Search

The mechanical environment of the myocardium has a potent effect on cardiomyocyte form and function, yet an understanding of the cardiomyocyte responses to extracellular stiffening remains incomplete. We therefore employed a cell culture substrate with tunable stiffness to define the cardiomyocyte responses to clinically relevant stiffness increments in the absence of cell-cell interactions. When cultured on substrates magnetically actuated to mimic the stiffness of diseased myocardium, isolated rat adult cardiomyocytes exhibited a time-dependent reduction of sarcomere shortening, characterized by slowed contraction and relaxation velocity, and alterations of the calcium transient. Cardiomyocytes cultured on stiff substrates developed increases in viscoelasticity and microtubule detyrosination in association with early increases in the α-tubulin detyrosinating enzyme vasohibin-2 (Vash2). We found that knockdown of Vash2 was sufficient to preserve contractile performance as well as calcium transient properties in the presence of extracellular substrate stiffening. Orthogonal prevention of detyrosination by overexpression of tubulin tyrosine ligase (TTL) was also able to preserve contractility and calcium homeostasis. These data demonstrate that a pathologic increment of extracellular stiffness induces early, cell-autonomous remodeling of adult cardiomyocytes that is dependent on detyrosination of α-tubulin.

INTRODUCTION: As many as 100 million people in the US have nonalcoholic fatty liver disease (NAFLD), characterized by increased liver lipid accumulation, which often leads to hepatocyte injury and fibrosis, characteristics of nonalcoholic steatohepatitis (NASH). NASH in turn can progress to cirrhosis and hepatocellular carcinoma. There are currently no US Food and Drug Administration–approved therapies for NAFLD or NASH. NAFLD occurs when there is disequilibrium between the processes of hepatic lipid synthesis and consumption. The nutrient sensor mechanistic target of rapamycin complex 1 (mTORC1) regulates several of these pathways. mTORC1 is thus an attractive target to modulate lipid homeostasis in the liver. However, mTORC1 also regulates numerous other cellular pathways, and blunting of mTORC1 modulation can lead to unexpected feedback loops and unwanted effects. RATIONALE: We hypothesized that selective modulation of hepatic mTORC1 signaling could benefit liver lipid metabolism and prevent NAFLD. In non-liver cell types, the protein folliculin (FLCN) has been shown to confer substrate specificity to mTORC1. Deletion of FLCN inhibits mTORC1-mediated phosphorylation of the transcription factor E3/B (TFE3/B) family of transcription factors, without affecting mTORC1-driven phosphorylation of its canonical substrates ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1). Unphosphorylated TFE3 translocates to the nucleus and activates genes that promote lysosomal biogenesis, mitochondrial biogenesis, and oxidative metabolism. We reasoned that suppression of FLCN in the liver might promote fatty acid oxidation and lipid clearance without untoward effects of generalized mTORC1 inhibition. RESULTS: Hepatocyte-specific genetic deletion of Flcn in adult mice selectively inhibited mTORC1-mediated cytoplasmic sequestration of TFE3, with little effect on other mTORC1 targets, including S6K, 4E-BP1, and Lipin1. Hepatocyte loss of Flcn protected mice from both NAFLD and NASH and partially reversed these processes when already established. The protection against NAFLD and NASH required TFE3, which activated lipid clearance. Unleashed TFE3 additionally suppressed de novo lipogenesis. The latter was mediated in part by TFE3-mediated induction of insulin-induced gene 2 (Insig2) to inhibit proteolytic activation of sterol regulatory element–binding protein-1c (SREBP-1c), a critical lipogenic transcription factor. CONCLUSION: Our data establish FLCN as a critical regulator of lipid homeostasis in the liver. Flcn deletion affords selective inhibition of mTORC1, leading to nuclear translocation and activation of the transcription factor TFE3, which coordinates hepatic lipid metabolic pathways to protect against NAFLD and NASH in mice. Thus, our data reveal FLCN as a promising target for the treatment of NAFLD and NASH. The data also illuminate previously published and seemingly conflicting data, which likely reflected different effects on each arm of mTORC1 signaling. There have been numerous attempts by many to develop disease-specific treatments for NAFLD and NASH, thus far without success. A recurrent problem has been the many compensatory responses by the liver to targeting any one pathway; for example, inhibitors of acetyl–coenzyme A carboxylase led to compensatory activation of SREBP-1c and consequent hyperlipidemia. Targeting FLCN is thus particularly attractive, in that loss of FLCN simultaneously and favorably affects multiple aspects of hepatic lipid homeostasis, including promoting fatty acid oxidation and lysosomal biogenesis and inhibiting de novo lipogenesis.

