Origin of congenital coronary arterio-ventricular fistulae from anomalous epicardial and myocardial development

Coronary Artery Fistulae (CAFs) are cardiac congenital anomalies consisting of an abnormal communication of a coronary artery with either a cardiac chamber or another cardiac vessel. In humans, these congenital anomalies can lead to complications such as myocardial hypertrophy, endocarditis, heart dilatation and failure. Unfortunately, despite their clinical relevance, the aetiology of CAFs remains unknown. In this work, we have used two different species (mouse and avian embryos) to experimentally model CAFs morphogenesis. Both conditional Itga4 (alpha 4 integrin) epicardial deletion in mice and cryocauterisation of chick embryonic hearts disrupted epicardial development and ventricular wall growth, two essential events in coronary embryogenesis. Additional transcriptomics and in vitro analyses were performed to better understand how arterio-ventricular connections are originated in the embryonic heart. Our results suggest myocardial discontinuities in the developing heart promote the formation of endocardial pouch-like structures resembling human CAF. The structure of these CAF-like anomalies was compared with histopathological data from a paediatric heart CAF, showing histomorphological and immunochemical similarities, including an accumulation of smooth muscle positive cells in the pouch-like structure wall. In vitro experiments showed the abnormal contact between the epicardium and the endocardium may promote the precocious differentiation of epicardial cells to smooth muscle. Our results suggest that myocardial discontinuities in the embryonic ventricular wall promote the early contact of the endocardium with epicardial-derived coronary progenitors at the cardiac surface, leading to ventricular endocardial extrusion, precocious differentiation of coronary smooth muscle cells, and the formation of pouch-like aberrant coronary-like structures in direct connection with the ventricular lumen. Our results may provide relevant information for the early diagnosis of these congenital anomalies and the molecular mechanisms that regulate their embryogenesis.

SIRT1 Inhibition Impairs Ca2+ Buffering in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

Background: SIRT1 is a deacetylase that has diverse roles in intracellular Ca2+ signaling, metabolism, and cardiovascular disease. SIRT1 increases sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity that is essential to buffer the increase in Ca2+ induced by release from the sarcoplasmic reticulum (SR). Our lab has shown that metabolic syndrome (MetS) impairs SERCA activity in coronary smooth muscle cells and causes coronary artery disease in Ossabaw miniature swine. We hypothesized that SIRT1 inhibition and MetS would impair Ca2+ buffering. Methods: CRISPR/Cas9 methods delivered a leucine to proline point mutation in SIRT1 (SIRT1L100P) into the Ossabaw swine genome to compare to wild type (WT) and mimic the naturally occurring mutation in humans and decrease SIRT1 activity. Four treatment groups of juvenile swine were based on genotype and diet: WT Lean, SIRT1 Lean, WT MetS, and SIRT1 MetS. Lean swine were fed normal chow and MetS were fed a hypercaloric, atherogenic diet for 7 months. The left anterior descending coronary artery was harvested and enzymatically digested to obtain cells. Fluorescence microscopy measured the Ca2+ indicator fura-2 in single cells. The cells were exposed to 5 mM caffeine to maximally release stores of Ca2+ from the SR. Ca2+ buffering capacity of each cell was analyzed after the caffeine-induced peak increase to assess Ca2+ efflux and SERCA activity. Results: MetS was confirmed by increased body weight, impaired glucose tolerance, hyperinsulinemia, and hypercholesterolemia. Coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging. The rapid phase of Ca2+ buffering due to Ca2+ efflux was not affected by SIRT1 mutation or MetS. The slower phase of Ca2+ buffering due to SERCA activity was impaired only by SIRT1 mutation (p<0.0005), not by MetS. Conclusion: SIRT1 mutation alone inhibited SERCA buffering of Ca2+ in coronary smooth muscle. (Support: NIH T35HL110854, DK120240, DK09751.)

Metabolic Syndrome and SIRT1 Mutation Impair Ca2+ Channels in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

