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Abnormal intracellular Ca(2+) cycling plays a key role in cardiac dysfunction, particularly during the setting of ischemia/reperfusion (I/R). During ischemia, there is an increase in cytosolic and sarcoplasmic reticulum (SR) Ca(2+). At the onset of reperfusion, there is a transient and abrupt increase in cytosolic Ca(2++), which occurs timely associated with reperfusion arrhythmias. However, little is known about the subcellular dynamics of Ca(2+) increase during I/R, and a possible role of the SR as a mechanism underlying this increase has been previously overlooked. The aim of the present work is to test two main hypotheses: (1) An increase diastolic Ca(2+) sparks frequency (cspf) constitutes a mayor substrate for the ischemia-induced diastolic Ca(2+) increase; (2) an increase in cytosolic Ca(2+) pro-arrhythmogenic events (Ca(2+) waves), mediates the abrupt diastolic Ca(2+) rise at the onset of reperfusion. We used confocal microscopy on mouse intact hearts loaded with Fluo-4. Hearts were submitted to global I/R (12/30 min) to assess epicardial Ca(2+) sparks in the whole heart. Intact heart sparks were faster than in isolated myocytes whereas cspf was not different. During ischemia, cspf significantly increased relative to preischemia (2.07±0.33 vs. 1.13±0.20 sp/s/100 μm, n=29/34, 7 hearts). Reperfusion significantly changed Ca(2+) sparks kinetics, by prolonging Ca(2+) sparks rise time and decreased cspf. However, it significantly increased Ca(2+) wave frequency relative to ischemia (0.71±0.14 vs. 0.38±0.06 w/s/100 μm, n=32/33, 7 hearts). The results show for the first time the assessment of intact perfused heart Ca(2+) sparks and provides direct evidence of increased Ca(2+) sparks in ischemia that transform into Ca(2+) waves during reperfusion. These waves may constitute a main trigger for reperfusion arrhythmias.

Background: Friedreich's ataxia (FRDA) is an autosomal recessive disease, whereby homozygous inheritance of an expanded GAA trinucleotide repeat expansion in the first intron of the FXN gene leads to transcriptional repression of the encoded protein frataxin. FRDA is a progressive neurodegenerative disorder, but the primary cause of death is heart disease which occurs in 60% of the patients. Several functions of frataxin have been proposed, but none of them fully explain why its deficiency causes the FRDA phenotypes nor why the most affected cell types are neurons and cardiomyocytes. Methods: To investigate, we generated iPSC‐derived neurons (iNs) and cardiomyocytes (iCMs) from an FRDA patient and upregulated FXN expression via lentivirus without altering genomic GAA repeats at the FXN locus. Results: RNA‐seq and differential gene expression enrichment analyses demonstrated that frataxin deficiency affected the expression of glycolytic pathway genes in neurons and extracellular matrix pathway genes in cardiomyocytes. Genes in these pathways were differentially expressed when compared to a control and restored to control levels when FRDA cells were supplemented with frataxin. Conclusions: These results offer novel insight into specific roles of frataxin deficiency pathogenesis in neurons and cardiomyocytes. [ABSTRACT FROM AUTHOR]

In most species androgens shorten the cardiac action potential and reduce the risk of afterdepolarizations. Despite the central role of the rat model in physiological studies, the effects of androgens on the rat heart are still inconclusive. We therefore performed electrophysiological studies on the perfused rat right ventricular free wall. We found a correlation between androgenic activity and a propensity to generate ventricular ectopic action potentials. We also found that the testosterone treatment increased action potential duration at 90 % of repolarization (APD90), while androgenic inhibition increased the time to peak and decreased APD90. We observed that the voltage-gated potassium channel Kv4.3 and the bi-directional membrane ion transporter NCX in the rat myocardium were regulated by androgenic hormones. One possible explanation for these findings is that due to the expression of specific ion channels in the rat myocardium, the action potential response to its hormonal background is different from those described in other experimental models. Our results indicate that androgenic control of NCX expression plays a key role in determining arrhythmogenicity in the rat heart. Keywords: Androgens, Arrhythmia, Rat, Na/Ca exchanger, Kv4.3, Author(s): Mariana Argenziano[sup.1], Gisela Tiscornia[sup.1], Rosalia Moretta[sup.1], Leonardo Casal[sup.1], Constanza Potilinski[sup.1,2], Carlos Amorena[sup.1,2] and Eduardo Garcia Gras[sup.1,2] Introduction Action potential duration (APD) is shortened by androgens in humans [1], Guinea [...]

