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IntroductionThe epicardium has an essential role in cardiac regeneration as seen in several animals and mammalian neonates. However, in adult mammals, including humans, the epicardium is reportedly quiescent. Researchers have been proposing that reactivating the epicardium may be a potential intervention but it is unclear what the endpoint may look like, or whether the adult heart will be able to properly harness the reactivated epicardium to once again drive regenerative processes.MethodsIn a recently published study, we used available and newly generated single-cell and single-nuclei RNA sequencing data from adult and fetal human hearts and compared the epicardial cells from both stages using an in-depth analysis pipeline. We also used immunohistochemistry and spatial transcriptomics to validate the position of identified cell populations.ResultsWe found a fibroblast-like population of epicardial cells only in the fetal hearts that appeared to express genes associated with EMT and angiogenesis alongside typical epicardial markers. Using spatial transcriptomics and immunohistochemistry, we showed that this epicardial population was further within the myocardium. However, the adult epicardium appeared to be confined to a mesothelial cluster of epicardial cells and was enriched for genes associated with immune, defense, and stress response. Interestingly, we predicted that adult hearts may still receive fetal epicardial communication through paracrine interactions and showed that adult endocardial and endothelial cells receive the majority of fetal-specific signals including a collection of WNT signaling molecules. We also found that human embryonic stem-cell derived epicardial cells were suitably similar to the human fetal epicardium and expressed fetal but not adult epicardial gene programs.Abstract BS71 Figure 1ConclusionsWe conclude that by comparing epicardial transcriptomics from both adult and fetal stages, we have revealed a potential endpoint for restoring epicardial activity, and by showing that adult hearts are still capable of reacting to these endpoints, we have reinforced the validity of regenerative strategies that administer or reactivate epicardial cells in situ.Conflict of InterestNone

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system 1 . The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug–target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG + and IgA + plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs. Single-cell and spatial transcriptomic analysis of eight human heart tissues reveals the cellular profiles and tissue architecture of niches including the cardiac conduction system, and a new tool, drug2cell, identifies drug target expression.

With advances in tissue engineering and bioelectronics, flexible electronic hydrogels that allow conformal tissue integration, online precision diagnosis, and simultaneous tissue regeneration are expected to be the next-generation platform for the treatment of myocardial infarction. Here, we report a functionalized polyaniline-based chronological adhesive hydrogel patch (CAHP) that achieves spatiotemporally selective and conformal embedded integration with a moist and dynamic epicardium surface. Significantly, CAHP has high adhesion toughness, rapid self-healing ability, and enhanced electrochemical performance, facilitating sensitive sensing of cardiac mechanophysiology-mediated microdeformations and simultaneous improvement of myocardial fibrosis-induced electrophysiology. As a result, the flexible CAHP platform monitors diastolic-systolic amplitude and rhythm in the infarcted myocardium online while effectively inhibiting ventricular remodeling, promoting vascular regeneration, and improving electrophysiological function through electrocoupling therapy. Therefore, this diagnostic and therapeutic integration provides a promising monitorable treatment protocol for cardiac disease. Flexible electronic hydrogels that allow conformal tissue integration, online precision diagnosis, and simultaneous tissue regeneration are desired for advancing the treatment of myocardial infarction. Here, the authors report a chronological adhesive hydrogel patch integrating diagnostic and therapeutic functions through mechanophysiological monitoring and electrocoupling therapy.

The epicardium plays an important role in cardiomyogenesis during development, while it becomes quiescent in adult heart during homeostasis. This study investigates the efficiency of thymosin β4 (Tβ4) release with RPRHQGVM conjugated to the C-terminus of RADA16-I (RADA-RPR), the functionalized self-assembling peptide (SAP), to activate the epicardium and repairing the infarcted myocardium. The functionalized SAP was constituted with self-assembling motif, Tβ4-binding site, and cell adhesive ligand. Myocardial infarction (MI) models of the transgenic mice were established by ligation of the left anterior descending coronary artery. At one week after intramyocardial injection of Tβ4-conjugated SAP, the activation of the epicardium was assessed. At four weeks after implantation, the migration and differentiation of epicardium-derived cells (EPDCs) as well as angiogenesis, lymphangiogenesis and myocardial regeneration were examined. We found that the designer RADA-RPR bound Tβ4 and adhered to EPDCs and that Tβ4 released from the functionalized SAP could effectively activate the epicardium and induce EPDCs to differentiate towards cardiovascular cells as well as lymphatic endothelial cells. Moreover, SAP-released Tβ4 (SAP-Tβ4) promoted proliferation of cardiomyocytes. Furthermore, angiogenesis, lymphangiogenesis and myocardial regeneration were enhanced in the MI models at 4 weeks after delivery of SAP-Tβ4 along with attenuation of adverse myocardial remodeling and significantly improved cardiac function. These results demonstrate that sustained release of Tβ4 from the functionalized SAP can activate the epicardium and effectively enhance the repair of infarcted myocardium. We believe the delivery of SAP-Tβ4 may be a promising strategy for MI therapy.

