The Cardiorenal Effects of Chronic Phosphodiesterase-V Inhibition in Preclinical Diastolic Dysfunction (Stage B Heart Failure).

OBJECTIVE: To determine whether chronic phosphodiesterase-V (PDEV) inhibition with tadalafil will improve urinary sodium excretion, glomerular filtration rate (GFR), plasma cyclic guanosine 3', 5'-monophosphate (cGMP), and urinary cGMP excretion in response to volume expansion (VE) in patients with preclinical diastolic dysfunction (PDD) or stage B heart failure. BACKGROUND: PDD is defined as abnormal diastolic function with normal systolic function, without clinical heart failure. PDD is predictive of development of heart failure and all-cause mortality. Impaired renal function and attenuated cGMP response to VE are hallmarks of PDD. METHODS: A double-blind, placebo-controlled, proof-of-concept study was conducted to compare 12 weeks of tadalafil 20 mg daily (n = 14) vs placebo (n = 7). Subjects underwent 2 study visits 12 weeks apart. Renal, neurohormonal and echocardiographic assessments were performed before and after intravascular VE (normal saline 0.25 mL/kg/min for 1 hour). RESULTS: Baseline characteristics were similar. There was no increase in GFR, plasma cGMP or urinary cGMP excretion in response to VE in either group at visit 1. At visit 2, tadalafil did not result in significant change in GFR but increased plasma cGMP and urinary cGMP excretion at baseline. In response to VE, tadalafil resulted in increased urine flow, urinary sodium excretion, GFR (7.00 [-1.0, 26.3] vs -9.00 [-24.5, 2.0] mL/min/1.73m2; P = 0.02) and plasma cGMP (0.50 [-0.1, 0.7] vs -0.25 [-0.6, -0.1] pmol/mL; P = 0.02). It did not improve urinary cGMP excretion after VE. CONCLUSION: In PDD, chronic PDEV inhibition with tadalafil improved renal response to VE through increased urine flow, urinary sodium excretion, GFR, and plasma cGMP. Further studies are required to determine whether this enhanced renal response can mitigate progression to clinical heart failure.

Evidence for Angiotensin II as a Naturally Existing Suppressor for the Guanylyl Cyclase A Receptor and Cyclic GMP Generation.

The natriuretic peptide system (NPS) and renin-angiotensin-aldosterone system (RAAS) function oppositely at multiple levels. While it has long been suspected that angiotensin II (ANGII) may directly suppress NPS activity, no clear evidence to date supports this notion. This study was designed to systematically investigate ANGII-NPS interaction in humans, in vivo, and in vitro. Circulating atrial, b-type, and c-type natriuretic peptides (ANP, BNP, CNP), cyclic guanosine monophosphate (cGMP), and ANGII were simultaneously investigated in 128 human subjects. Prompted hypothesis was validated in vivo to determine the influence of ANGII on ANP actions. The underlying mechanisms were further explored via in vitro approaches. In humans, ANGII demonstrated an inverse relationship with ANP, BNP, and cGMP. In regression models predicting cGMP, adding ANGII levels and the interaction term between ANGII and natriuretic peptides increased the predictive accuracy of the base models constructed with either ANP or BNP, but not CNP. Importantly, stratified correlation analysis further revealed a positive association between cGMP and ANP or BNP only in subjects with low, but not high, ANGII levels. In rats, co-infusion of ANGII even at a physiological dose attenuated cGMP generation mediated by ANP infusion. In vitro, we found the suppressive effect of ANGII on ANP-stimulated cGMP requires the presence of ANGII type-1 (AT1) receptor and mechanistically involves protein kinase C (PKC), as this suppression can be substantially rescued by either valsartan (AT1 blocker) or Go6983 (PKC inhibitor). Using surface plasmon resonance (SPR), we showed ANGII has low binding affinity to the guanylyl cyclase A (GC-A) receptor compared to ANP or BNP. Our study reveals ANGII is a natural suppressor for the cGMP-generating action of GC-A via AT1/PKC dependent manner and highlights the importance of dual-targeting RAAS and NPS in maximizing beneficial properties of natriuretic peptides in cardiovascular protection.

LANA oligomeric architecture is essential for KSHV nuclear body formation and viral genome maintenance during latency.