Background: Changes in Ca2+ regulation have been implicated in various pathologies such as coronary artery disease and metabolic syndrome (MetS), thereby potentiating these diseases. Our lab has shown that MetS decreases voltage-gated Ca2+ channel (VGCC) activity and sarcoplasmic reticulum (SR) Ca2+ release in coronary smooth muscle cells and increases coronary artery disease in Ossabaw miniature swine. Furthermore, decreased SIRT1 enzyme function can impair Ca2+ signaling and increase coronary disease and MetS. We hypothesized that impaired SIRT1 and MetS would decrease VGCC function and SR calcium store. Methods: CRISPR/Cas9 methods delivered a leucine to proline point mutation in SIRT1 (SIRT1L100P) into the Ossabaw swine genome to compare to wild type (WT), mimicking the naturally occurring mutation in humans which decreases SIRT1 activity. Four treatment groups of juvenile swine were based on genotype and diet: WT Lean, SIRT1 Lean, WT MetS, and SIRT1 MetS. Lean swine were fed normal chow and MetS were fed a hypercaloric, atherogenic diet for 7 months. The left anterior descending coronary artery was harvested and enzymatically digested to obtain cells. Fluorescence microscopy measured the Ca2+ indicator fura-2 in single cells. Depolarization of cells with perfusion of 80 mM K+ was used to elicit Ca2+ influx through VGCC. Caffeine (5 mM) exposure activated the Ca2+ release channel (ryanodine receptor) on the SR. Results: MetS was confirmed by increased body weight, impaired glucose tolerance, hyperinsulinemia, and hypercholesterolemia. Coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging. A two-way analysis of variance revealed statistically significant overall effects of genotype (p=0.02), diet (p<0.0001), and an interaction (p<0.0001) between these variables to decrease VGCC function. In contrast, no effect was observed on SR Ca2+ release. Conclusion and Potential Impact: SIRT1 inhibition and MetS decreased VGCC function independently, but not additively or synergistically. (Support: NIH T35HL110854, DK120240, DK09751.)

Extracellular inflammasomes as novel endogenous danger signals that exert pro-inflammatory signaling on vascular cells

Background and aims As part of the innate immune response, NLRP3 inflammasomes are involved in the process of sterile inflammation, IL-1β release and are key mediators of inflammation-related vascular diseases, such as atherosclerosis. Recent data showed the existence of extracellular inflammasomes released from monocytes during pyroptotic cell death. Their biological function in the vascular system is still not known. Here, we established a method to detect extracellular inflammasomes and tested the hypothesis that extracellular NLRP3 inflammasomes can be internalized by vascular cells, such as macrophages, endothelial cells and coronary smooth muscle cells and induce pro-inflammatory signaling. Methods and result Stimulation of THP1 monocytes and of isolated primary human monocytes with Lipopolysaccharide and Nigericin activated the NLRP3 inflammasome and induced pyroptosis and the release of inflammasome complexes. Extracellular inflammasomes were isolated from cell-free supernatant and identified as inflammasome complexes (oligomers) by immunoblot. For functional characterization, isolated fluorescent-labeled NLRP3 inflammasome complexes were shown to be internalized by THP1 macrophages (19.7±9.7% pos. cells), primary endothelial cells (HUVEC: 9.0±2.3% pos. cells, coronary artery endothelial cells: 11.0% ± 2.3% pos. cells) and coronary smooth muscle cells (42.8±9.9% pos.cells) using immunofluorescence staining, Z-stacks and imaging flow cytometric analysis. Extracellular NLRP3 inflammasomes (eNLRP3) induced pro-inflammatory signaling in macrophages by increasing IL1b and Tnfa gene expression (3.0- fold) as well as IL-1β release (con: 1.9 pg/ml vs. eNLRP3: 191.0 pg/ml). In coronary smooth muscle cells, treatment with extracellular inflammasomes increased endogenous Nlrp3 and IL1b gene expression as well as upregulation of Cell adhesion molecule 1 (Cadm1). Coronary artery endothelial cells showed also increased protein level of surface adhesion marker Intercellular Adhesion Molecule 1 (ICAM1). Conclusion Upon canonical NLRP3 inflammasome activation, mononuclear cells release inflammasome complexes into the extracellular space. Macrophages, endothelial cells and smooth muscle cells are able to internalize these extracellular inflammasome complexes that exert pro-inflammatory effects. These findings support the concept that cell-free NLRP3 inflammasomes act as extracellular signal molecules triggering pro-atherogenic signaling mechanisms in vascular cells. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Leipzig University, Medical Faculty

Quantitation analysis by flow cytometry shows that Wt1 is required for development of the proepicardium and epicardium

ABSTRACTThe epicardium is a cell layer found on the external surface of the heart. During development it has an epithelial identity and contains progenitor cells for coronary smooth muscle and cardiac fibroblasts. The epicardium has been suggested to have therapeutic potential in cardiac repair. Study of epicardial development has been difficult because it is dynamic and morphologically complex. We developed a flow cytometry-based method to quantify cardiac development including the epicardial lineage. This provided accurate and sensitive analysis of (1) the emergence of epicardial progenitors within the proepicardium (2) their transfer to the heart to form the epicardium, and (3) their epithelial-to-mesenchymal transition (EMT) to create the subepicardium. Platelet-derived growth factor alpha (Pdgfra) and Wilms tumor protein (Wt1) have both been reported to be pro-mesenchymal during epicardial EMT. Quantitative analysis with flow cytometry confirmed a pro-mesenchymal role for Pdgfra but not for Wt1. Analysis of Wt1 null embryos showed that they had (1) poor formation of proepicardial villi, (2) reduced transfer of proepicardial cells to the heart, (3) a discontinuous epicardium with poor epithelial identity, and (4) a proportionally excessive number of mesenchymal-like cells. This data shows that Wt1 is essential for epicardial formation and maintenance rather than being pro-mesenchymal.