Background: The risk for late‐onset Alzheimer disease (AD) in APOEε4 carriers differs between ancestral groups, where APOEε4's odds ratio for AD risk is lower in African (AFR) homozygous carriers than in non‐Hispanic White (NHW) or Japanese (JPT) carriers (odds ratio ∼2‐5 vs >15). Local ancestry (LA) analyses in APOEε4 carrier populations have shown the protective effect in AFR relative to EUR/JPT is due to noncoding factors lying in the LA surrounding APOEε4. Thus, regulatory differences between risk and protective LA haplotypes are most likely involved in the differential risk effect seen for APOEε4 on different backgrounds. Methods: We identified 56 significant sequence differences between AFR and EUR/JPT APOEε4 haplotypes from the 1000 genomes in the immediate topologically associated domain surrounding APOE. We performed two different Massively Parallel Reporter Assay (MPRA) designs; one assessing small haplotype (∼900bp) effects and one based upon single variant effects. We supplemented these results with single fragment luciferase reporter assays. All assays were performed in at least duplicate in HMC3 (microglia), U118 (astrocytes) and SH‐SY5Y (neurons) cell lines. Additionally, we integrated chromatin interaction information from promoter capture C chromatin conformation assays in the same cell types. Results: We identified a region in the first introns of TOMM40 with increased EUR/JPT enhancer activity, supported by both MPRA analyses and APOE promoter interaction in astrocytes and microglia. Two additional regions with differential enhancer activity in neurons, but no promoter interaction, were identified; downstream of APOE and in PVRL2 introns upstream of APOE presenting with higher EUR or higher JPT haplotype variant enhancer activity compared to AFR, respectively. Conclusions: Our results indicate several areas of differential regulation in this LA region on APOEε4 haplotypes dependent on cell type. As APOE is mostly expressed in glial cells, the data in TOMM40 introns points to this region as having the biggest impact on APOE expression in our study and thus highly supports the involvement of this region in the differential risk effects seen for APOEε4. Follow‐up of the identified regulatory regions is currently ongoing using in‐house iPSC derived cell lines. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) research has principally focused on neurons due to their role in neurodegeneration. In contrast, recent studies suggest that genetic mechanisms drive microglia to prolonged inflammation in AD brains, exacerbating neurodegeneration. Indeed, recent genome‐wide association studies (GWAS) of AD have identified multiple loci near genes related to microglial function, such as TREM2 and CR1. However, GWAS does not have the sensitivity to identify causal variants or effector genes. We used a combination of ATAC‐seq and high‐resolution promoter‐focused Capture‐C in two human microglial cell models to map interactions between GWAS‐implicated variants and their putative effector genes. We then validated an observed interaction at the 'CASS4' locus using CRISPR‐Cas9 genome editing. Method: Improving on the relatively low resolution of available Hi‐C data, we employed high resolution Capture‐C to characterize the physical genome‐wide interactions of all human promoters. We performed this in the human microglial cell line HMC3 and human iPSC‐derived microglia (iMg). We confirmed the enhancer activity of a SNP we elected to pursue and its associated regulatory element using dual‐luciferase assays. To confirm the regulatory interaction with the Capture‐C implicated effector gene, we used lentiviral CRISPR‐Cas9 to delete an approximately 300bp region containing the SNP in HMC3 cells. We then confirmed changes in RNA and protein expression using RNA‐seq and Western blotting, respectively. Result: Variant‐to‐gene mapping in both microglial cell models identified 67 putative effector genes (51 coding) across both cell types, with 14 observed in both models. We identified a novel proxy SNP, rs6024870 (r2 = 0.93 to sentinel SNP rs6014724), at the 'CASS4' locus, which coincided with open chromatin and directly contacted the promoter of RTFDC1, a gene not previously implicated in AD. Deletion of the putative enhancer region harboring rs6024870 by CRISPR‐Cas9 in HMC3 reduced the expression of RTFDC1 at both the mRNA and protein level. We also note that CASS4 levels were modestly influenced by this CRISPR‐mediated perturbation. Conclusion: We implicate RTFDC1 as a novel effector gene at the AD 'CASS4' GWAS locus. Further efforts will characterize the phenotypic effect of this variant in microglial cell models, including on inflammation and phagocytic activity. [ABSTRACT FROM AUTHOR]