The molecular basis for the formation of functional, higher-ordered macro-molecular domains is not completely known. The Kaposi's Sarcoma-Associated Herpesvirus (KSHV) genome forms a super-molecular domain structure during latent infection that is strictly dependent on the DNA binding of the viral nuclear antigen LANA to the viral terminal repeats (TR). LANA is known to form oligomeric structures that have been implicated in viral episome maintenance. In this study, we show that the LANA oligomerization interface is required for the formation of higher-order nuclear bodies that partially colocalize with DAXX, EZH2, H3K27me3, and ORC2 but not with PML. These nuclear bodies assemble at the periphery of condensed cellular chromosomes during mitotic cell division. We demonstrate that the LANA oligomerization interface contributes to the cooperative DNA binding at the viral TR and the recruitment of ORC to the viral episome. Oligomerization mutants failed to auto-regulate LANA/ORF73 transcription, and this correlated with the loss of a chromosome conformational DNA-loop between the TR and LANA promoter. Viral genomes with LANA oligomerization mutants were subject to genome rearrangements including the loss of subgenomic DNA. Our data suggests that LANA oligomerization drives stable binding to the TR and formation of an epigenetically stable chromatin architecture resulting in higher-order LANA nuclear bodies important for viral genome integrity and long-term episome persistence.

Correction: Deregulation of KSHV latency conformation by ER-stress and caspase-dependent RAD21-cleavage.

[This corrects the article DOI: 10.1371/journal.ppat.1006596.].

Long-term blood pressure lowering and cGMP-activating actions of the novel ANP analog MANP.

Based on the cardiac hormone atrial natriuretic peptide (ANP) and its seminal role in blood pressure (BP) homeostasis, we investigated the chronic BP lowering actions of a novel ANP analog currently entering clinical trials for hypertension. Previous reports demonstrate that this analog MANP activates the guanylyl cyclase A receptor (GC-A) and results in more potent biological actions compared with ANP; thus, it may represent a new therapeutic drug for hypertension. A major goal of this study was to establish that chronic subcutaneous delivery of MANP is feasible and hypotensive together with cGMP effects. We investigated the BP-lowering and cGMP-activating actions of acute and chronic subcutaneous delivery in normal and hypertensive rats. Furthermore, we explored vascular mechanisms of MANP in human aortic smooth muscle cells (HASMC) and ex vivo in isolated arteries. In normal rats with a single subcutaneous injection, MANP promoted robust dose-dependent BP-lowering actions and natriuresis, together with cGMP activation. Most importantly in hypertensive rats, once-a-day subcutaneous injection of MANP for 7 days induced cGMP elevation and long-term BP reduction compared with vehicle. Mechanistically, in HASMC, MANP activated cGMP and attenuated angiotensin II-mediated increases in intracellular Ca2+ levels while directly vasorelaxing arterial rings. Our study demonstrates for the first time the effectiveness of subcutaneous administration of MANP for 7 days and provides innovative, vascular mechanisms of BP regulation supporting its continued development as a novel therapeutic for hypertension.

Annexin A1 is a Potential Novel Biomarker of Congestion in Acute Heart Failure.

OBJECTIVES: This study sought to identify the role of annexin A1 (AnxA1) as a congestion marker in acute heart failure (AHF) and to identify its putative role in predicting clinical outcomes. BACKGROUND: AnxA1 is a protein that inhibits inflammation following ischemia-reperfusion injury in cardiorenal tissues. Because AHF is a state of tissue hypoperfusion, we hypothesized that plasma AnxA1 levels are altered in AHF. METHODS: In the Renal Optimization Strategies Evaluation (ROSE) trial, patients hospitalized for AHF with kidney injury were randomized to receive dopamine, nesiritide, or placebo for 72 hours in addition to diuresis. In a subanalysis, plasma AnxA1 levels were measured at baseline and at 72 hours in 275 patients. Participants were divided into 3 tertiles based on their baseline AnxA1 levels. RESULTS: The prevalence of peripheral edema 2+ increased with increasing AnxA1 levels (P < .007). Cystatin C, blood urea nitrogen, and kidney injury molecule-1 plasma levels were higher among participants in tertile 3 vs tertiles 1 or 2 (P< .05). Patients with a congestion score of 4 had a mean baseline AnxA1 level 8.63 units higher than those with a congestion score of 0 (P = .03). Patients in tertiles 2 and 3 were twice as likely to experience creatinine elevation as patients in tertile 1 (P = .03). Patients in tertiles 2 and 3 were at a higher risk of 60-day all-cause mortality or heart failure hospitalization and 180-day all-cause mortality (P < .05). CONCLUSIONS: Among patients hospitalized for AHF with impaired kidney function, elevated AnxA1 levels are associated with worse congestion, higher risk for further creatinine elevation, and higher rates of 60-day morbidity or all-cause mortality and 180-day all-cause mortality. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01132846.