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.

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.

New directions in material applications have allowed for the fresh insight into the coordination of biophysical cues and regulators. Although the role of the mechanical microenvironment on cell responses and mechanics is often studied, most analyses only consider static environments and behavior, however, cells and tissues are themselves dynamic materials that adapt in myriad ways to alterations in their environment. Here, we introduce an approach, through the addition of magnetic inclusions into a soft poly(dimethylsiloxane) elastomer, to fabricate a substrate that can be stiffened nearly instantaneously in the presence of cells through the use of a magnetic gradient to investigate short-term cellular responses to dynamic stiffening or softening. This substrate allows us to observe time-dependent changes, such as spreading, stress fiber formation, Yes-associated protein translocation, and sarcomere organization. The identification of temporal dynamic changes on a short time scale suggests that this technology can be more broadly applied to study targeted mechanisms of diverse biologic processes, including cell division, differentiation, tissue repair, pathological adaptations, and cell-death pathways. Our method provides a unique in vitro platform for studying the dynamic cell behavior by better mimicking more complex and realistic microenvironments. This platform will be amenable to future studies aimed at elucidating the mechanisms underlying mechanical sensing and signaling that influence cellular behaviors and interactions.

Background: Drivers of adaptive and pathologic human right ventricular (RV) remodeling are poorly defined, but there is some evidence that WNT signaling contributes to these processes. Methods: Using a candidate gene approach, we sought to identify genes specifically expressed in RV failure by assessing the expression of 28 WNT-related genes in the RVs of three groups: human explanted nonfailing donor hearts (NF, n = 29), pre-transplant cardiomyopathy hearts with preserved RV function (pRV, n = 78), and pre-transplant cardiomyopathy hearts with RV failure (RVF, n = 35). Results: We identified the noncanonical WNT receptor ROR2 as transcriptionally strongly upregulated in RVF compared to pRV and NF (p < 0.05, adjusted for multiple hypothesis testing using Benjamin-Hochberg). By western blot, ROR2 protein expression correlated to mRNA expression (R 2 = 0.41, p = 8.1 x 10-18) and increased linearly with higher right atrial to pulmonary capillary wedge ratio in RVF (R 2 =0.40, p = 3.0 x 10-5). Using trichrome, immunohistochemistry, and RNAscope, we identified preferential ROR2 mRNA and protein expression in fibrotic regions by both cardiomyocytes and noncardiomyocytes. High ROR2 expression correlated with increased expression of a known ROR2 target, the WNT/Ca2+ responsive protease calpain and its target FLNA (R 2 0.25 and 0.67, respectively, p < 0.05), as well as with increased relative phosphorylation of FLNA (R 2 0.62, p < 0.05), a marker of its activation by ROR2. Finally, mice with a range of RVF severity, generated by pulmonary artery constriction, demonstrated a robust increase in ROR2 and FLNA expression compared to sham-treated animals, and ROR2 and FLNA expression correlated strongly with each other (R 2 0.89, p = 6.9 x 10-5) and with increasing RV weight normalized to body weight (R 2 ~0.8, p < 0.001, both). Conclusion: ROR2 expression in the RV is dramatically increased in humans and mice with severe RVF, and ROR2 expression correlates with worsening RV hemodynamics, calpain expression, FLNA cleavage, and FLNA phosphorylation. Taken together, the data reveal the robust activation of noncanonical WNT signaling in human RVF, and identify ROR2-mediated cytoskeleton protein cleavage as a potential novel target of human pathologic RV remodeling.

