Ankyrin-B (encoded by ANK2), originally identified as a key cytoskeletal-associated protein in the brain, is highly expressed in the heart and plays critical roles in cardiac physiology and cell biology. In the heart, ankyrin-B plays key roles in the targeting and localization of key ion channels and transporters, structural proteins, and signaling molecules. The role of ankyrin-B in normal cardiac function is illustrated in animal models lacking ankyrin-B expression, which display significant electrical and structural phenotypes and life-threatening arrhythmias. Further, ankyrin-B dysfunction has been associated with cardiac phenotypes in humans (now referred to as “ankyrin-B syndrome”) including sinus node dysfunction, heart rate variability, atrial fibrillation, conduction block, arrhythmogenic cardiomyopathy, structural remodeling, and sudden cardiac death. Here, we review the diverse roles of ankyrin-B in the vertebrate heart with a significant focus on ankyrin-B-linked cell- and molecular-pathways and disease.

Rationale: Coronary artery disease (CAD) is a pervasive and critical healthcare problem. Elevated high density lipoprotein-associated cholesterol (HDL-C) is associated with improved atherosclerotic cardiovascular disease (ASCVD) outcomes on a population level, but clinical trials aimed at HDL-C elevation have not succeeded in improving ASCVD event risk. Nevertheless, human variants in the HDL receptor, encoded by SCARB1, are associated with dyslipidemia, suggesting that HDL metabolism, not HDL-C, is a suitable target for therapy. However, variants in SCARB1 have never been directly attributed to CAD by Mendelian inheritance. Objective: To determine if compound heterozygous variants in SCARB1 cause disease in two brothers with severe, early-onset CAD. Methods and Results: Using whole exome sequencing, we have identified rare, compound heterozygous variants in SCARB1 that segregate with severe, premature CAD, following patterns of Mendelian inheritance. Using induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs) from the proband, we discovered the maternal variant (c.754_755delinsC) to be the first identified SCARB1 null allele, characterized by the absence of RNA and protein expression. Further, we demonstrate that the variant on the paternal allele (c.956G>T (p.G319V)) results in decreased cholesterol uptake, decreased SR-BI:HDL binding, and increased affinity for SR-BI dimerization. Finally, we generated a p.G319V knock-in mouse model that displays nearly 100% homozygous lethality and elevated plasma cholesterol in heterozygous animals, confirming pathogenicity of this variant. Conclusions: In summary, our data provide the first molecular mechanism to show the Mendelian inheritance of CAD as a result of human SCARB1 variants. The rarity of these variants supports pathogenicity in this family. Furthermore, SR-BI p.G319V, which has previously been reported benign in the context of heterozygosity, was uniquely presented alongside a null allele, demonstrating the disease-contributing capability of loss-of-function SCARB1 variants within the population.

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RATIONALE: The purpose of this study was to determine whether conventionally used ILC2 markers, ST2 and CD127, are sufficient to include all Th2-cytokine producing ILCs. We hypothesized that ST2-and CD127-negative ILC2 populations exist in mouse lung and that these nontraditional populations might be activated by the clinically relevant fungal allergen Alternaria, a known potent inducer of IL-33. METHODS: Mice were challenged intranasally with Alternaria alternata for three consecutive days, and 24 hours after the last challenge brochoalveolar lavage fluid and lungs were harvested for flow cytometric analysis, ELISA, and lung section staining. ILCs were identified by flow cytometry as CD45+Lineage-Thy1.2+ lymphocyte-sized cells and were separated into four subsets based on ST2 and CD127 expression. Intracellular cytokine and transcription factor staining were performed on the ILC populations. RESULTS: Th2 cytokine-producing ILCs were identified in all four subpopulations, regardless of ST2 and CD127 expression, as evidenced by high GATA-3, IL-5, IL-13 and ILC2 surface marker expression. Further, ILC2 cytokines and surface markers were upregulated following Alternaria challenge, irrespective of ST2 and CD127 expression. The four ILC subpopulations were also found in RAG2 knockout mice, which is supportive of their presence being T-cell independent. Some of the unconventional ILC2s also produced IFNg and IL-17A suggesting the existence of ILC2 plasticity in these populations. CONCLUSIONS: Unconventional ST2-and CD127-negative ILC2 populations exist in mouse lung and are further induced by Alternaria. Thus, future mouse ILC2 investigations should be aware of ILC2 populations that do not express ST2 and CD127.