C53: A novel particulate guanylyl cyclase B receptor activator that has sustained activity in vivo with anti-fibrotic actions in human cardiac and renal fibroblasts.

The native particulate guanylyl cyclase B receptor (pGC-B) activator, C-type natriuretic peptide (CNP), induces anti-remodeling actions in the heart and kidney through the generation of the second messenger 3', 5' cyclic guanosine monophosphate (cGMP). Indeed fibrotic remodeling, particularly in cardiorenal disease states, contributes to disease progression and thus, has been a key target for drug discovery and development. Although the pGC-B/cGMP system has been perceived as a promising anti-fibrotic pathway, its therapeutic potential is limited due to the rapid degradation and catabolism of CNP by neprilysin (NEP) and natriuretic peptide clearance receptor (NPRC). The goal of this study was to bioengineer and test in vitro and in vivo a novel pGC-B activator, C53. Here we established that C53 selectively generates cGMP via the pGC-B receptor and is highly resistant to NEP and has less interaction with NPRC in vitro. Furthermore in vivo, C53 had enhanced cGMP-generating actions that paralleled elevated plasma CNP-like levels, thus indicating a longer circulating half-life compared to CNP. Importantly in human cardiac fibroblasts (HCFs) and renal fibroblasts (HRFs), C53 exerted robust cGMP-generating actions, inhibited TGFß-1 stimulated HCFs and HRFs proliferation chronically and suppressed the differentiation of HCFs and HRFs to myofibroblasts. The current findings advance innovation in drug discovery and highlight C53 as a novel pGC-B activator with sustained in vivo activity and anti-fibrotic actions in vitro. Future studies are warranted to explore the efficacy and therapeutic opportunity of C53 targeting fibrosis in cardiorenal disease states and beyond.

Worsening Renal Function in Patients With Acute Heart Failure Undergoing Aggressive Diuresis Is Not Associated With Tubular Injury.

BACKGROUND: Worsening renal function (WRF) in the setting of aggressive diuresis for acute heart failure treatment may reflect renal tubular injury or simply indicate a hemodynamic or functional change in glomerular filtration. Well-validated tubular injury biomarkers, N-acetyl-ß-d-glucosaminidase, neutrophil gelatinase-associated lipocalin, and kidney injury molecule 1, are now available that can quantify the degree of renal tubular injury. The ROSE-AHF trial (Renal Optimization Strategies Evaluation-Acute Heart Failure) provides an experimental platform for the study of mechanisms of WRF during aggressive diuresis for acute heart failure because the ROSE-AHF protocol dictated high-dose loop diuretic therapy in all patients. We sought to determine whether tubular injury biomarkers are associated with WRF in the setting of aggressive diuresis and its association with prognosis. METHODS: Patients in the multicenter ROSE-AHF trial with baseline and 72-hour urine tubular injury biomarkers were analyzed (n=283). WRF was defined as a ≥20% decrease in glomerular filtration rate estimated with cystatin C. RESULTS: Consistent with protocol-driven aggressive dosing of loop diuretics, participants received a median 560 mg IV furosemide equivalents (interquartile range, 300-815 mg), which induced a urine output of 8425 mL (interquartile range, 6341-10 528 mL) over the 72-hour intervention period. Levels of N-acetyl-ß-d-glucosaminidase and kidney injury molecule 1 did not change with aggressive diuresis (both P>0.59), whereas levels of neutrophil gelatinase-associated lipocalin decreased slightly (-8.7 ng/mg; interquartile range, -169 to 35 ng/mg; P<0.001). WRF occurred in 21.2% of the population and was not associated with an increase in any marker of renal tubular injury: neutrophil gelatinase-associated lipocalin (P=0.21), N-acetyl-ß-d-glucosaminidase (P=0.46), or kidney injury molecule 1 (P=0.22). Increases in neutrophil gelatinase-associated lipocalin, N-acetyl-ß-d-glucosaminidase, and kidney injury molecule 1 were paradoxically associated with improved survival (adjusted hazard ratio, 0.80 per 10 percentile increase; 95% confidence interval, 0.69-0.91; P=0.001). CONCLUSIONS: Kidney tubular injury does not appear to have an association with WRF in the context of aggressive diuresis of patients with acute heart failure. These findings reinforce the notion that the small to moderate deteriorations in renal function commonly encountered with aggressive diuresis are dissimilar from traditional causes of acute kidney injury.

