SnoRNAs in cardiovascular development, function, and disease.

Small nucleolar RNAs (snoRNAs) are emerging as important regulators of cardiovascular (patho)biology. Several roles of snoRNAs have recently been identified in heart development and congenital heart diseases, as well as their dynamic regulation in hypertrophic and dilated cardiomyopathies, coronary heart disease (CHD), myocardial infarction (MI), cardiac fibrosis, and heart failure. Furthermore, reports of changes in vesicular snoRNA expression and altered levels of circulating snoRNAs in response to cardiac stress suggest that snoRNAs also function in cardiac signaling and intercellular communication. In this review, we summarize and discuss key findings and outline the clinical potential of snoRNAs considering current challenges and gaps in the field of cardiovascular diseases (CVDs).

Perlecan (HSPG2) promotes structural, contractile, and metabolic development of human cardiomyocytes.

Perlecan (HSPG2), a heparan sulfate proteoglycan similar to agrin, is key for extracellular matrix (ECM) maturation and stabilization. Although crucial for cardiac development, its role remains elusive. We show that perlecan expression increases as cardiomyocytes mature in vivo and during human pluripotent stem cell differentiation to cardiomyocytes (hPSC-CMs). Perlecan-haploinsuffient hPSCs (HSPG2+/-) differentiate efficiently, but late-stage CMs have structural, contractile, metabolic, and ECM gene dysregulation. In keeping with this, late-stage HSPG2+/- hPSC-CMs have immature features, including reduced ⍺-actinin expression and increased glycolytic metabolism and proliferation. Moreover, perlecan-haploinsuffient engineered heart tissues have reduced tissue thickness and force generation. Conversely, hPSC-CMs grown on a perlecan-peptide substrate are enlarged and display increased nucleation, typical of hypertrophic growth. Together, perlecan appears to play the opposite role of agrin, promoting cellular maturation rather than hyperplasia and proliferation. Perlecan signaling is likely mediated via its binding to the dystroglycan complex. Targeting perlecan-dependent signaling may help reverse the phenotypic switch common to heart failure.

Integrating analog and digital modes of gene expression at Arabidopsis FLC.

Quantitative gene regulation at the cell population level can be achieved by two fundamentally different modes of regulation at individual gene copies. A 'digital' mode involves binary ON/OFF expression states, with population-level variation arising from the proportion of gene copies in each state, while an 'analog' mode involves graded expression levels at each gene copy. At the Arabidopsis floral repressor FLOWERING LOCUS C (FLC), 'digital' Polycomb silencing is known to facilitate quantitative epigenetic memory in response to cold. However, whether FLC regulation before cold involves analog or digital modes is unknown. Using quantitative fluorescent imaging of FLC mRNA and protein, together with mathematical modeling, we find that FLC expression before cold is regulated by both analog and digital modes. We observe a temporal separation between the two modes, with analog preceding digital. The analog mode can maintain intermediate expression levels at individual FLC gene copies, before subsequent digital silencing, consistent with the copies switching OFF stochastically and heritably without cold. This switch leads to a slow reduction in FLC expression at the cell population level. These data present a new paradigm for gradual repression, elucidating how analog transcriptional and digital epigenetic memory pathways can be integrated.

Circulating intermediate monocytes CD14++CD16+ are increased after elective percutaneous coronary intervention.