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Dyslipidemia is a cardiovascular risk factor for coronary artery disease and atherosclerosis that is characterized by elevated serum cholesterol and lipid levels. Although high-density lipoprotein-associated cholesterol (HDL-C) is associated with reduced risk of cardiovascular events, targeted therapy to increase HDL-C levels have been unsuccessful in altering outcomes of associated atherosclerotic disease. Single nucleotide polymorphisms in SCARB1 , the gene that encodes HDL receptor Scavenger Receptor B1 (SR-BI), are associated with dyslipidemia and atherosclerotic cardiovascular disease. We were the first to identify inherited mutations in SCARB1 that segregate with disease in a family with severe coronary artery disease and dyslipidemia, including elevated HDL. Our findings suggest that HDL function (vs. HDL-C concentration) may be a promising target for cholesterol-based therapy. Here, we performed an unbiased high throughput drug screen with 788 FDA-approved compounds, using HepG2 cells to measure endogenous HDL binding. We identified five compounds that significantly increased endogenous HDL binding: imatinib, trimethoprim, eszopiclone, clemastine, and mepenzolate, of which, imatinib was the only compound to increase SR-BI expression. Imatinib is a tyrosine kinase inhibitor that is a chemotherapeutic agent designed to treat individuals with chronic myeloid leukemia. Limited clinical evidence suggests a reduction in total cholesterol with 400mg/day imatinib. Additionally, imatinib treatment (150mg/kg) in mouse models of atherosclerosis reduces total cholesterol. Yet, no data is available on the effects of imatinib on HDL and reverse cholesterol transport. We have found that imatinib promotes HDL binding and SR-BI expression in vitro . Furthermore, in wildtype C57Bl/6 mice on a high fat, high cholesterol diet, imatinib treatment (50mg/kg) was sufficient to decrease plasma total cholesterol, HDL-C and triglyceride levels and elevate hepatic SR-BI expression. In summary, our data supports the exploration of imatinib-mediated SR-BI regulation, HDL metabolism, and RCT pathway to identify new therapeutic targets for dyslipidemia.

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IntroductionWe have previously reported that lung epithelial tissue factor (TF) knockout (TFΔEpi) mice have increased alveolar capillary barrier permeability. Ultrastructurally, these mouse lungs have profound morphological defects in epithelial attachment to the basement membrane. Adhesion/migration are regulated in part by integrins which bind the extracellular matrix proteins to modulate cytoskeletal dynamics. In cancer cells, TF binds β1 integrin and increases cell migration, a finding that suggests a role for non‐coagulative mechanisms of TF in cell adhesion and migration. We hypothesize that TF‐β1 integrin binding promotes alveolar epithelial barrier integrity through cell adhesion and migration during acute lung injury (ALI).MethodTF overexpressing (TF+Epi) and knockout (TFΔEpi) mice, along with TF knockout A549 cells (A549ΔTF) were used to test whether lung epithelial TF is critical for the integrity of the alveolar epithelial barrier. TF+Epi mice were treated with intratracheal PBS or LPS and lung permeability was measured by bronchoalveolar lavage (BAL) fluid protein level. Alveolar type II epithelial cells (ATII) were isolated from TFΔEpi and TF+Epi mice to measure β1 integrin protein levels. A549ΔTF cells were treated with LPS (100ug/mL) or PBS for 24 hours before western blot of β1 integrin expression. Migration was assessed in wild‐type and A549ΔTF cells using Electric Cell‐substrate Impedance Sensing (ECIS) on cell monolayers. Adhesion was assayed by plating wild‐type and A549ΔTF cells on matrigel and quantifying the absorbance of crystal violet stained adherent cells.ResultsTF+Epi mice had decreased lung permeability (BAL protein 303 μg/mL +/− 62 versus 416 +/− 137 in control, p=0.025) following LPS challenge. A549ΔTF cells had significantly decreased cell adhesion (Figure) (absorbance 0.308 +/− 0.119 versus 0.603 +/− 0.246 in wild type, *p<0.001) along with decreased migration (p=0.017) compared to control A549 cells with wild type TF expression levels. A549ΔTF cells had decreased β1 integrin protein levels (relative expression 0.017 +/− 0.01 versus 0.61 +/− 0.02 control, p=0.036) following LPS challenge. ATII cells isolated from TFΔEpi mice also have decreased β1 integrin protein levels.ConclusionsTF contributes to alveolar barrier integrity in part through a mechanism dependent on β1 integrin. These data provide support that lung epithelial TF may play a non‐coagulative role in protection during ALI. This novel role for TF in maintaining alveolar epithelial barrier integrity offers a potential therapeutic target during ALI.Support or Funding InformationVanderbilt University Initiative for Maximizing Student Diversity funding through the NIH, Nashville Veterans Affairs Medical Center, Funding: HL090785, HL103836This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.