Background:We previously performed a strain intercross between atherosclerosis resistant AKR Apoe -/- mice and atherosclerosis sensitive DBA/2 Apoe -/- mice and identified the Ath28 quantitative trait locus (QTL) on the distal end of chromosome 2. Congenic strain fine mapping identified the Ath28 . 1 QTL atherosclerosis modifying subregion, encompassing 217 Kb, containing for only three protein-coding genes, Zbp1, Pck1 , and Pmepa1 , encoding respectively, Z-DNA binding protein 1, phosphoenolpyruvate carboxykinase 1, and prostate transmembrane protein androgen induced 1. Methods:The effect of macrophage-specific knockout of Pck1 (KO) was tested using the AAV2 transduced proprotein convertase subtilisin kexin type 9 (PCSK9) overexpression mouse model of hyperlipidemia and atherosclerosis. Results:Unexpectedly, macrophage Pck1 deficiency lowered body weight, liver weight, and HDL-cholesterol levels in both sexes, while total and non-HDL cholesterol levels were only decreased in male mice. Aortic root lesion area and necrotic lesion area were unchanged in KO mice of both sexes. Conclusion:Pck1 was not confirmed as an atherosclerosis modifier gene.

OBJECTIVE:High-density lipoprotein-cholesterol (HDL-C) has recently garnered attention in lung transplantation studies for its anti-inflammatory properties. Herein, we aimed to study the association between basal HDL-C serum levels and outcomes of lung transplantation with respect to sex. METHODS:In this retrospective cohort study in a tertiary center between 2009 and 2019, 1063 patients that underwent single or double lung transplant were included. Continuous variables are presented as median [25th - 75th percentiles] and analyzed using the Wilcoxon rank-sum test. Primary outcome was primary graft dysfunction (PGD) at 72 h. Survival was evaluated using Kaplan-Meier analysis. Multivariable logistic regression was used to identify independent risk factors for long term mortality, PGD at 72 h, and chronic lung allograft dysfunction. RESULTS:Our study had 349 females and 714 males with a median age of 59 [49 - 63] years and 62 [55-67] years, respectively. Recipients with HDL-C >69 mg/dL had significantly better survival (HR 0.73 [95% CI 0.58-0.91, p = 0.03). Dividing the cohort based on sex, high HDL-C in female recipients was associated with significantly better survival, lower PGD grade, and shorter hospital stay. In male recipients, the same trends were observed but did not reach statistical significance. Higher baseline HDL-C level was associated with better survival (HR 0.46, 95% CI 0.22-0.96; p = 0.040), lower risk for PGD (OR 0.611, 95% CI 0.562-0.86, p = 0.002), chronic allograft dysfunction (OR 0.91, 95% CI 0.84-0.98; p=0.012). CONCLUSION:Higher baseline High-density lipoprotein-cholesterol levels before lung transplant were associated with better outcomes, including lower primary graft dysfunction grades, less graft failure, and better survival. The association between higher high-density lipoprotein-cholesterol levels and better outcomes after lung transplantation in females warrants further studies to better characterize its influence on lung transplantation outcomes.

Large-scale multiancestry genome-wide association studies have identified hundreds of loci associated with type 2 diabetes (T2D) and glycemic traits, yet imputed genotyping arrays limit the detection of low-frequency and rare variants. Whole-genome sequencing (WGS) offers a more complete view of genetic variation, especially across diverse populations. We analyzed high-coverage (38×) WGS data from 21,913 T2D case subjects, 61,036 control subjects, and up to 50,011 individuals with no diabetes with fasting glucose, fasting insulin, and HbA1c from the National Heart, Lung, and Blood Institute Trans-Omics for Precision Medicine Program. We performed single-variant association testing, conditional analysis, fine-mapping, and Bayesian colocalization to identify genetic signals and assess regulatory relevance in diabetes-related tissues. We identified 76 distinct association signals across 34 loci, including novel variants at DUSP9 for T2D, and ROBO1, NDN, and MYT1 for HbA1c. Fine-mapping narrowed credible sets and improved causal variant resolution. Colocalization highlighted 80 expression signals in diabetes-related tissues, linking genetic associations to functional regulatory mechanisms. Our findings demonstrate the utility of WGS to uncover novel variants in diverse populations, enhance locus resolution, and link regulatory variation to disease-relevant tissues. This work refines the genetic architecture of T2D and glycemic traits and supports precision medicine efforts targeting diverse populations. ARTICLE HIGHLIGHTS:We aimed to improve understanding of the genetic architecture of type 2 diabetes and glycemic traits by leveraging whole-genome sequencing in diverse populations. Our goal was to identify novel variants, refine known loci, and link genetic signals to regulatory mechanisms through colocalization with expression quantitative trait loci. We discovered novel variants, significantly improved fine-mapping resolution, and identified 80 regulatory colocalization signals in diabetes-relevant tissues. These findings support precision medicine approaches by connecting genetic variation to functional biology in type 2 diabetes.