Background: No medical therapies exist to treat right ventricular (RV) remodeling and RV failure (RVF), in large part because molecular pathways that are specifically activated in pathologic human RV remodeling remain poorly defined. Murine models have suggested involvement of Wnt signaling, but this has not been well-defined in human RVF. Methods: Using a candidate gene approach, we sought to identify genes specifically expressed in human pathologic RV remodeling by assessing the expression of 28 WNT-related genes in the RVs of three groups: explanted nonfailing donors (NF, n = 29), explanted dilated and ischemic cardiomyopathy, obtained at the time of cardiac transplantation, either with preserved RV function (pRV, n = 78) or with RVF (n = 35). Results: We identified the noncanonical WNT receptor ROR2 as transcriptionally strongly upregulated in RVF compared to pRV and NF (Benjamini-Hochberg adjusted P < 0.05). ROR2 protein expression correlated linearly to mRNA expression (R 2 = 0.41, P = 8.1 × 10-18) among all RVs, and to higher right atrial to pulmonary capillary wedge ratio in RVF (R 2 = 0.40, P = 3.0 × 10-5). Utilizing Masson's trichrome and ROR2 immunohistochemistry, we identified preferential ROR2 protein expression in fibrotic regions by both cardiomyocytes and noncardiomyocytes. We compared RVF with high and low ROR2 expression, and found that high ROR2 expression was associated with increased expression of the WNT5A/ROR2/Ca2+ responsive protease calpain-μ, cleavage of its target FLNA, and FLNA phosphorylation, another marker of activation downstream of ROR2. ROR2 protein expression as a continuous variable, correlated strongly to expression of calpain-μ (R 2 = 0.25), total FLNA (R 2 = 0.67), calpain cleaved FLNA (R 2 = 0.32) and FLNA phosphorylation (R 2 = 0.62, P < 0.05 for all). Conclusion: We demonstrate robust reactivation of a fetal WNT gene program, specifically its noncanonical arm, in human RVF characterized by activation of ROR2/calpain mediated cytoskeleton protein cleavage.

Endothelial function may be deranged in heart failure with preserved ejection fraction (HFpEF). Serum NO-derived metabolites (NO m ) might provide a biochemical surrogate of endothelial function in patients with heart failure (HF). We measured serum NO m in 415 participants in the Penn HF Study. Participants with HFpEF (n = 82) and those whose EF had recovered (Recovered-HF, n = 125) were matched 1:1 to heart failure with reduced ejection fraction (HFrEF) participants based on age, gender, race, tobacco use, and eGFR. Serum NO m levels were quantified after chemical reduction coupled with gas-phase chemiluminescence detection. After adjustment for matching covariates and BMI, HFpEF (34.5 μM; interquartile range [IQR] 25.0, 51.5) participants had lower NO m levels than HFrEF (41.0 μM; IQR 28.3, 58.0; ratio of HFpEF:HFrEF 0.82, 95% confidence interval [CI] 0.67 to 0.99; p = 0.04), which further decreased when adjusted for covariates that affect endothelial function (ratio 0.79, 95% CI 0.65 to 0.98; p = 0.03). There were no differences between HFrEF (34.0; IQR 25.3, 49.0) and matched Recovered-HF (36.0 μM; IQR 25.0, 55.0) or HFpEF and Recovered-HF. Age (+21%/10-year increase, p m in HFpEF, whereas age (+11%/10-year increase, p = 0.03), current tobacco use (+67%, p = 0.01), and eGFR (p = 0.01) associated with NO m in Recovered-HF. In conclusion, HFpEF participants have reduced NO m compared with HFrEF in this matched cohort. This might suggest either compromised endothelial function or poor dietary intake. Black race was associated with lower NO m in HFpEF.

Background: A greater understanding of the gene programs that drive adaptive and pathologic right ventricle (RV) remodeling are needed given the poor response of the RV to standard reverse remodeling agents and the absence of RV-specific therapies. Left ventricular failure (LVF) provides an ideal model to study human RV remodeling given the spectrum of secondary RV involvement. Methods: Dilated (DCM) and ischemic cardiomyopathy (ICM) hearts in the Penn Human Heart Tissue Bank were identified as having preserved RV function (pRV) or RVF using preexplant right atrial to wedge pressure ratio. Using RT-PCR and a candidate gene approach, we assessed RV differential expression of 28 WNT -related genes and NPPA/NPPB as positive controls between nonfailing (NF, n= 29), DCM-pRV (n = 47), DCM-RVF (n = 26), ICM-pRV (n = 31), and ICM-RVF (n = 10). Genes which met significance for differential expression between NF, pRV, and RVF for both DCM and ICM using Kruskal-Wallis were further assessed by Mann Whitney U comparing pRV to RVF after combining ICM and DCM groups (Benjamini-Hochberg p < 0.05 for significance). Results: Most (20/28) WNT -related genes were differentially expressed in at least one cardiomyopathy group, but only 13, as well as NPPA, were significant for both DCM and ICM. Only, CREBBP, NFATC2, and ROR2 were differentially expressed between pRV and RVF (Table). Discussion: WNT -related genes show significant DGE in RV remodeling. But, of those tested, only CREBBP, NFATC2, and ROR2 appear to be implicated in the progression from adaptive to pathologic RV remodeling. Additional studies are underway to confirm differential protein expression.