Despite great progress in engineering functional tissues for organ repair, including the heart, an invasive surgical approach is still required for their implantation. Here, we designed an elastic and microfabricated scaffold using a biodegradable polymer (poly(octamethylene maleate (anhydride) citrate)) for functional tissue delivery via injection. The scaffold’s shape memory was due to the microfabricated lattice design. Scaffolds and cardiac patches (1 cm × 1 cm) were delivered through an orifice as small as 1 mm, recovering their initial shape following injection without affecting cardiomyocyte viability and function. In a subcutaneous syngeneic rat model, injection of cardiac patches was equivalent to open surgery when comparing vascularization, macrophage recruitment and cell survival. The patches significantly improved cardiac function following myocardial infarction in a rat, compared with the untreated controls. Successful minimally invasive delivery of human cell-derived patches to the epicardium, aorta and liver in a large-animal (porcine) model was achieved. Cardiac repair usually requires highly invasive interventional procedures. Here, the authors develop an injectable shape-memory cardiac patch and demonstrated its applicability in a myocardial infarction model.

Cardiac patches are an effective way to deliver therapeutics to the heart. However, such procedures are normally invasive and difficult to perform. Here, we develop and test a method to utilize the pericardial cavity as a natural “mold” for in situ cardiac patch formation after intrapericardial injection of therapeutics in biocompatible hydrogels. In rodent models of myocardial infarction, we demonstrate that intrapericardial injection is an effective and safe method to deliver hydrogels containing induced pluripotent stem cells-derived cardiac progenitor cells or mesenchymal stem cells-derived exosomes. After injection, the hydrogels form a cardiac patch-like structure in the pericardial cavity, mitigating immune response and increasing the cardiac retention of the therapeutics. With robust cardiovascular repair and stimulation of epicardium-derived cells, the delivered therapeutics mitigate cardiac remodeling and improve cardiac functions post myocardial infarction. Furthermore, we demonstrate the feasibility of minimally-invasive intrapericardial injection in a clinically-relevant porcine model. Collectively, our study establishes intrapericardial injection as a safe and effective method to deliver therapeutic-bearing hydrogels to the heart for cardiac repair. Current routes to deliver therapeutics to the heart are normally invasive or difficult to perform. Here the authors develop intrapericardial injection as an efficient, easy-to-perform and minimally invasive method to deliver therapeutics for cardiac repair.

The adult zebrafish heart has a high capacity for regeneration following injury. However, the composition of the regenerative niche has remained largely elusive. Here, we dissected the diversity of activated cell states in the regenerating zebrafish heart based on single-cell transcriptomics and spatiotemporal analysis. We observed the emergence of several transient cell states with fibroblast characteristics following injury, and we outlined the proregenerative function of collagen-12-expressing fibroblasts. To understand the cascade of events leading to heart regeneration, we determined the origin of these cell states by high-throughput lineage tracing. We found that activated fibroblasts were derived from two separate sources: the epicardium and the endocardium. Mechanistically, we determined Wnt signalling as a regulator of the endocardial fibroblast response. In summary, our work identifies specialized activated fibroblast cell states that contribute to heart regeneration, thereby opening up possible approaches to modulating the regenerative capacity of the vertebrate heart. Single-cell RNA sequencing and spatiotemporal analysis of the regenerating zebrafish heart identify transient proregenerative fibroblast-like cells that are derived from the epicardium and the endocardium. Wnt signalling regulates the endocardial fibroblast response.

The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels. We then use epicardioids to investigate the functional cross-talk between cardiac cell types, gaining new insights into the role of IGF2/IGF1R and NRP2 signaling in human cardiogenesis. Finally, we show that epicardioids mimic the multicellular pathogenesis of congenital or stress-induced hypertrophy and fibrotic remodeling. As such, epicardioids offer a unique testing ground of epicardial activity in heart development, disease and regeneration. The lineage of human epicardium is studied using time course single-cell analysis of epicardioids.

During cardiogenesis, the epicardium grows from the proepicardial organ to form the outermost layer of the early heart. Part of the epicardium undergoes epithelial-mesenchymal transformation, and migrates into the myocardium. These epicardium- derived cells differentiate into interstitial fibroblasts, coronary smooth muscle cells, and perivascular fibroblasts. Moreover, epicardium-derived cells are important regulators of formation of the compact myocardium, the coronary vasculature, and the Purkinje fiber network, thus being essential for proper cardiac development. The fibrous structures of the heart such as the fibrous heart skeleton and the semilunar and atrioventricular valves also depend on a contribution of these cells during development. We hypothesise that the essential properties of epicardium-derived cells can be recapitulated in adult diseased myocardium. These cells can therefore be considered as a novel source of adult stem cells useful in clinical cardiac regeneration therapy. [PUBLICATION ABSTRACT]

Heart development is a continuous process involving significant remodeling during embryogenesis and neonatal stages. To date, several groups have used single-cell sequencing to characterize the heart transcriptomes but failed to capture the progression of heart development at most stages. This has left gaps in understanding the contribution of each cell type across cardiac development. Here, we report the transcriptional profile of the murine heart from early embryogenesis to late neonatal stages. Through further analysis of this dataset, we identify several transcriptional features. We identify gene expression modules enriched at early embryonic and neonatal stages; multiple cell types in the left and right atriums are transcriptionally distinct at neonatal stages; many congenital heart defect-associated genes have cell type-specific expression; stage-unique ligand-receptor interactions are mostly between epicardial cells and other cell types at neonatal stages; and mutants of epicardium-expressed genes Wt1 and Tbx18 have different heart defects. Assessment of this dataset serves as an invaluable source of information for studies of heart development. A detailed multi-staged single cell atlas of heart development could improve our understanding of cell type diversification during cardiac development. Here, the authors generated a large dataset with cells from embryonic and neonatal hearts to identify the stage and chamber specific features in heart development.