Gut microbiota-derived trimethylamine N-oxide (TMAO) plays a role in the pathogenesis of cardiovascular disease, but its role in the pathogenesis of atrial fibrillation (AF) remains uncertain. TMAO levels were quantified in plasma from serial subjects undergoing elective cardiac catheterizations and shown to independently associate with prevalent AF following adjustment for risk factors. Human cAMP response element modulator isoform IbΔC-X transgenic mice (CREM-IbΔC-X) supplemented with a TMAO diet developed AF sooner. C57BL/6J mice on and off a TMAO diet had more inducible AF via a transesophageal pacing study compared with chow-fed controls. Dietary choline supplementation increased circulating TMAO levels and significantly accelerated AF onset in CREM-IbΔC-X mice. Iodomethylcholine (IMC) reduced circulating TMAO levels and choline-induced AF onset. Cecal metagenomic analyses showed that choline supplementation induced changes in microbial communities associated with AF, while many of these changes were attenuated by IMC. Choline supplementation promoted overall adverse atrial remodeling with left atrial dilation. Optical mapping studies showed that mice supplemented with choline exhibited reduced conduction velocity, shortened action potential duration at 80% repolarization, and decreased wavelength. TMAO inhibited muscarinic receptor 2, resulting in autonomic dysfunction that promotes AF. In summary, TMAO, independently associated with AF risk in subjects, enhanced AF in multiple mouse models via autonomic dysfunction and is a therapeutic target for preventing AF.

Rare coding genetic variants may exert large effects on risk of common disease, yet their contribution to disease architecture and their utility in gene prioritization remain limited by inadequate sample sizes. Here, we performed a massive-scale rare variant association study (RVAS), analyzing over 1.1 million sequenced participants among which 130,000 had atrial fibrillation (AF). Through a multi-mask burden testing approach, we identified 15 genes significantly associated with AF through rare large-effect variation. Integrative analyses revealed strong convergence between genes implicated by rare and common variation, and highlighted instances where RVAS data may aid in GWAS prioritization. Nevertheless, several RVAS genes were not among GWAS loci ( FAM189A2 , ACTC1 , FNIP1 , FBN1 ), or were not nominated through contemporary GWAS prioritization ( KDM5B , ZFP36L2 ). Finally, we observed that ultra-rare protein-disrupting variants - concentrated in a small number of large-effect size genes - explained at least 2% of AF susceptibility across European and African ancestry groups. These findings refine the genetic architecture of AF, while highlighting the value and cost of RVAS for genomic discovery in common disease.

Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis, with symptoms including pruritus, pancreatitis, fat malabsorption, intestinal inflammation, and failure to thrive. High-throughput studies showed interconnection between ATP8B1 and phosphoinositide (PIPs), but the mechanism linking ATP8B1, lipid metabolism, and inflammation remains unclear. Atp8b1 G308V/G308V mouse model, unbiased RNAseq, high-resolution-stimulation emission depltion (STED)-microscopy, and Crispr-Cas9 generated ATP8B1 −/− knockouts in hepatocytes/monocytes/macrophages were used to determine role of ATP8B1 in phosphatidylinositol,4-5-bisphosphate (PIP2) trafficking and inflammation. Human ATP8B1, purified from Sf9 insect cells and reconstituted in proteoliposomes, was used to test cell-free PIP2 flip. Various in-vitro techniques were used for testing direct interaction between PIP2 and ATP8B1. ATP8B1 maintains PIP2 at the inner leaflet of plasma membrane (PM). ATP8B1 flips PIP2 in cells, without altering flip of PE or bulk-endocytosis. ATP8b1 flips PIP2 in a cell-free system. ATP8B1 deletion promotes bile-salt-mediated cholesterol extraction from hepatocytes in a PIP2-dependent manner. PIP2 directly binds to the P-loop of ATP8B1. Unbiased RNAseq showed upregulation of inflammatory cytokines in ATP8b1 −/− immune cells. ATP8B1 −/− monocytes/macrophages showed aberrant lipopolysaccharide (LPS)-induced cleavage of GSDMD, formation of GSDMD pores, and interleukin-1beta (IL1β) release. Inflammation-resolving efferocytosis was impaired in ATP8B1 −/− macrophages. Biophysical properties of PM were altered in ATP8b1 −/− cells, with the mechanism being disrupted localization of PIP2. Atp8b1 G308V/G308V mice exposed to LPS showed higher plasma IL1β and lower survival rates vs. WT mice. ATP8B1 maintains PIP2 at the inner leaflet of PM. ATP8b1 directly flips and binds PIP2. ATP8B1 regulates LPS-induced GsdmD cleavage, formation of GsdmD pores, IL1β release, and mortality in mice.

Atrial fibrillation (AF) is a prevalent and morbid abnormality of the heart rhythm with a strong genetic component. Here, we meta-analyzed genome and exome sequencing data from 36 studies that included 52,416 AF cases and 277,762 controls. In burden tests of rare coding variation, we identified novel associations between AF and the genes MYBPC3, LMNA, PKP2, FAM189A2 and KDM5B. We further identified associations between AF and rare structural variants owing to deletions in CTNNA3 and duplications of GATA4. We broadly replicated our findings in independent samples from MyCode, deCODE and UK Biobank. Finally, we found that CRISPR knockout of KDM5B in stem-cell-derived atrial cardiomyocytes led to a shortening of the action potential duration and widespread transcriptomic dysregulation of genes relevant to atrial homeostasis and conduction. Our results highlight the contribution of rare coding and structural variants to AF, including genetic links between AF and cardiomyopathies, and expand our understanding of the rare variant architecture for this common arrhythmia.

Isolation of the pulmonary veins (PV) is a primary goal of ablation procedures to treat atrial fibrillation (AF), and the top genetic risk locus for AF is near PITX2, implicated in formation of the PVs. However, the challenges in obtaining PV tissues have limited progress in transcriptomic and mechanistic insights. Human PV and left atrial appendage (LAA) tissues, obtained from unused transplant donors, were used for spatial transcriptomic studies. Multiple cells and cell types may reside in each 55 µm diameter spatial area. Seurat clustering yielded 15 different clusters. Cell-type specific marker genes were used to determine the dominant cell types in these clusters, identifying several clusters enriched for cardiomyocytes, while others were enriched for additional cell types including fibroblasts, vascular smooth muscle cells, endothelial cells, and adipocytes. Spatial transcriptomics clearly resolved the venous, cardiomyocyte, and epicardial regions of the PV tissues, as well as fibrotic regions in LAAs and PVs. Spatial expression of the AF-associated genes PITX2, SHOX2, and HCN4 confirmed presence in LAA and PVs with apparently higher expression of the cardiac master transcription factor SHOX2 in the PV vs. LAA tissues, implicating the potential importance of SHOX2 regulation in the PVs.

A hallmark event in the development of atherosclerotic plaque is the accumulation of lipid-laden macrophages in the subendothelial layers of affected blood vessels. Macrophages are key players in all stages of atherogenesis, including plaque initiation, growth, and rupture, as well as healing of ruptured plaques. In this context, macrophages are the principal innate immune cells that modulate atherogenesis by engaging in various processes, such as inflammation, extracellular matrix degradation, phagocytosis, and efferocytosis. In the current study, Kruppel-like transcription factor 6 (KLF6) deficiency attenuated proinflammatory gene expression in macrophages and experimentally induced atherosclerotic plaque development. In vivo studies showed that myeloid-KLF6 deficiency on Apoe-null background significantly curtailed high-fat/high-cholesterol diet-induced atherosclerotic lesion formation and macrophage abundance in atherosclerotic plaques. Integrated transcriptomics and Gene Set Enrichment Analysis showed that KLF6 deficiency significantly curtailed a large number of tumor necrosis factor (TNF)-induced gene targets, TNF-induced interferon-γ response, interferon-α response, and inflammatory response signaling in macrophages. At the molecular level, KLF6 promoted interferon regulatory factor 1 (IRF1) signaling to enhance TNF-induced proinflammatory gene expression in macrophages. Collectively, study results show that KLF6 promoted proinflammatory gene expression in macrophages and enhanced experimentally induced atherosclerotic plaque formation in vivo.

