated for burden of truncating and missense variants. The impact of TTNtvs on the severity of clinical presentation, and on clinical outcomes, was evaluated. Results: Four hundred sixty-nine women met inclusion criteria. Of the women with PPCM, 10.4% bore TTNtvs (odds ratio=9.4 compared with 1.2% in the reference population; Bonferroni-corrected P [ P *]=1.2×10 –46 ). We additionally identified overrepresentation of truncating variants in FLNC (odds ratio=24.8, P *=7.0×10 –8 ), DSP (odds ratio=14.9, P *=1.0×10 –8 ), and BAG3 (odds ratio=53.1, P *=0.02), genes not previously associated with PPCM. This profile is highly similar to that found in nonischemic dilated cardiomyopathy. Women with TTNtvs had lower left ventricular ejection fraction on presentation than did women without TTNtvs (23.5% versus 29%, P =2.5×10 –4 ), but did not differ significantly in timing of presentation after delivery, in prevalence of preeclampsia, or in rates of clinical recovery. Conclusions: This study provides the first extensive genetic and phenotypic landscape of PPCM and demonstrates that predisposition to heart failure is an important risk factor for PPCM. The work reveals a degree of genetic similarity between PPCM and dilated cardiomyopathy, suggesting that gene-specific therapeutic approaches being developed for dilated cardiomyopathy may also apply to PPCM, and that approaches to genetic testing in PPCM should mirror those taken in dilated cardiomyopathy. Last, the clarification of genotype/phenotype associations has important implications for genetic counseling.

Background Inherited cardiomyopathies display variable penetrance and expression, and a component of phenotypic variation is genetically determined. To evaluate the genetic contribution to this variable expression, we compared protein coding variation in the genomes of those with hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Methods and Results Nonsynonymous single‐nucleotide variants (nsSNVs) were ascertained using whole genome sequencing from familial cases of HCM (n=56) or DCM (n=70) and correlated with echocardiographic information. Focusing on nsSNVs in 102 genes linked to inherited cardiomyopathies, we correlated the number of nsSNVs per person with left ventricular measurements. Principal component analysis and generalized linear models were applied to identify the probability of cardiomyopathy type as it related to the number of nsSNVs in cardiomyopathy genes. The probability of having DCM significantly increased as the number of cardiomyopathy gene nsSNVs per person increased. The increase in nsSNVs in cardiomyopathy genes significantly associated with reduced left ventricular ejection fraction and increased left ventricular diameter for individuals carrying a DCM diagnosis, but not for those with HCM. Resampling was used to identify genes with aberrant cumulative allele frequencies, identifying potential modifier genes for cardiomyopathy. Conclusions Participants with DCM had more nsSNVs per person in cardiomyopathy genes than participants with HCM. The nsSNV burden in cardiomyopathy genes did not correlate with the probability or manifestation of left ventricular measures in HCM. These findings support the concept that increased variation in cardiomyopathy genes creates a genetic background that predisposes to DCM and increased disease severity.

Background Diurnal variation of natriuretic peptide (NP) levels and its relationship with 24-h blood pressure (BP) rhythm has not been established. Obese individuals have a relative NP deficiency and disturbed BP rhythmicity. Objectives This clinical trial evaluated the diurnal rhythmicity of NPs (B-type natriuretic peptide [BNP], mid-regional pro-atrial natriuretic peptide [MR-proANP], N-terminal pro–B-type natriuretic peptide [NT-proBNP]) and the relationship of NP rhythm with 24-h BP rhythm in healthy lean and obese individuals. Methods On the background of a standardized diet, healthy, normotensive, lean (body mass index 18.5 to 25 kg/m2) and obese (body mass index 30 to 45 kg/m2) individuals, age 18 to 40 years, underwent 24-h inpatient protocol involving ambulatory BP monitoring starting 24 h prior to the visit, controlled light intensity, and repeated blood draws for assessment of analytes. Cosinor analysis of normalized NP levels (normalized to 24-h mean value) was conducted to assess the diurnal NP rhythm and its relationship with systolic BP. Results Among 52 participants screened, 40 participants (18 lean, 22 obese; 50% women; 65% Black) completed the study. The median range spread (percentage difference between the minimum and maximum values) over 24 h for MR-proANP, BNP, and NT-proBNP levels was 72.0% (interquartile range [IQR]: 50.9% to 119.6%), 75.5% (IQR: 50.7% to 106.8%), and 135.0% (IQR: 66.3% to 270.4%), respectively. A cosine wave-shaped 24-h oscillation of normalized NP levels (BNP, MR-proANP, and NT-proBNP) was noted both in lean and obese individuals (prhythmicity Conclusions This human physiological trial elucidates evidence of diurnal NP rhythmicity and the presence of an NP-BP rhythm axis. There exists a misalignment of the NP-BP diurnal rhythm in the obese, which may contribute to the disturbed diurnal BP pattern observed among obese individuals. (The Diurnal Rhythm in Natriuretic Peptide Levels; NCT03834168 )