Background: The effects of thyroid dysfunction in patients with preexisting heart failure have not been adequately studied. We examined the prevalence of thyroid dysfunction and associations with cardiovascular outcomes in a large, prospective cohort of outpatients with preexisting heart failure. Methods and Results: We examined associations between thyroid dysfunction and New York Heart Association class, atrial fibrillation, and a composite end point of ventricular assist device placement, heart transplantation, or death in 1365 participants with heart failure enrolled in the Penn Heart Failure Study. Mean age was 57 years, 35% were women, and the majority had New York Heart Association class II (45%) or III (32%) symptoms. More severe heart failure was associated with higher thyroid-stimulating hormone (TSH), higher free thyroxine (FT4), and lower total triiodothyronine (TT3) concentrations ( P P ≤0.01 all models). There were 462 composite end points over a median 4.2 years of follow-up. In adjusted models, compared with euthyroidism, subclinical hypothyroidism (TSH 4.51–19.99 mIU/L with normal FT4) was associated with an increased risk of the composite end point overall (hazard ratio, 1.82; 95% CI, 1.27–2.61; P =0.001) and in the subgroup with TSH ≥7.00 mIU/L (hazard ratio, 3.25; 95% CI, 1.96–5.39; P P =0.34). Isolated low T3 was also associated with the composite end point (hazard ratio, 2.12; 95% CI, 1.65–2.72; P Conclusions: In patients with preexisting heart failure, subclinical hypothyroidism with TSH ≥7 mIU/L and isolated low T3 levels are associated with poor prognosis. Clinical trials are needed to explore therapeutic effects of T4 and T3 administration in heart failure.

Clinical studies have suggested the prognostic value of galectin-3, a marker of fibrosis, in chronic heart failure. However, the specific role of galectin-3, compared with established biomarkers, remains uncertain.The Penn Heart Failure Study was an ambulatory heart failure cohort that included 1385 participants with reduced (1141), preserved (106), and recovered (138) left ventricular ejection fraction (LVEF). Cox regression models determined the association between galectin-3 and risk of all-cause mortality, cardiac transplantation, or placement of a ventricular assist device. Receiver operating characteristic curves compared the prognostic accuracy of galectin-3, high-sensitivity soluble Toll-like receptor 2 (ST2), troponin I, and B-type natriuretic peptide (BNP) at 1 and 5 years. Higher galectin-3 levels were associated with an increased risk of adverse events (adjusted hazard ratio of 1.96 for each doubling in galectin-3; P .001). This association was most pronounced among participants with preserved LVEF (adjusted hazard ratio 3.30; P .001). At 5 years, galectin-3 was the most accurate discriminator of risk among participants with preserved LVEF (area under the curve 0.782; P = .81 vs high-sensitivity ST2; P = .029 vs troponin I; P = .35 vs BNP). BNP was most accurate among participants with reduced and recovered LVEF (areas under the curves 0.716 and 0.728, respectively).Galectin-3 could have prognostic value for long-term events among patients with heart failure and preserved ejection fraction.