BET-Inhibitors Disrupt Rad21-Dependent Conformational Control of KSHV Latency.

Kaposi's Sarcoma-associated Herpesvirus (KSHV) establishes stable latent infection in B-lymphocytes and pleural effusion lymphomas (PELs). During latency, the viral genome persists as an epigenetically constrained episome with restricted gene expression programs. To identify epigenetic regulators of KSHV latency, we screened a focused small molecule library containing known inhibitors of epigenetic factors. We identified JQ1, a Bromodomain and Extended Terminal (BET) protein inhibitor, as a potent activator of KSHV lytic reactivation from B-cells carrying episomal KSHV. We validated that JQ1 and other BET inhibitors efficiently stimulated reactivation of KSHV from latently infected PEL cells. We found that BET proteins BRD2 and BRD4 localize to several regions of the viral genome, including the LANA binding sites within the terminal repeats (TR), as well as at CTCF-cohesin sites in the latent and lytic control regions. JQ1 did not disrupt the interaction of BRD4 or BRD2 with LANA, but did reduce the binding of LANA with KSHV TR. We have previously demonstrated a cohesin-dependent DNA-loop interaction between the latent and lytic control regions that restrict expression of ORF50/RTA and ORF45 immediate early gene transcripts. JQ1 reduced binding of cohesin subunit Rad21 with the CTCF binding sites in the latency and lytic control regions. JQ1 also reduced DNA-loop interaction between latent and lytic control regions. These findings implicate BET proteins BRD2 and BRD4 in the maintenance of KSHV chromatin architecture during latency and reveal BET inhibitors as potent activators of KSHV reactivation from latency.

Deregulation of KSHV latency conformation by ER-stress and caspase-dependent RAD21-cleavage.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a human gammaherpesvirus recognized as the principal causative agent of KS and primary effusion lymphoma (PEL). KSHV establishes persistent latent infection in B lymphocytes where viral gene expression is restricted, in part, by a cohesin-dependent chromosome conformation. Here, we show that endoplasmic reticulum (ER) stress induces a rapid, caspase-dependent cleavage of cohesin subunit RAD21. ER stress-induced cleavage of RAD21 correlated with a rapid and strong viral lytic transcriptional activation. This effect was observed in several KSHV positive PEL cells, but not in other B-cells or non-B-cell models of KSHV latency. The cleaved-RAD21 does not dissociate from viral genomes, nor disassemble from other components of the cohesin complex. However, RAD21 cleavage correlated with the disruption of the latency genome conformation as revealed by chromosome conformation capture (3C). Ectopic expression of C-terminal RAD21 cleaved form could partially induce KSHV lytic genes transcription in BCBLI cells, suggesting that ER-stress induced RAD21 cleavage was sufficient to induce KSHV reactivation from latency in PEL cells. Taken together our results reveal a novel aspect for control and maintenance of KSHV genome latency conformation mediated by stress-induced RAD21 cleavage. Our studies also suggest that RAD21 cleavage may be a general regulatory mechanism for rapid alteration of cellular chromosome conformation and cohesin-dependent transcription regulation.

Cardiac resynchronization therapy improves myocardial conduction.

