Characterization of heterozygous and homozygous mouse models with the most common hypertrophic cardiomyopathy mutation MYBPC3c.2373InsG in the Netherlands.

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in the cardiac myosin binding protein-C (cMyBP-C) encoding gene MYBPC3. In the Netherlands, approximately 25% of patients carry the MYBPC3c.2373InsG founder mutation. Most patients are heterozygous (MYBPC3+/InsG) and have highly variable phenotypic expression, whereas homozygous (MYBPC3InsG/InsG) patients have severe HCM at a young age. To improve understanding of disease progression and genotype-phenotype relationship based on the hallmarks of human HCM, we characterized mice with CRISPR/Cas9-induced heterozygous and homozygous mutations. At 18-28 weeks of age, we assessed the cardiac phenotype of Mybpc3+/InsG and Mybpc3InsG/InsG mice with echocardiography, and performed histological analyses. Cytoskeletal proteins and cardiomyocyte contractility of 3-4 week old and 18-28 week old Mybpc3c.2373InsG mice were compared to wild-type (WT) mice. Expectedly, knock-in of Mybpc3c.2373InsG resulted in the absence of cMyBP-C and our 18-28 week old homozygous Mybpc3c.2373InsG model developed cardiac hypertrophy and severe left ventricular systolic and diastolic dysfunction, whereas HCM was not evident in Mybpc3+/InsG mice. Mybpc3InsG/InsG cardiomyocytes also presented with slowed contraction-relaxation kinetics, to a greater extent in 18-28 week old mice, partially due to increased levels of detyrosinated tubulin and desmin, and reduced cardiac troponin I (cTnI) phosphorylation. Impaired cardiomyocyte contraction-relaxation kinetics were successfully normalized in 18-28 week old Mybpc3InsG/InsG cardiomyocytes by combining detyrosination inhibitor parthenolide and ß-adrenergic receptor agonist isoproterenol. Both the 3-4 week old and 18-28 week old Mybpc3InsG/InsG models recapitulate HCM, with a severe phenotype present in the 18-28 week old model.

The long non-coding RNA MEG8 induces an endothelial barrier through regulation of microRNA-370 and -494 processing.

The 14q32 locus is an imprinted region in the human genome which contains multiple non-coding RNAs. We investigated the role of the long non-coding RNA maternally expressed gene 8 (MEG8) in endothelial function and its underlying mechanism. A 5-fold increase in MEG8 was observed with increased passage number in human umbilical vein endothelial cells (HUVECs), suggesting MEG8 is induced during aging. MEG8 knockdown resulted in a 1.8-fold increase in senescence, suggesting MEG8 might be protective during aging. The endothelial barrier was also impaired after MEG8 silencing. MEG8 knockdown resulted in reduced expression of microRNA (miRNA)-370 and -494 but not -127, -487b and -410. Overexpression of miRNA-370 or -494 partially rescued the MEG8-silencing-induced barrier loss. Mechanistically, MEG8 regulates expression of miRNA-370 and -494 at the mature miRNA level through interaction with the RNA-binding proteins cold-inducible RNA-binding protein (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multi-enzyme complex subunit ß (HADHB). Mature miRNA-370 and miRNA-494 were found to interact with CIRBP, whereas precursor miRNA-370 and miRNA-494 were found to interact with HADHB. Individual CIRBP and 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.

MEG8 regulates Tissue Factor Pathway Inhibitor 2 (TFPI2) expression in the endothelium.

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.

Ultrarapid Delayed Rectifier K+ Channelopathies in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

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 Kv1.5 α-subunit of the ion channel carrying the atrial-specific ultrarapid delayed rectifier K+ current (IKur). 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 IKur 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 IKur, 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, IKur blockade had minimal effects on AP parameters. Next, we used dynamic clamp to inject various amounts of a virtual IKur, with characteristics as in freshly isolated human atrial myocytes, into 11 atrial-like and 10 ventricular-like hiPSC-CMs, in which native IKur was blocked. Injection of IKur with 100% density shortened the APs, with its effect being strongest on the AP duration at 20% repolarization (APD20) of atrial-like hiPSC-CMs. At IKur densities < 100% (compared to 100%), simulating loss-of-function mutations, significant AP prolongation and raise of plateau were observed. At IKur densities > 100%, simulating gain-of-function mutations, APD20 was decreased in both atrial- and ventricular-like hiPSC-CMs, but only upon a strong increase in IKur. In ventricular-like hiPSC-CMs, lowering of the plateau resulted in AP shortening. We conclude that a decrease in IKur, 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 IKur modulation on atrial-like hiPSC-CMs are less pronounced than effects of virtual IKur injection because IKur density of atrial-like hiPSC-CMs is substantially smaller than that of freshly isolated human atrial myocytes.