Background— The failing human heart is characterized by metabolic abnormalities, but these defects remains incompletely understood. In animal models of heart failure there is a switch from a predominance of fatty acid utilization to the more oxygen-sparing carbohydrate metabolism. Recent studies have reported decreases in myocardial lipid content, but the inclusion of diabetic and nondiabetic patients obscures the distinction of adaptations to metabolic derangements from adaptations to heart failure per se. Methods and Results— We performed both unbiased and targeted myocardial lipid surveys using liquid chromatography-mass spectroscopy in nondiabetic, lean, predominantly nonischemic, advanced heart failure patients at the time of heart transplantation or left ventricular assist device implantation. We identified significantly decreased concentrations of the majority of myocardial lipid intermediates, including long-chain acylcarnitines, the primary subset of energetic lipid substrate for mitochondrial fatty acid oxidation. We report for the first time significantly reduced levels of intermediate and anaplerotic acyl-coenzyme A (CoA) species incorporated into the Krebs cycle, whereas the myocardial concentration of acetyl-CoA was significantly increased in end-stage heart failure. In contrast, we observed an increased abundance of ketogenic β-hydroxybutyryl-CoA, in association with increased myocardial utilization of β-hydroxybutyrate. We observed a significant increase in the expression of the gene encoding succinyl-CoA:3-oxoacid-CoA transferase, the rate-limiting enzyme for myocardial oxidation of β-hydroxybutyrate and acetoacetate. Conclusions— These findings indicate increased ketone utilization in the severely failing human heart independent of diabetes mellitus, and they support the role of ketone bodies as an alternative fuel and myocardial ketone oxidation as a key metabolic adaptation in the failing human heart.

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.

Background: Inhibition of PKC-α (protein kinase C-α) enhances contractility and cardioprotection in animal models, but effects in humans are unknown. Genotypes at rs9912468 strongly associate with PRKCA expression in the left ventricle, enabling genetic approaches to measure effects of reduced PKC-α in human populations. Methods and Results: We analyzed the cis expression quantitative trait locus for PRKCA marked by rs9912468 using 313 left ventricular specimens from European Ancestry patients. The forward strand minor allele (G) at rs9912468 is associated with reduced PKC-α transcript abundance (1.7-fold reduction in minor allele homozygotes, P =1×10 −41 ). This association was cardiac specific in expression quantitative trait locus data sets that span 16 human tissues. Cardiac epigenomic data revealed a predicted enhancer in complete ( R 2 =1.0) linkage disequilibrium with rs9912468 within intron 2 of PRKCA. We cloned this region and used reporter constructs to verify cardiac-specific enhancer activity in vitro in cardiac and noncardiac cells and in vivo in zebrafish. The PRKCA enhancer contains 2 common genetic variants and 4 haplotypes; the haplotype correlated with the rs9912468 PKC-α–lowering allele (G) showed lowest activity. In contrast to previous reports in animal models, the PKC-α–lowering allele is associated with adverse left ventricular remodeling (higher mass, larger diastolic dimension), reduced fractional shortening, and higher risk of dilated cardiomyopathy in human populations. Conclusions: These findings support PKC-α as a regulator of the human heart but suggest that PKC-α inhibition may adversely affect the left ventricle depending on timing and duration. Pharmacological studies in human subjects are required to discern potential benefits and harms of PKC-α inhibitors as an approach to treat heart disease.

Heart failure is a complex clinical syndrome and has become the most common reason for adult hospitalization in developed countries. Two subtypes of heart failure, ischemic heart disease (ISCH) and dilated cardiomyopathy (DCM), have been studied using microarray platforms. However, microarray has limited resolution. Here we applied RNA sequencing (RNA-Seq) to identify gene signatures for heart failure from six individuals, including three controls, one ISCH and two DCM patients. Using genes identified from this small RNA-Seq dataset, we were able to accurately classify heart failure status in a much larger set of 313 individuals. The identified genes significantly overlapped with genes identified via genome-wide association studies for cardiometabolic traits and the promoters of those genes were enriched for binding sites for transcriptions factors. Our results indicate that it is possible to use RNA-Seq to classify disease status for complex diseases such as heart failure using an extremely small training dataset.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain similar to 8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD.