BACKGROUND: Cardiac resynchronization therapy (CRT) reverses left ventricular remodeling and improves left ventricular systolic function. However, little is known about whether CRT improves ventricular conduction. OBJECTIVE: To determine the relationship between CRT response and QRS narrowing. METHODS: The study included consecutive patients who underwent CRT with defibrillator (CRT-D) implantation between January 2002 and December 2012 and had subsequent generator replacement (GR). At the time of GR, super-responder was defined as those who had left ventricular ejection fraction (LVEF) of 50% or more; patients who had an LVEF of 36-49% with an increase of more than 5% were considered responders, and the others were nonresponders. All patients were assessed 6 months after CRT-D implantation and at the time of GR. RESULTS: Of 114 study patients, 58 (50.9%) were nonresponders, 29 (25.4%) responders, and 27 (23.7%) super-responders. QRS narrowing at 6 months was significant in super-responders (175.4 ± 21.4 to 159.7 ± 20.7 ms, P = 0.001) and responders (169.0 ± 21.3 to 157.7 ± 17.6 ms, P = 0.008). At time of GR, only super-responders had further QRS narrowing (159.7 ± 20.7 to 146.3 ± 19.2 ms, P < 0.001). QRS duration change after 6 months was independently associated with super-response (hazard ratio: 1.20 for every 5 ms QRS duration narrowing; 95% confidence interval, 1.06-1.38; P = 0.005), and QRS narrowing of 10 ms or more was associated with lower risk of all-cause mortality after GR. CONCLUSION: Continuous QRS narrowing after CRT, as an electrical marker, is associated with a super-response to CRT. Further QRS narrowing 6 months after CRT implantation was associated with lower risk of mortality after GR.

Isosorbide Mononitrate in Heart Failure with Preserved Ejection Fraction.

BACKGROUND: Nitrates are commonly prescribed to enhance activity tolerance in patients with heart failure and a preserved ejection fraction. We compared the effect of isosorbide mononitrate or placebo on daily activity in such patients. METHODS: In this multicenter, double-blind, crossover study, 110 patients with heart failure and a preserved ejection fraction were randomly assigned to a 6-week dose-escalation regimen of isosorbide mononitrate (from 30 mg to 60 mg to 120 mg once daily) or placebo, with subsequent crossover to the other group for 6 weeks. The primary end point was the daily activity level, quantified as the average daily accelerometer units during the 120-mg phase, as assessed by patient-worn accelerometers. Secondary end points included hours of activity per day during the 120-mg phase, daily accelerometer units during all three dose regimens, quality-of-life scores, 6-minute walk distance, and levels of N-terminal pro-brain natriuretic peptide (NT-proBNP). RESULTS: In the group receiving the 120-mg dose of isosorbide mononitrate, as compared with the placebo group, there was a nonsignificant trend toward lower daily activity (-381 accelerometer units; 95% confidence interval [CI], -780 to 17; P=0.06) and a significant decrease in hours of activity per day (-0.30 hours; 95% CI, -0.55 to -0.05; P=0.02). During all dose regimens, activity in the isosorbide mononitrate group was lower than that in the placebo group (-439 accelerometer units; 95% CI, -792 to -86; P=0.02). Activity levels decreased progressively and significantly with increased doses of isosorbide mononitrate (but not placebo). There were no significant between-group differences in the 6-minute walk distance, quality-of-life scores, or NT-proBNP levels. CONCLUSIONS: Patients with heart failure and a preserved ejection fraction who received isosorbide mononitrate were less active and did not have better quality of life or submaximal exercise capacity than did patients who received placebo. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT02053493.).

EBNA2 Drives Formation of New Chromosome Binding Sites and Target Genes for B-Cell Master Regulatory Transcription Factors RBP-jκ and EBF1.

Epstein-Barr Virus (EBV) transforms resting B-lymphocytes into proliferating lymphoblasts to establish latent infections that can give rise to malignancies. We show here that EBV-encoded transcriptional regulator EBNA2 drives the cooperative and combinatorial genome-wide binding of two master regulators of B-cell fate, namely EBF1 and RBP-jκ. Previous studies suggest that these B-cell factors are statically bound to target gene promoters. In contrast, we found that EBNA2 induces the formation of new binding for both RBP-jκ and EBF1, many of which are in close physical proximity in the cellular and viral genome. These newly induced binding sites co-occupied by EBNA2-EBF1-RBP-jκ correlate strongly with transcriptional activation of linked genes that are important for B-lymphoblast function. Conditional expression or repression of EBNA2 leads to a rapid alteration in RBP-jκ and EBF1 binding. Biochemical and shRNA depletion studies provide evidence for cooperative assembly at co-occupied sites. These findings reveal that EBNA2 facilitate combinatorial interactions to induce new patterns of transcription factor occupancy and gene programming necessary to drive B-lymphoblast growth and survival.

Effect of Inorganic Nitrite vs Placebo on Exercise Capacity Among Patients With Heart Failure With Preserved Ejection Fraction: The INDIE-HFpEF Randomized Clinical Trial.

