Discovery of Genetic Variation on Chromosome 5q22 Associated with Mortality in Heart Failure

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

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

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 ∼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

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Polycomb Group Proteins Set the Stage for Early Lineage Commitment

Precise control of gene expression patterns is critical for the specification of cellular diversity during metazoan development. Polycomb group (PcG) proteins comprise a class of transcriptional modifiers that have dynamic and essential roles in regulating a number of key processes including lineage commitment. How this is accomplished during mammalian development is incompletely understood. Here, we discuss recent studies in embryonic stem cells (ESCs) that provide critical new insights into how PcG proteins may be targeted to genomic sites as well as the mechanisms by which these regulators influence gene expression and multilineage differentiation in mammals.Massachusetts Life Sciences CenterSmith Family Foundation (Excellence in Biomedical Research

Transcriptional Reversion of Cardiac Myocyte Fate During Mammalian Cardiac Regeneration

Rationale: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level. Objective: The objectives of our study were to determine whether myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process. Methods and Results: We derived a core transcriptional signature of injury-induced cardiac myocyte (CM) regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo CM differentiation, in vitro CM explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of CM differentiation processes, including reactivation of latent developmental programs similar to those observed during destabilization of a mature CM phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13, which induced CM cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of interleukin 13 signaling in CMs. These downstream signaling molecules are also modulated in the regenerating mouse heart. Conclusions: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration. Keywords: cardiac myocyte; gene expression; growth factors/cytokines; myogenesis regenerationNational Institutes of Health (U.S.) (Grant F32HL104913)National Institutes of Health (U.S.) (Grant K99HL122514)National Institutes of Health (U.S.) (Grant U01HL098179)Natioanal Science Foundation (U.S.) (Grant CBET-0939511

Failed Progenitor Specification Underlies the Cardiopharyngeal Phenotypes in a Zebrafish Model of 22q11.2 Deletion Syndrome

Microdeletions involving TBX1 result in variable congenital malformations known collectively as 22q11.2 deletion syndrome (22q11.2DS). Tbx1-deficient mice and zebrafish recapitulate several disease phenotypes, including pharyngeal arch artery (PAA), head muscle (HM), and cardiac outflow tract (OFT) deficiencies. In zebrafish, these structures arise from nkx2.5⁺ progenitors in pharyngeal arches 2–6. Because pharyngeal arch morphogenesis is compromised in Tbx1-deficient animals, the malformations were considered secondary. Here, we report that the PAA, HM, and OFT phenotypes in tbx1 mutant zebrafish are primary and arise prior to pharyngeal arch morphogenesis from failed specification of the nkx2.5⁺ pharyngeal lineage. Through in situ analysis and lineage tracing, we reveal that nkx2.5 and tbx1 are co-expressed in this progenitor population. Furthermore, we present evidence suggesting that gdf3-ALK4 signaling is a downstream mediator of nkx2.5⁺ pharyngeal lineage specification. Collectively, these studies support a cellular mechanism potentially underlying the cardiovascular and craniofacial defects observed in the 22q11.2DS population. Microdeletions encompassing the TBX1 locus cause 22q11.2 deletion syndrome (DS), which is characterized by congenital heart, aorta, and craniofacial malformations. Using a zebrafish model of 22q11.2DS, Guner-Ataman et al. demonstrate that tbx1-mutant animals fail to specify the nkx2.5⁺ progenitor population that gives rise to the affected structures. Keywords: Tbx1; cardiopharyngeal; zebrafish; nkx2.5; heart; DiGeorge; 22q11; progenitor; arch arter

Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (βOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes βOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous βOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, βOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through βOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of βOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury. Ketone body metabolites inform intestinal stem cell decisions in response to diverse diets.National Institutes of Health (U.S.) (Grants R00 AG045144, R01CA211184, R01CA034992, U54-CA163109