Background/Objectives: Macrophages play a pivotal role in various pathogenic processes, necessitating the development of efficient differentiation techniques to meet the high demand for these cells in research and therapy. Human macrophages can be obtained via culturing peripheral blood monocytes; however, this source has limited yields and requires patient contact for each proposed use. In addition, it would be difficult to perform gene editing on peripheral blood monocytes. The objectives of this study are to define a robust and consistent method for the differentiation of induced pluripotent stem cells (iPSCs) into macrophages that can address these needs for recurrent studies with high yields and the potential for gene editing. Methods: We refined the traditional embryoid body-based differentiation strategy to create a novel three-phase method that optimizes yield, consistent quality, and reproducibility. This approach incorporates microwell plates and cell filtration to standardize the production of embryoid bodies and subsequent macrophage progenitors. Using up to five independent iPSC donors, we performed several assays for macrophage functions and polarization, such as marker protein staining by flow cytometry, lipoprotein uptake, phagocytosis, cytokine release, inflammasome activation, and the effects of M1-like and M2-like polarization. RNA sequencing was performed to determine the segregation of cells at different stages of differentiation and by iPSC donor, as well as to identify marker genes for each stage of differentiation. Results: The iPSC-derived macrophages generated through this method exhibit characteristic features and cell marker proteins, as well as classical macrophage activities, including lipoprotein uptake, bacterial phagocytosis, cytokine release, and inflammasome activation. We demonstrate the effects of M1-like and M2-like polarization on cytokine release. The first three principal components of the RNA sequencing data showed clear clustering by differentiation stage. In contrast, the fourth and fifth principal components clustered the differentiated macrophages by their respective iPSC donor. Marker genes were identified for each stage of differentiation and polarization. Conclusions: The methods provide an optimized and simplified procedure to produce iPSC-derived macrophages. Our results demonstrate the reproducibility of this method in generating high-quality macrophages suitable for a variety of biomedical applications.

Background and aims An AKRxDBA/2 mouse strain intercross identified an atherosclerosis modifier gene within a small region on chromosome 2, containing only three protein coding genes, including Z-DNA binding protein 1 (Zbp1), which plays a role in non-canonical necroptosis. We aimed to determine if knockout (KO) of Zpb1 decreased lesion and necrotic areas in mice. Methods Zpb1 isoform specific expression was assessed by RNAseq in mouse macrophages and aortic arches. Necroptosis was assessed in cultured mouse macrophages. Hyperlipidemia was induced by weekly injection of an antisense oligonucleotide targeting the mouse LDL receptor gene, combined with feeding a Western-type diet. Lesions were assessed in the aortic root and brachiocephalic artery in both sexes separately and after adjusting for sex in combined analyses. Results Both AKR and DBA/2 strains express two isoforms of Zbp1 mRNA with higher expression in the DBA/2 atherosclerosis-sensitive strain. Zbp1 KO and wildtype (WT) macrophages were equally susceptible to canonical and non-canonical necroptosis. There were some sex specific effects of the KO in body weight, organ weights, and plasma cholesterol values. In both sexes, KO mice trended towards, or had significantly smaller, lesion areas and necrotic cores, which were significant in combined sex analyses. Lesion cell compositions were qualitatively similar in KO and WT mice. Conclusions Zbp1 is an atherosclerosis modifier gene, similar to the canonical necroptosis genes Ripk1 and Mlkl. Higher expression of Zbp1 in DBA/2 mice may play a role in this strain having the largest lesions of all strains tested in the Apoe-deficient background.