Importance: There are few effective treatments for heart failure with preserved ejection fraction (HFpEF). Short-term administration of inorganic nitrite or nitrate preparations has been shown to enhance nitric oxide signaling, which may improve aerobic capacity in HFpEF. Objective: To determine the effect of 4 weeks' administration of inhaled, nebulized inorganic nitrite on exercise capacity in HFpEF. Design, Setting, and Participants: Multicenter, double-blind, placebo-controlled, 2-treatment, crossover trial of 105 patients with HFpEF. Participants were enrolled from July 22, 2016, to September 12, 2017, at 17 US sites, with final date of follow-up of January 2, 2018. Interventions: Inorganic nitrite or placebo administered via micronebulizer device. During each 6-week phase of the crossover study, participants received no study drug for 2 weeks (baseline/washout) followed by study drug (nitrite or placebo) at 46 mg 3 times a day for 1 week followed by 80 mg 3 times a day for 3 weeks. Main Outcomes and Measures: The primary end point was peak oxygen consumption (mL/kg/min). Secondary end points included daily activity levels assessed by accelerometry, health status as assessed by the Kansas City Cardiomyopathy Questionnaire (score range, 0-100, with higher scores reflecting better quality of life), functional class, cardiac filling pressures assessed by echocardiography, N-terminal fragment of the prohormone brain natriuretic peptide levels, other exercise indices, adverse events, and tolerability. Outcomes were assessed after treatment for 4 weeks. Results: Among 105 patients who were randomized (median age, 68 years; 56% women), 98 (93%) completed the trial. During the nitrite phase, there was no significant difference in mean peak oxygen consumption as compared with the placebo phase (13.5 vs 13.7 mL/kg/min; difference, -0.20 [95% CI, -0.56 to 0.16]; P = .27). There were no significant between-treatment phase differences in daily activity levels (5497 vs 5503 accelerometry units; difference, -15 [95% CI, -264 to 234]; P = .91), Kansas City Cardiomyopathy Questionnaire Clinical Summary Score (62.6 vs 61.9; difference, 1.1 [95% CI, -1.4 to 3.5]; P = .39), functional class (2.5 vs 2.5; difference, 0.1 [95% CI, -0.1 to 0.2]; P = .43), echocardiographic E/e' ratio (16.4 vs 16.6; difference, 0.1 [95% CI, -1.2 to 1.3]; P = .93), or N-terminal fragment of the prohormone brain natriuretic peptide levels (520 vs 533 pg/mL; difference, 11 [95% CI, -53 to 75]; P = .74). Worsening heart failure occurred in 3 participants (2.9%) during the nitrite phase and 8 (7.6%) during the placebo phase. Conclusions and Relevance: Among patients with HFpEF, administration of inhaled inorganic nitrite for 4 weeks, compared with placebo, did not result in significant improvement in exercise capacity. Trial Registration: ClinicalTrials.gov Identifier: NCT02742129.

Experimental cardiac radiation exposure induces ventricular diastolic dysfunction with preserved ejection fraction.

Breast cancer radiotherapy increases the risk of heart failure with preserved ejection fraction (HFpEF). Cardiomyocytes are highly radioresistant, but radiation specifically affects coronary microvascular endothelial cells, with subsequent microvascular inflammation and rarefaction. The effects of radiation on left ventricular (LV) diastolic function are poorly characterized. We hypothesized that cardiac radiation exposure may result in diastolic dysfunction without reduced EF. Global cardiac expression of the sodium-iodide symporter (NIS) was induced by cardiotropic gene (adeno-associated virus serotype 9) delivery to 5-wk-old rats. SPECT/CT (125I) measurement of cardiac iodine uptake allowed calculation of the 131I doses needed to deliver 10- or 20-Gy cardiac radiation at 10 wk of age. Radiated (Rad; 10 or 20 Gy) and control rats were studied at 30 wk of age. Body weight, blood pressure, and heart rate were similar in control and Rad rats. Compared with control rats, Rad rats had impaired exercise capacity, increased LV diastolic stiffness, impaired LV relaxation, and elevated filling pressures but similar LV volume, EF, end-systolic elastance, preload recruitable stroke work, and peak +dP/dt Pathology revealed reduced microvascular density, mild concentric cardiomyocyte hypertrophy, and increased LV fibrosis in Rad rats compared with control rats. In the Rad myocardium, oxidative stress was increased and in vivo PKG activity was decreased. Experimental cardiac radiation exposure resulted in diastolic dysfunction without reduced EF. These data provide insight into the association between cardiac radiation exposure and HFpEF risk and lend further support for the importance of inflammation-related coronary microvascular compromise in HFpEF.NEW & NOTEWORTHY Cardiac radiation exposure during radiotherapy increases the risk of heart failure with preserved ejection fraction. In a novel rodent model, cardiac radiation exposure resulted in coronary microvascular rarefaction, oxidative stress, impaired PKG signaling, myocardial fibrosis, mild cardiomyocyte hypertrophy, left ventricular diastolic dysfunction, and elevated left ventricular filling pressures despite preserved ejection fraction.

