Atrial fibrillation (AF) is the most common cardiac arrhythmia. About 5–15% of AF patients have a mutation in a cardiac gene, including mutations in KCNA5 , encoding the K v 1.5 α-subunit of the ion channel carrying the atrial-specific ultrarapid delayed rectifier K + current (I Kur ). Both loss-of-function and gain-of-function AF-related mutations in KCNA5 are known, but their effects on action potentials (APs) of human cardiomyocytes have been poorly studied. Here, we assessed the effects of wild-type and mutant I Kur on APs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We found that atrial-like hiPSC-CMs, generated by a retinoic acid-based differentiation protocol, have APs with faster repolarization compared to ventricular-like hiPSC-CMs, resulting in shorter APs with a lower AP plateau. Native I Kur , measured as current sensitive to 50 μM 4-aminopyridine, was 1.88 ± 0.49 (mean ± SEM, n = 17) and 0.26 ± 0.26 pA/pF ( n = 17) in atrial- and ventricular-like hiPSC-CMs, respectively. In both atrial- and ventricular-like hiPSC-CMs, I Kur blockade had minimal effects on AP parameters. Next, we used dynamic clamp to inject various amounts of a virtual I Kur , with characteristics as in freshly isolated human atrial myocytes, into 11 atrial-like and 10 ventricular-like hiPSC-CMs, in which native I Kur was blocked. Injection of I Kur with 100% density shortened the APs, with its effect being strongest on the AP duration at 20% repolarization (APD 20 ) of atrial-like hiPSC-CMs. At I Kur densities < 100% (compared to 100%), simulating loss-of-function mutations, significant AP prolongation and raise of plateau were observed. At I Kur densities > 100%, simulating gain-of-function mutations, APD 20 was decreased in both atrial- and ventricular-like hiPSC-CMs, but only upon a strong increase in I Kur . In ventricular-like hiPSC-CMs, lowering of the plateau resulted in AP shortening. We conclude that a decrease in I Kur , mimicking loss-of-function mutations, has a stronger effect on the AP of hiPSC-CMs than an increase, mimicking gain-of-function mutations, whereas in ventricular-like hiPSC-CMs such increase results in AP shortening, causing their AP morphology to become more atrial-like. Effects of native I Kur modulation on atrial-like hiPSC-CMs are less pronounced than effects of virtual I Kur injection because I Kur density of atrial-like hiPSC-CMs is substantially smaller than that of freshly isolated human atrial myocytes.
A large portion of the genome is transcribed into non-coding RNA, which does not encode protein. Many long non-coding RNAs (lncRNAs) have been shown to be involved in important regulatory processes such as genomic imprinting and chromatin modification. The 14q32 locus contains many non-coding RNAs such as Maternally Expressed Gene 8 (MEG8). We observed an induction of this gene in ischemic heart disease. We investigated the role of MEG8 specifically in endothelial function as well as the underlying mechanism. We hypothesized that MEG8 plays an important role in cardiovascular disease via epigenetic regulation of gene expression. Experiments were performed in human umbilical vein endothelial cells (HUVECs). In vitro silencing of MEG8 resulted in impaired angiogenic sprouting. More specifically, total sprout length was reduced as was proliferation, while migration was unaffected. We performed RNA sequencing to assess changes in gene expression after loss of MEG8. The most profoundly regulated gene, Tissue Factor Pathway Inhibitor 2 (TFPI2), was fivefold increased following MEG8 silencing. TFPI2 has previously been described as an inhibitor of angiogenesis. Mechanistically, MEG8 silencing resulted in a reduction of the inhibitory histone modification H3K27me3 at the TFPI2 promoter. Interestingly, additional silencing of TFPI2 partially restored angiogenic sprouting capacity but did not affect proliferation of MEG8 silenced cells. In conclusion, silencing of MEG8 impairs endothelial function, suggesting a potential beneficial role in maintaining cell viability. Our study highlights the MEG8/TFPI2 axis as potential therapeutic approach to improve angiogenesis following ischemia.
The 14q32 locus is an imprinted region in the human genome which contains multiple non-coding RNAs. We investigated the role of Maternally Expressed Gene 8 (MEG8) in endothelial function and the underlying mechanism. A 5-fold increase in MEG8 was observed with increased passage number in Human Umbilical Vein Endothelial Cells, suggesting MEG8 is induced during aging. MEG8 knockdown resulted in a 1.8-fold increase in senescence, suggesting MEG8 might be protective during aging. Endothelial barrier was impaired after MEG8 silencing. MEG8 knockdown resulted in reduced expression of miRNA-370 and -494 but not -127, -487b and -410. Overexpression of miRNA-370/-494 partially rescued MEG8-silencing-induced barrier loss. Mechanistically, MEG8 regulates expression of miRNA-370 and -494 at the mature miRNA level through interaction with RNA binding proteins Cold Inducible RNA Binding Protein (CIRBP) and Hydroxyacyl-CoA Dehydrogenase Trifunctional Multi-enzyme Complex Subunit Beta (HADHB). Precursor and mature miRNA-370/-494 were shown to interact with HADHB and CIRBP respectively. CIRBP/HADHB silencing resulted in downregulation of miRNA-370 and induction of miRNA-494. These results suggest MEG8 interacts with CIRBP and HADHB and contributes to miRNA processing at the post-transcriptional level.
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