Allergic asthma is an inflammatory disorder characterized by infiltration of the airway wall with inflammatory cells driven mostly by activation of Th2- lymphocytes, eosinophils and mast cells. There is a link between increased allergy and a reduction of some infections in Western countries. Epidemiological data also show that respiratory allergy is less frequent in people exposed to orofecal and foodborne microbes such as Toxoplasma gondii. We previously showed that both acute and chronic parasite T. gondii infection substantially blocked development of airway inflammation in adult BALB/c mice. Based on the high levels of IFN-γ along with the reduction of Th2 phenotype, we hypothesized that the protective effect might be related to the strong Th1 immune response elicited against the parasite. However, other mechanisms could also be implicated. The possibility that regulatory T cells inhibit allergic diseases has received growing support from both animal and human studies. Here we investigated the cellular mechanisms involved in T. gondii induced protection against allergy. Our results show for the first time that thoracic lymph node cells from mice sensitized during chronic T. gondii infection have suppressor activity. Suppression was detected both in vitro, on allergen specific T cell proliferation and in vivo, on allergic lung inflammation after adoptive transference from infected/sensitized mice to previously sensitized animals. This ability was found to be contact-independent and correlated with high levels of TGF-β and CD4+ FoxP3+ cells. [ABSTRACT FROM AUTHOR]

Late-onset Alzheimer's disease (LOAD) research has principally focused on neurons over the years due to their known role in the production of amyloid beta plaques and neurofibrillary tangles. In contrast, recent genomic studies of LOAD have implicated microglia as culprits of the prolonged inflammation exacerbating the neurodegeneration observed in patient brains. Indeed, recent LOAD genome-wide association studies (GWAS) have reported multiple loci near genes related to microglial function, including TREM2 , ABI3 , and CR1 . However, GWAS alone cannot pinpoint underlying causal variants or effector genes at such loci, as most signals reside in non-coding regions of the genome and could presumably confer their influence frequently via long-range regulatory interactions. We elected to carry out a combination of ATAC-seq and high-resolution promoter-focused Capture-C in two human microglial cell models (iPSC-derived microglia and HMC3) in order to physically map interactions between LOAD GWAS-implicated candidate causal variants and their corresponding putative effector genes. Notably, we observed consistent evidence that rs6024870 at the GWAS CASS4 locus contacted the promoter of nearby gene, RTFDC1 . We subsequently observed a directionallly consistent decrease in RTFDC1 expression with the the protective minor A allele of rs6024870 via both luciferase assays in HMC3 cells and expression studies in primary human microglia. Through CRISPR-Cas9-mediated deletion of the putative regulatory region harboring rs6024870 in HMC3 cells, we observed increased pro-inflammatory cytokine secretion and decreased DNA double strand break repair related, at least in part, to RTFDC1 expression levels. Our variant-to-function approach therefore reveals that the rs6024870-harboring regulatory element at the LOAD ' CASS4' GWAS locus influences both microglial inflammatory capacity and DNA damage resolution, along with cumulative evidence implicating RTFDC1 as a novel candidate effector gene., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.