A role for CTCF and cohesin in subtelomere chromatin organization, TERRA transcription, and telomere end protection.

The contribution of human subtelomeric DNA and chromatin organization to telomere integrity and chromosome end protection is not yet understood in molecular detail. Here, we show by ChIP-Seq that most human subtelomeres contain a CTCF- and cohesin-binding site within ∼1-2 kb of the TTAGGG repeat tract and adjacent to a CpG-islands implicated in TERRA transcription control. ChIP-Seq also revealed that RNA polymerase II (RNAPII) was enriched at sites adjacent to the CTCF sites and extending towards the telomere repeat tracts. Mutation of CTCF-binding sites in plasmid-borne promoters reduced transcriptional activity in an orientation-dependent manner. Depletion of CTCF by shRNA led to a decrease in TERRA transcription, and a loss of cohesin and RNAPII binding to the subtelomeres. Depletion of either CTCF or cohesin subunit Rad21 caused telomere-induced DNA damage foci (TIF) formation, and destabilized TRF1 and TRF2 binding to the TTAGGG proximal subtelomere DNA. These findings indicate that CTCF and cohesin are integral components of most human subtelomeres, and important for the regulation of TERRA transcription and telomere end protection.

Evaluation of a provocative dyspnea severity score in acute heart failure.

BACKGROUND: The acute heart failure (AHF) Syndromes International Working Group proposed that dyspnea be assessed under standardized, incrementally provocative maneuvers and called for studies to assess the feasibility of this approach. We sought to assess the feasibility and statistical characteristics of a novel provocative dyspnea severity score (pDS) versus the traditional dyspnea visual analog scale (DVAS) in an AHF trial. METHODS: At enrollment, 24, 48 and 72hours, 230 ROSE-AHF patients completed a DVAS. Dyspnea was then assessed with 5-point Likert dyspnea scales administered during 4 stages (A: upright-with O2, B: upright-without O2, C: supine-without O2 and D: exercise-without O2). Patients with moderate or less dyspnea were eligible for the next stage. RESULTS: At enrollment, oxygen withdrawal and supine provocation were highly feasible (≥97%), provoking more severe dyspnea (≥1 Likert point) in 24% and 42% of eligible patients, respectively. Exercise provocation had low feasibility with 38% of eligible patients unable to exercise due to factors other than dyspnea. A pDS was constructed from Likert scales during the 3 feasible assessment conditions (A-C). Relative to DVAS, the distribution of the pDS was more skewed with a high "ceiling effect" at enrollment (23%) limiting sensitivity to change. Change in pDS was not related to decongestion or 60-day outcomes. CONCLUSIONS: Although oxygen withdrawal and supine provocation are feasible and elicit more severe dyspnea, exercise provocation had unacceptable feasibility in this AHF cohort. The statistical characteristics of a pDS based on feasible provocation measures do not support its potential as a robust dyspnea assessment tool in AHF.

Intensification of Medication Therapy for Cardiorenal Syndrome in Acute Decompensated Heart Failure.

BACKGROUND: Worsening renal function in heart failure may be related to increased venous congestion, decreased cardiac output, or both. Diuretics are universally used in acute decompensated heart failure, but they may be ineffective and may lead to azotemia. We aimed to compare the decongestive properties of a urine output-guided diuretic adjustment and standard therapy for the management of cardiorenal syndrome in acute decompensated heart failure. METHODS AND RESULTS: Data were pooled from subjects randomized to the stepwise pharmacologic care algorithm (SPCA) in the CARRESS-HF trial and those who developed cardiorenal syndrome (rise in creatinine >0.3 mg/dL) in the DOSE-AHF and ROSE-AHF trials. Patients treated with SPCA (n = 94) were compared with patients treated with standard decongestive therapy (SDT) that included intravenous loop diuretic use (DOSE-AHF and ROSE-AHF; n = 107) at the time of cardiorenal syndrome and followed for net fluid balance, weight loss, and changing renal function. The SPCA group had higher degrees of jugular venous pressure (P < .0001) at the time of cardiorenal syndrome. The group that received SPCA had more weight change (-3.4 ± 5.2 lb) and more net fluid loss (1.705 ± 1.417 L) after 24 hours than the SDT group (-0.8 ± 3.4 lb and 0.892 ± 1.395 L, respectively; P < .001 for both) with a slight improvement in renal function (creatinine change -0.1 ± 0.3 vs 0.0 ± 0.3 mg/dL, respectively; P = .03). CONCLUSIONS: Compared with SDT, patients who received an intensification of medication therapy for treating persisting congestion had greater net fluid and weight loss without being associated with renal compromise.