Visual Abstract, Highlights • DM is a significant risk factor for major adverse cardiovascular events in patients with HFpEF. • Patients with diabetes with HFpEF have a distinct proteome compared with patients without diabetes with HFpEF. • Proteomics analysis identified higher levels of alpha-1-microglobulin/bikunin precursor protein in patients with diabetes with HFpEF and lower levels of CILP2 and Apo M. • Lower Apo M levels mediate most of the association between diabetes and major adverse cardiovascular events in HFpEF., Summary Diabetes mellitus (DM) is associated with a higher risk of heart failure hospitalization and mortality in patients with heart failure with preserved ejection fraction (HFpEF). Using SomaScan assays and proteomics analysis of plasma from participants in the TOPCAT (Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist) trial and the Penn Heart Failure Study, this study identified 10 proteins with significantly different expression in patients with HFpEF and DM. Of these, apolipoprotein M was found to mediate 72% (95% CI: 36% to 100%; p

Background: Obese individuals have disturbed blood pressure (BP) rhythmicity and relative natriuretic peptide (NP) deficiency. The relationship of diurnal variation in NP levels and 24h BP rhythm is not known. Furthermore, mechanisms behind difference in circulating NPs in healthy obese and lean individuals has not been explored. We conducted a prospective clinical trial to evaluate 1) the diurnal rhythmicity of NPs and its relationship with 24-hour BP rhythm between healthy lean and obese individuals, and 2) elucidate mechanism behind NP deficiency in obese. Methods: Healthy, normotensive, lean (BMI:18.5-25 kg/m 2 ) and obese (BMI:30-45 kg/m 2 ) individuals aged 18-40 years, underwent 24-hour standardized inpatient protocol involving ambulatory BP monitoring (ABPM), controlled light intensity, and 10 blood draws, following 5-days of standardized diet. NP gene expression was evaluated in a cohort of 37 healthy donor heart tissues obtained from the MAGNet repository. Results: Among 52 participants screened, a total of 40 participants (18 lean; 22 obese) were enrolled. Diurnal variation in MRproANP levels was seen in both lean and obese individuals (p for rhythmicity=0.001). The mesor of the NP rhythm was 15% (8.5-21.6%) lower in obese. The diurnal variation in MRproANP was in antiphase with diurnal variation of systolic BP (pFigure ). Obese participants had lower 24h renin levels (p=0.06) and higher nocturnal sodium excretion (p=0.08). Among obese, there was lower expression of NP production genes ( NPPA, NPPB) (p, and higher expression of clearance gene ( NPR3 ) (p Conclusions: In a mechanistic human trial, we elucidate key neurohormonal differences in rhythm and evidence of poor salt handling in obese. Decreased production and increased clearance may contribute to the NP deficiency in obese. Targeting the diurnal NP-BP rhythm axis may reduce the cardiovascular risk burden, specifically in obese individuals.

BACKGROUND: The failing heart is characterized by changes in gene expression. However, the regulatory regions of the genome that drive these gene expression changes have not been well defined in human hearts. METHODS: To define genomewide enhancer and promoter use in heart failure, Cap Analysis of Gene Expression (CAGE sequencing) was applied to three healthy and four failed human hearts to identify promoter and enhancer regions used in left ventricles. Healthy hearts were derived from donors unused for transplantation and failed hearts were obtained as discarded tissue after transplantation. RESULTS: CAGE sequencing identified a combined potential for ~23,000 promoters and ~5000 enhancers active in human left ventricles. Of these, 17,000 promoters and 1800 enhancers had additional support for their regulatory function. Comparing promoter usage between healthy and failed hearts highlighted promoter shifts which altered amino-terminal protein sequences. Enhancer usage between healthy and failed hearts identified a majority of differentially utilized heart failure enhancers were intronic and primarily localized within the first intron, revealing this position as a common feature associated with tissue-specific gene expression changes in the heart. CONCLUSIONS: This dataset defines the dynamic genomic regulatory landscape underlying heart failure and serves as an important resource for understanding genetic contributions to cardiac dysfunction. Additionally, regulatory changes contributing to heart failure are attractive therapeutic targets for controlling ventricular remodeling and clinical progression.