Genome-wide association studies have identified a locus on chromosome 10q22, where many co-inherited single nucleotide polymorphisms (SNPs) are associated with atrial fibrillation (AF). This study seeks to identify the impact of this locus on gene expression at the transcript isoform level in human left atria and to gain insight into potential causal variants. Bulk RNA sequencing was analyzed to identify myozenin 1 (MYOZ1) and synaptopodin 2-like (SYNPO2L) transcript isoforms and the association of common SNPs in this region with transcript isoform expression levels. Chromatin marks were used to suggest candidate regulatory SNPs in this region. Protein amino acid changes were examined for predicted functional consequences. Transfection of MYOZ1 and two SYNPO2L isoforms were performed to localize their encoded proteins in cardiomyocytes derived from stem cells. We identified one MYOZ1 transcript isoform and four SYNPO2L transcript isoforms, two of which encode proteins, while the other two encode long noncoding RNAs (lncRNAs). The risk allele of the strongest AF susceptibility SNP on chromosome 10q22 is associated with decreased MYOZ1 expression and increased expression of the two SNYPO2L lncRNA isoforms. There are many SNPs co-inherited with the top AF-associated SNP due to linkage disequilibrium (LD), including rs11000728, which we propose as the MYOZ1 regulatory SNP, confirmed by reporter gene transfection. In addition, this LD block includes three missense SNPs in the SYNPO2L gene, with the minor protective haplotype predicted to be detrimental to protein function. MYOZ1 and both protein isoforms of SYNPO2L were localized to the sarcomere. This is a complex locus with the potential for several SNPs in a haplotype to alter AF susceptibility by opposing effects on MYOZ1 and SYNPO2L lncRNA expression, along with effects on SYNPO2L protein function.

BACKGROUND:Atrial fibrillation GWAS (genome-wide association studies) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding Nesprin-2, which connects the nuclear membrane with the cytoskeleton. METHODS:Reporter gene vector transfection and CRISPR-Cas9 editing were used to identify the causal variant regulating the expression of SYNE2 alpha 1. After SYNE2 knockdown or SYNE2 alpha 1 overexpression in human stem cell-derived cardiomyocytes, nuclear phenotypes were assessed by imaging and atomic force microscopy. Gene expression was assessed by RNAseq and gene set enrichment analysis. Fura-2 AM staining assessed calcium transients. Optical mapping assessed action potential duration and conduction velocity. RESULTS:The risk allele of rs1152591 had lower promoter and enhancer activity and was significantly associated with lower expression of the short SYNE2 alpha 1 isoform in human stem cell-derived cardiomyocytes, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2 alpha 1 overexpression had dominant negative effects on the nucleus with its overexpression or SYNE2 knockdown leading to increased nuclear area and decreased nuclear stiffness. Gene expression results from SYNE2 alpha 1 overexpression demonstrated both concordant and nonconcordant effects with SYNE2 knockdown. SYNE2 alpha 1 overexpression had a gain of function on electrophysiology, leading to significantly faster calcium reuptake and decreased assessed action potential duration, while SYNE2 knockdown showed both shortened assessed action potential duration and decreased conduction velocity. CONCLUSIONS:rs1152591 was identified as a causal atrial fibrillation variant, with the risk allele decreasing SYNE2 alpha 1 expression. Downstream effects of SYNE2 alpha 1 overexpression include changes in nuclear stiffness and electrophysiology, which may contribute to the mechanism for the risk allele's association with AF.

Migraine in elderly patients requires an individualized approach, with unique considerations and challenges in both diagnosing and managing the disorder. Aging brains differ from younger ones in many ways, with distinct trajectories impacting brain volume, neurotransmitter systems, and functional systems, such as the descending pain inhibitory system. In this chapter, we will deconstruct migraine in the elderly by discussing the definitions of the elderly and migraine, the prevalence of migraine, and the management of migraine including the challenges posed by its comorbidities, limitations of treatments, and its effects on cognition. Studying, quantifying, characterizing, diagnosing, or managing migraine in the elderly is a challenge. However, it is clear that migraine in the elderly is not an uncommon occurrence, and providers should be aware that many elderly patients will not present with the complete phenotypic profile. Fortunately, the weight of evidence has not established migraine as a risk factor for dementia in the elderly, although migraine and its pharmacologic treatments may adversely impact cognition. Successful management requires understanding the interactions of migraine with comorbidities in the elderly and the adverse impacts of polypharmacy.