Ultrafiltration in decompensated heart failure with cardiorenal syndrome.

BACKGROUND: Ultrafiltration is an alternative strategy to diuretic therapy for the treatment of patients with acute decompensated heart failure. Little is known about the efficacy and safety of ultrafiltration in patients with acute decompensated heart failure complicated by persistent congestion and worsened renal function. METHODS: We randomly assigned a total of 188 patients with acute decompensated heart failure, worsened renal function, and persistent congestion to a strategy of stepped pharmacologic therapy (94 patients) or ultrafiltration (94 patients). The primary end point was the bivariate change from baseline in the serum creatinine level and body weight, as assessed 96 hours after random assignment. Patients were followed for 60 days. RESULTS: Ultrafiltration was inferior to pharmacologic therapy with respect to the bivariate end point of the change in the serum creatinine level and body weight 96 hours after enrollment (P=0.003), owing primarily to an increase in the creatinine level in the ultrafiltration group. At 96 hours, the mean change in the creatinine level was -0.04±0.53 mg per deciliter (-3.5±46.9 µmol per liter) in the pharmacologic-therapy group, as compared with +0.23±0.70 mg per deciliter (20.3±61.9 µmol per liter) in the ultrafiltration group (P=0.003). There was no significant difference in weight loss 96 hours after enrollment between patients in the pharmacologic-therapy group and those in the ultrafiltration group (a loss of 5.5±5.1 kg [12.1±11.3 lb] and 5.7±3.9 kg [12.6±8.5 lb], respectively; P=0.58). A higher percentage of patients in the ultrafiltration group than in the pharmacologic-therapy group had a serious adverse event (72% vs. 57%, P=0.03). CONCLUSIONS: In a randomized trial involving patients hospitalized for acute decompensated heart failure, worsened renal function, and persistent congestion, the use of a stepped pharmacologic-therapy algorithm was superior to a strategy of ultrafiltration for the preservation of renal function at 96 hours, with a similar amount of weight loss with the two approaches. Ultrafiltration was associated with a higher rate of adverse events. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT00608491.).

Epigenetic deregulation of the LMP1/LMP2 locus of Epstein-Barr virus by mutation of a single CTCF-cohesin binding site.

The chromatin regulatory factors CTCF and cohesin have been implicated in the coordinated control of multiple gene loci in Epstein-Barr virus (EBV) latency. We have found that CTCF and cohesin are highly enriched at the convergent and partially overlapping transcripts for the LMP1 and LMP2A genes, but it is not yet known how CTCF and cohesin may coordinately regulate these transcripts. We now show that genetic disruption of this CTCF binding site (EBVΔCTCF166) leads to a deregulation of LMP1, LMP2A, and LMP2B transcription in EBV-immortalized B lymphocytes. EBVΔCTCF166 virus-immortalized primary B lymphocytes showed a decrease in LMP1 and LMP2A mRNA and a corresponding increase in LMP2B mRNA. The reduction of LMP1 and LMP2A correlated with a loss of euchromatic histone modification H3K9ac and a corresponding increase in heterochromatic histone modification H3K9me3 at the LMP2A promoter region in EBVΔCTCF166. Chromosome conformation capture (3C) revealed that DNA loop formation with the origin of plasmid replication (OriP) enhancer was eliminated in EBVΔCTCF166. We also observed that the EBV episome copy number was elevated in EBVΔCTCF166 and that this was not due to increased lytic cycle activity. These findings suggest that a single CTCF binding site controls LMP2A and LMP1 promoter selection, chromatin boundary function, DNA loop formation, and episome copy number control during EBV latency.