Rationale: Nitrate-rich beetroot juice has been shown to improve exercise capacity in heart failure with preserved ejection fraction, but studies using pharmacological preparations of inorganic nitrate are lacking. Objectives: To determine (1) the dose–response effect of potassium nitrate (KNO 3 ) on exercise capacity; (2) the population-specific pharmacokinetic and safety profile of KNO 3 in heart failure with preserved ejection fraction. Methods and Results: We randomized 12 subjects with heart failure with preserved ejection fraction to oral KNO 3 (n=9) or potassium chloride (n=3). Subjects received 6 mmol twice daily during week 1, followed by 6 mmol thrice daily during week 2. Supine cycle ergometry was performed at baseline (visit 1) and after each week (visits 2 and 3). Quality of life was assessed with the Kansas City Cardiomyopathy Questionnaire. The primary efficacy outcome, peak O 2 -uptake, did not significantly improve ( P =0.13). Exploratory outcomes included exercise duration and quality of life. Exercise duration increased significantly with KNO 3 (visit 1: 9.87, 95% confidence interval [CI] 9.31–10.43 minutes; visit 2: 10.73, 95% CI 10.13–11.33 minute; visit 3: 11.61, 95% CI 11.05–12.17 minutes; P =0.002). Improvements in the Kansas City Cardiomyopathy Questionnaire total symptom (visit 1: 58.0, 95% CI 52.5–63.5; visit 2: 66.8, 95% CI 61.3–72.3; visit 3: 70.8, 95% CI 65.3–76.3; P =0.016) and functional status scores (visit 1: 62.2, 95% CI 58.5–66.0; visit 2: 68.6, 95% CI 64.9–72.3; visit 3: 71.1, 95% CI 67.3–74.8; P =0.01) were seen after KNO 3 . Pronounced elevations in trough levels of nitric oxide metabolites occurred with KNO 3 (visit 2: 199.5, 95% CI 98.7–300.2 μmol/L; visit 3: 471.8, 95% CI 377.8–565.8 μmol/L) versus baseline (visit 1: 38.0, 95% CI 0.00–132.0 μmol/L; P 3 did not lead to clinically significant hypotension or methemoglobinemia. After 6 mmol of KNO 3 , systolic blood pressure was reduced by a maximum of 17.9 (95% CI −28.3 to −7.6) mm Hg 3.75 hours later. Peak nitric oxide metabolites concentrations were 259.3 (95% CI 176.2–342.4) μmol/L 3.5 hours after ingestion, and the median half-life was 73.0 (interquartile range 33.4–232.0) minutes. Conclusions: KNO 3 is potentially well tolerated and improves exercise duration and quality of life in heart failure with preserved ejection fraction. This study reinforces the efficacy of KNO 3 and suggests that larger randomized trials are warranted. Clinical Trial Registration : URL: http://www.clinicaltrials.gov . Unique identifier: NCT02256345

