JAZF1, A Novel p400/TIP60/NuA4 Complex Member, Regulates H2A.Z Acetylation at Regulatory Regions

Histone variants differ in amino acid sequence, expression timing and genomic localization sites from canonical histones and convey unique functions to eukaryotic cells. Their tightly controlled spatial and temporal deposition into specific chromatin regions is accomplished by dedicated chaperone and/or remodeling complexes. While quantitatively identifying the chaperone complexes of many human H2A variants by using mass spectrometry, we also found additional members of the known H2A.Z chaperone complexes p400/TIP60/NuA4 and SRCAP. We discovered JAZF1, a nuclear/nucleolar protein, as a member of a p400 sub-complex containing MBTD1 but excluding ANP32E. Depletion of JAZF1 results in transcriptome changes that affect, among other pathways, ribosome biogenesis. To identify the underlying molecular mechanism contributing to JAZF1's function in gene regulation, we performed genome-wide ChIP-seq analyses. Interestingly, depletion of JAZF1 leads to reduced H2A.Z acetylation levels at > 1000 regulatory sites without affecting H2A.Z nucleosome positioning. Since JAZF1 associates with the histone acetyltransferase TIP60, whose depletion causes a correlated H2A.Z deacetylation of several JAZF1-targeted enhancer regions, we speculate that JAZF1 acts as chromatin modulator by recruiting TIP60's enzymatic activity. Altogether, this study uncovers JAZF1 as a member of a TIP60-containing p400 chaperone complex orchestrating H2A.Z acetylation at regulatory regions controlling the expression of genes, many of which are involved in ribosome biogenesis

Randomized, double blind study of non-excitatory, cardiac contractility modulation electrical impulses fr symptomatic heart failure

AIMS: We performed a randomized, double blind, crossover study of cardiac contractility modulation (CCM) signals in heart failure patients. METHODS AND RESULTS: One hundred and sixty-four subjects with ejection fraction (EF) < 35% and NYHA Class II (24%) or III (76%) symptoms received a CCM pulse generator. Patients were randomly assigned to Group 1 (n = 80, CCM treatment 3 months, sham treatment second 3 months) or Group 2 (n = 84, sham treatment 3 months, CCM treatment second 3 months). The co-primary endpoints were changes in peak oxygen consumption (VO2,peak) and Minnesota Living with Heart Failure Questionnaire (MLWHFQ). Baseline EF (29.3 +/- 6.7% vs. 29.8 +/- 7.8%), VO2,peak (14.1 +/- 3.0 vs. 13.6 +/- 2.7 mL/kg/min), and MLWHFQ (38.9 +/- 27.4 vs. 36.5 +/- 27.1) were similar between the groups. VO2,peak increased similarly in both groups during the first 3 months (0.40 +/- 3.0 vs. 0.37 +/- 3.3 mL/kg/min, placebo effect). During the next 3 months, VO2,peak decreased in the group switched to sham (-0.86 +/- 3.06 mL/kg/min) and increased in patients switched to active treatment (0.16 +/- 2.50 mL/kg/min). MLWHFQ trended better with treatment (-12.06 +/- 15.33 vs. -9.70 +/- 16.71) during the first 3 months, increased during the second 3 months in the group switched to sham (+4.70 +/- 16.57), and decreased further in patients switched to active treatment (-0.70 +/- 15.13). A comparison of values at the end of active treatment periods vs. end of sham treatment periods indicates statistically significantly improved VO2,peak and MLWHFQ (P = 0.03 for each parameter). CONCLUSION: In patients with heart failure and left ventricular dysfunction, CCM signals appear safe; exercise tolerance and quality of life (MLWHFQ) were significantly better while patients were receiving active treatment with CCM for a 3-month period

Cellular Imaging of Human Atherosclerotic Lesions by Intravascular Electric Impedance Spectroscopy

Background: Newer techniques are required to identify atherosclerotic lesions that are prone to rupture. Electric impedance spectroscopy (EIS) is able to provide information about the cellular composition of biological tissue. The present study was performed to determine the influence of inflammatory processes in type Va (lipid core, thick fibrous cap) and Vc (abundant fibrous connective tissue while lipid is minimal or even absent) human atherosclerotic lesions on the electrical impedance of these lesions measured by EIS. Methods and Results: EIS was performed on 1 aortic and 3 femoral human arteries at 25 spots with visually heavy plaque burden. Severely calcified lesions were excluded from analysis. A highly flexible micro-electrode mounted onto a balloon catheter was placed on marked regions to measure impedance values at 100 kHz. After paraffin embedding, visible marked cross sections (n = 21) were processed. Assessment of lesion types was performed by Movats staining. Immunostaining for CD31 (marker of neovascularisation), CD36 (scavenger cells) and MMP-3 (matrix metalloproteinase-3) was performed. The amount of positive cells was assessed semi-quantitatively. 15 type Va lesions and 6 type Vc lesions were identified. Lesions containing abundant CD36-, CD31- and MMP-3-positive staining revealed significantly higher impedance values compared to lesions with marginal or without positive staining (CD36+455650 V vs. CD36- 346653 V, p = 0.001; CD31+436643 V vs. CD31- 340655 V, p = 0.001; MMP-3+ 400668 V vs. MMP-3- 323633 V, p = 0.03)

Hydroxylation of the NOTCH1 intracellular domain regulates Notch signaling dynamics

Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor - the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response

Studying Brugada syndrome with an SCN1B variants in human-induced pluripotent stem cell-derived cardiomyocytes

BACKGROUND: Among rare channelopathies BrS patients are at high risk of sudden cardiac death (SCD). SCN5A mutations are found in a quarter of patients. Other rare gene mutations including SCN1B have been implicated to BrS. Studying the human cellular phenotype of BrS associated with rare gene mutation remains lacking. OBJECTIVES: We sought to study the cellular phenotype of BrS with the SCN1B gene variants using human-induced pluripotent stem cell (hiPSCs)–derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: A BrS patient suffering from recurrent syncope harboring a two variants (c.629T > C and c.637C > A) in SCN1B, which encodes the function-modifying sodium channel beta1 subunit, and three independent healthy subjects were recruited and their skin biopsies were used to generate hiPSCs, which were differentiated into cardiomyocytes (hiPSC-CMs) for studying the cellular electrophysiology. A significantly reduced peak and late sodium channel current (I(Na)) and a shift of activation curve to more positive potential as well as a shift of inactivation curve to more negative potential were detected in hiPSC-CMs of the BrS patient, indicating that the SCN1B variants impact the function of sodium channels in cardiomyocytes. The reduced I(Na) led to a reduction of amplitude (APA) and upstroke velocity (V(max)) of action potentials. Ajmaline, a sodium channel blocker, showed a stronger effect on APA and Vmax in BrS cells as compared to cells from healthy donors. Furthermore, carbachol was able to increase arrhythmia events and the beating frequency in BrS. CONCLUSION: Our hiPSC-CMs from a BrS-patient with two variants in SCN1B recapitulated some key phenotypic features of BrS and can provide a platform for studies on BrS with SCN1B variants

Epicardial adipose tissue in patients with heart failure

<p>Abstract</p> <p>Purpose</p> <p>The aim of this study was to evaluate the extent of epicardial adipose tissue (EAT) and its relationship with left ventricular (LV) parameters assessed by cardiovascular magnetic resonance (CMR) in patients with congestive heart failure (CHF) and healthy controls.</p> <p>Background</p> <p>EAT is the true visceral fat deposited around the heart which generates various bioactive molecules. Previous studies found that EAT is related to left ventricular mass (LVM) in healthy subjects. Further studies showed a constant EAT to myocardial mass ratio in normal, ischemic and hypertrophied hearts.</p> <p>Methods</p> <p>CMR was performed in 66 patients with CHF due to ischemic cardiomyopathy (ICM), or dilated cardiomyopathy (DCM) and 32 healthy controls. Ventricular volumes, dimensions and LV function were assessed. The amount of EAT was determined volumetrically and expressed as mass indexed to body surface area. Additionally, the EAT/LVM and the EAT/left ventricular remodelling index (LVRI) ratios were calculated.</p> <p>Results</p> <p>Patients with CHF had less indexed EAT mass than controls (22 ± 5 g/m²versus 34 ± 4 g/m², p < 0.0001). In the subgroup analysis there were no significant differences in indexed EAT mass between patients with ICM and DCM (21 ± 4 g/m²versus 23 ± 6 g/m², p = 0.14). Linear regression analysis showed that with increasing LV end-diastolic diameter (LV-EDD) (r = 0.42, p = 0.0004) and LV end-diastolic mass (LV-EDM) (r = 0.59, p < 0.0001), there was a significantly increased amount of EAT in patients with CHF. However, the ratio of EAT mass/LV-EDM was significantly reduced in patients with CHF compared to healthy controls (0.54 ± 0.1 versus 0.21 ± 0.1, p < 0.0001). In CHF patients higher indexed EAT/LVRI-ratios in CHF patients correlated best with a reduced LV-EF (r = 0.49, p < 0.0001).</p> <p>Conclusion</p> <p>Patients with CHF revealed significantly reduced amounts of EAT. An increase in LVM is significantly related to an increase in EAT in both patients with CHF and controls. However, different from previous reports the EAT/LVEDM-ratio in patients with CHF was significantly reduced compared to healthy controls. Furthermore, the LV function correlated best with the indexed EAT/LVRI ratio in CHF patients. Metabolic abnormalities and/or anatomic alterations due to disturbed cardiac function and geometry seem to play a key role and are a possible explanation for these findings.</p

Med5(Nut1) and Med17(Srb4) Are Direct Targets of Mediator Histone H4 Tail Interactions

The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. In addition to its canonical role in transcriptional activation, recent studies have demonstrated that S. cerevisiae Mediator can interact directly with nucleosomes, and their histone tails. Mutations in Mediator subunits have shown that Mediator and certain chromatin structures mutually impact each other structurally and functionally in vivo. We have taken a UV photo cross-linking approach to further delineate the molecular basis of Mediator chromatin interactions and help determine whether the impact of certain Mediator mutants on chromatin is direct. Specifically, by using histone tail peptides substituted with an amino acid analog that is a UV activatible crosslinker, we have identified specific subunits within Mediator that participate in histone tail interactions. Using Mediator purified from mutant yeast strains we have evaluated the impact of these subunits on histone tail binding. This analysis has identified the Med5 subunit of Mediator as a target for histone tail interactions and suggests that the previously observed effect of med5 mutations on telomeric heterochromatin and silencing is direct