ACE2 (angiotensin-converting enzyme 2) is a key component of the renin-angiotensin-aldosterone system. Yet, little is known about the clinical and biologic correlates of circulating ACE2 levels in humans. We assessed the clinical and proteomic correlates of plasma (soluble) ACE2 protein levels in human heart failure. We measured plasma ACE2 using a modified aptamer assay among PHFS (Penn Heart Failure Study) participants (n=2248). We performed an association study of ACE2 against ≈5000 other plasma proteins measured with the SomaScan platform. Plasma ACE2 was not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 was associated with older age, male sex, diabetes mellitus, a lower estimated glomerular filtration rate, worse New York Heart Association class, a history of coronary artery bypass surgery, and higher pro-BNP (pro-B-type natriuretic peptide) levels. Plasma ACE2 exhibited associations with 1011 other plasma proteins. In pathway overrepresentation analyses, top canonical pathways associated with plasma ACE2 included clathrin-mediated endocytosis signaling, actin cytoskeleton signaling, mechanisms of viral exit from host cells, EIF2 (eukaryotic initiation factor 2) signaling, and the protein ubiquitination pathway. In conclusion, in humans with heart failure, plasma ACE2 is associated with various clinical factors known to be associated with severe coronavirus disease 2019 (COVID-19), including older age, male sex, and diabetes mellitus, but is not associated with ACE inhibitor and angiotensin-receptor blocker use. Plasma ACE2 protein levels are prominently associated with multiple cellular pathways involved in cellular endocytosis, exocytosis, and intracellular protein trafficking. Whether these have a causal relationship with ACE2 or are relevant to novel coronavirus-2 infection remains to be assessed in future studies.

Background: Apo (apolipoprotein) M mediates the physical interaction between high-density lipoprotein (HDL) particles and sphingosine-1-phosphate (S1P). Apo M exerts anti-inflammatory and cardioprotective effects in animal models. Methods: In a subset of PHFS (Penn Heart Failure Study) participants (n=297), we measured apo M by Enzyme-Linked ImmunoSorbent Assay (ELISA). We also measured total S1P by liquid chromatography–mass spectrometry and isolated HDL particles to test the association between apo M and HDL-associated S1P. We confirmed the relationship between apo M and outcomes using modified aptamer-based apo M measurements among 2170 adults in the PHFS and 2 independent cohorts: the Washington University Heart Failure Registry (n=173) and a subset of TOPCAT (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial; n=218). Last, we examined the relationship between apo M and ≈5000 other proteins (SomaScan assay) to identify biological pathways associated with apo M in heart failure. Results: In the PHFS, apo M was inversely associated with the risk of death (standardized hazard ratio, 0.56 [95% CI, 0.51–0.61]; P P P P =0.001) in models that adjusted for multiple confounders. This association was present in both heart failure with reduced and preserved ejection fraction and was replicated in the Washington University cohort and a cohort with heart failure with preserved ejection fraction only (TOPCAT). The S1P and apo M content of isolated HDL particles strongly correlated ( R =0.81, P Conclusions: Reduced circulating apo M is independently associated with adverse outcomes across the spectrum of human heart failure. Further research is needed to assess whether the apo M/S1P axis is a suitable therapeutic target in heart failure.

SummaryWe present the results of the largest genome wide association study (GWAS) performed so far in dilated cardiomyopathy (DCM), a leading cause of systolic heart failure and cardiovascular death, with 2,719 cases and 4,440 controls in the discovery population. We identified and replicated two new DCM-associated loci, one on chromosome 3p25.1 (lead SNP rs62232870, p = 8.7 × 10−11 and 7.7 × 10−4 in the discovery and replication step, respectively) and the second on chromosome 22q11.23 (lead SNP rs7284877, p = 3.3 × 10−8 and 1.4 × 10−3 in the discovery and replication step, respectively) while confirming two previously identified DCM loci on chromosome 10 and 1, BAG3 and HSPB7. The genetic risk score constructed from the number of lead risk-alleles at these four DCM loci revealed that individuals with 8 risk-alleles were at a 27% increased risk of DCM compared to individuals with 5 risk alleles (median of the referral population). We estimated the genome wide heritability at 31% ± 8%.In silico annotation and functional 4C-sequencing analysis on iPSC-derived cardiomyocytes strongly suggest SLC6A6 as the most likely DCM gene at the 3p25.1 locus. This gene encodes a taurine and beta-alanine transporter whose involvement in myocardial dysfunction and DCM is supported by recent observations in humans and mice. Although less easy to discriminate the better candidate at the 22q11.23 locus, SMARCB1 appears as the strongest one.This study provides both a better understanding of the genetic architecture of DCM and new knowledge on novel biological pathways underlying heart failure, with the potential for a therapeutic perspective.