Failure of the human heart to maintain sufficient output of blood for the demands of the body, heart failure, is a common condition with high mortality even with modern therapeutic alternatives. To identify molecular determinants of mortality in patients with new-onset heart failure, we performed a meta-analysis of genome-wide association studies and follow-up genotyping in independent populations. We identified and replicated an association for a genetic variant on chromosome 5q22 with 36% increased risk of death in subjects with heart failure (rs9885413, P = 2.7x10⁻⁹. We provide evidence from reporter gene assays, computational predictions and epigenomic marks that this polymorphism increases activity of an enhancer region active in multiple human tissues. The polymorphism was further reproducibly associated with a DNA methylation signature in whole blood (P = 4.5x10⁻⁴⁰) that also associated with allergic sensitization and expression in blood of the cytokine TSLP (P = 1.1x10⁻⁴). Knockdown of the transcription factor predicted to bind the enhancer region (NHLH1) in a human cell line (HEK293) expressing NHLH1 resulted in lower TSLP expression. In addition, we observed evidence of recent positive selection acting on the risk allele in populations of African descent. Our findings provide novel genetic leads to factors that influence mortality in patients with heart failure., National Heart, Lung, and Blood Institute (HHSN268201100005C), National Heart, Lung, and Blood Institute (HHSN268201100006C), National Heart, Lung, and Blood Institute (HHSN268201100007C), National Heart, Lung, and Blood Institute (HHSN268201100008C), National Heart, Lung, and Blood Institute (HHSN268201100009C), National Heart, Lung, and Blood Institute (HHSN268201100010C), National Heart, Lung, and Blood Institute (HHSN268201100011C), National Heart, Lung, and Blood Institute (HHSN268201100012C), National Heart, Lung, and Blood Institute (N01-HC-55015), National Heart, Lung, and Blood Institute (N01-HC-55016), National Heart, Lung, and Blood Institute (N01-HC-55018), National Heart, Lung, and Blood Institute (N01-HC-55019), National Heart, Lung, and Blood Institute (N01-HC-55020), National Heart, Lung, and Blood Institute (N01-HC-55021), National Heart, Lung, and Blood Institute (N01-HC-55022), National Heart, Lung, and Blood Institute (R01HL087641), National Heart, Lung, and Blood Institute (R01HL59367), National Heart, Lung, and Blood Institute (R01HL086694), National Human Genome Research Institute (U.S.) (U01HG004402), United States. National Institutes of Health (HHSN268200625226C), United States. National Institutes of Health (UL1RR025005), National Heart, Lung, and Blood Institute (HHSN268201200036C), National Heart, Lung, and Blood Institute (N01HC55222), National Heart, Lung, and Blood Institute (HHSN268200800007C), National Heart, Lung, and Blood Institute (N01HC85079), National Heart, Lung, and Blood Institute (N01HC85080), National Heart, Lung, and Blood Institute (N01HC85081), National Heart, Lung, and Blood Institute (N01HC85082), National Heart, Lung, and Blood Institute (N01HC85083), National Heart, Lung, and Blood Institute (N01HC85086), National Heart, Lung, and Blood Institute (U01HL080295), National Science Foundation (U.S.) (R01HL087652), National Heart, Lung, and Blood Institute (R01HL105756), National Heart, Lung, and Blood Institute (R01HL103612), National Heart, Lung, and Blood Institute (R01HL120393), National Institute on Aging (R01AG023629), National Center for Advancing Translational Sciences (U.S.) (UL1TR000124), National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (DK063491), National Heart, Lung, and Blood Institute (N01-HC-25195), National Heart, Lung, and Blood Institute (2K24HL04334), National Heart, Lung, and Blood Institute (R01HL077477), National Heart, Lung, and Blood Institute (R01HL093328), National Heart, Lung, and Blood Institute (NIH R01HL105993), National Institute on Aging (N01AG62101), National Heart, Lung, and Blood Institute (N01AG62103), National Heart, Lung, and Blood Institute (N01AG62106), National Institute on Aging (1R01AG032098-01A1), United States. National Institutes of Health (HHSN268200782096C), National Cancer Institute (U.S.) (CA-34944), National Cancer Institute (U.S.) (CA-40360), National Cancer Institute (U.S.) (CA-097193), National Heart, Lung, and Blood Institute (HL-26490), National Heart, Lung, and Blood Institute (HL-34595)

The IGF system plays a key role in mammary gland growth and development. Our lab previously reported that IGF-I primarily regulates IGF-binding protein (BP)-3 in bovine mammary epithelial cells (MEC) and IGFBP-5 in mammary fibroblasts (MF). Presently, we examined the signaling pathways used by IGF-I to elicit this distinct, cell-type specific regulation. The phosphatidylinositol-3 kinase pathway was required for IGF-I to increase IGFBP-3 and -5 in MF and IGFBP-3 in MEC. Surprisingly, inhibiting the mitogen-activated protein kinase (MAPK) pathway in MEC increased IGFBP-5 mRNA levels 2- to 4-fold under basal conditions and 8- to 12-fold in cells treated with IGF-I within 4 h. Similar patterns of IGFBP-3 and -5 regulation were observed in murine MEC. Cells treated with IGF-I in the presence of MAPK inhibitors secreted more IGFBP-5 protein into conditioned media relative to cells treated with IGF-I alone; however, IGFBP-5 protein was not detected in conditioned media of cells treated with only a MAPK inhibitor. The IGFBP-5 mRNA response to MAPK inhibitors was specific for MEC, as blocking MAPK activity decreased the ability of IGF-I to induce IGFBP-5 in MF. In addition, no other IGFBP was increased in either cell type when MAPK activity was inhibited. These increases in IGFBP-5 expression in response to inhibition of the MAPK pathway corresponded with the induction of apoptosis. In conclusion, we report the novel observation that the MAPK/extracellular signal regulated kinase (ERK) pathway specifically represses IGFBP-5 expression in MEC. The corresponding changes in apoptosis and IGFBP-5 expression support a role for this specific IGFBP in mammary gland involution.

Advanced heart failure (HF) is morbid and costly with 700,000 patients estimated to reach end-stage HF and be considered for ventricular assist device (VAD) or heart transplantation (HT). The transition to VAD or HT is informed by functional class, exercise capacity, and markers of end-organ