AIM: Inflammation plays a central role in the pathogenesis of atherosclerosis and in the sequelae of percutaneous coronary intervention (PCI). Previous work demonstrated that intermediate monocytes (CD14++CD16+) are associated with adverse cardiovascular events, yet monocyte subset response following elective PCI has not been described. This article explores the changes in monocyte subset and humoral response after elective PCI. METHODS: This prospective study included 30 patients without inflammatory diseases being referred for elective PCI. We included patients treated with drug coated balloons or 2nd generation drug eluting stents. Patients underwent blood tests at baseline (prior to PCI), four hours, two weeks and two months later. Analyses were performed in terms of monocyte subsets (classical CD14++CD16-, intermediate CD14++CD16+ and non-classical CD14+CD16++), gene expression of CD14+ leucocytes and humoral biomarkers. RESULTS: Intermediate monocytes decreased significantly four hours after PCI, were recovered at two weeks, and increased significantly at two months post elective, uncomplicated PCI. They remain significantly elevated in the DES group but not in the DCB group. Gene expression analysis of CD14+ leucocytes showed IL18 had decreased expression at two weeks, CXCR4 and IL1ß decreased at two months, while pentraxin 3 increased at two weeks and two months. In terms of humoral biomarkers, hsTnI remains elevated up to two weeks post PCI while IL6 and TNFα remain elevated till two months post PCI. CONCLUSION: Intermediate monocytes increase significantly two months following elective, uncomplicated PCI. They remain significantly elevated in the DES group but not in the DCB group suggesting that the PCI strategy could be one of the ways to modulate the inflammatory response post PCI.

Transcriptomic Analysis of Cardiomyocyte Extracellular Vesicles in Hypertrophic Cardiomyopathy Reveals Differential snoRNA Cargo.

Hypertrophic cardiomyopathy (HCM) is characterized by increased left ventricular wall thickness that can lead to devastating conditions such as heart failure and sudden cardiac death. Despite extensive study, the mechanisms mediating many of the associated clinical manifestations remain unknown and human models are required. To address this, human-induced pluripotent stem cell (hiPSC) lines were generated from patients with a HCM-associated mutation (c.ACTC1G301A) and isogenic controls created by correcting the mutation using CRISPR/Cas9 gene editing technology. Cardiomyocytes (hiPSC-CMs) were differentiated from these hiPSCs and analyzed at baseline, and at increased contractile workload (2 Hz electrical stimulation). Released extracellular vesicles (EVs) were isolated and characterized after a 24-h culture period and transcriptomic analysis performed on both hiPSC-CMs and released EVs. Transcriptomic analysis of cellular mRNA showed the HCM mutation caused differential splicing within known HCM pathways, and disrupted metabolic pathways. Analysis at increasing contraction frequency showed further disruption of metabolic gene expression, with an additive effect in the HCM background. Intriguingly, we observed differences in snoRNA cargo within HCM released EVs that specifically altered when HCM hiPSC-CMs were subjected to increased workload. These snoRNAs were predicted to have roles in post-translational modifications and alternative splicing, processes differentially regulated in HCM. As such, the snoRNAs identified in this study may unveil mechanistic insight into unexplained HCM phenotypes and offer potential future use as HCM biomarkers or as targets in future RNA-targeting therapies.

Modelling Metabolic Shifts during Cardiomyocyte Differentiation, Iron Deficiency and Transferrin Rescue Using Human Pluripotent Stem Cells.

Cardiomyocytes rely on specialised metabolism to meet the high energy demand of the heart. During heart development, metabolism matures and shifts from the predominant utilisation of glycolysis and glutamine oxidation towards lactate and fatty acid oxidation. Iron deficiency (ID) leads to cellular metabolism perturbations. However, the exact alterations in substrate metabolism during ID are poorly defined. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), the present study investigated changes in major metabolic substrate utilisation in the context of ID or upon transferrin rescue. Typically, during hiPSC-CM differentiation, the greatest increase in total metabolic output and rate was seen in fatty acid metabolism. When ID was induced, hiPSC-CMs displayed increased reliance on glycolytic metabolism, and six TCA cycle, five amino acid, and four fatty acid substrates were significantly impaired. Transferrin rescue was able to improve TCA cycle substrate metabolism, but the amino acid and fatty acid metabolism remained perturbed. Replenishing iron stores partially reverses the adverse metabolic changes that occur during ID. Understanding the changes in metabolic substrate utilisation and their modification may provide potential for discovery of new biomarkers and therapeutic targets in cardiovascular diseases.