Isolation of Cardiac Endothelial Cells for Transcriptomic Analysis of the Zebrafish and Mouse Heart.

Isolation of high quality cardiac endothelial cells is a prerequisite for successful bulk and single cell sequencing for RNA (scRNA-seq). We describe a protocol using both enzymatic and mechanical dissociation and fluorescence-activated cell sorting (FACS) to isolate endothelial cells from larval and adult zebrafish hearts and from healthy and ischemic adult mouse hearts. Endothelial cells with high viability and purity can be obtained using this method for downstream transcriptional analyses applications.

Hooked on heart regeneration: the zebrafish guide to recovery.

While humans lack sufficient capacity to undergo cardiac regeneration following injury, zebrafish can fully recover from a range of cardiac insults. Over the past two decades, our understanding of the complexities of both the independent and co-ordinated injury responses by multiple cardiac tissues during zebrafish heart regeneration has increased exponentially. Although cardiomyocyte regeneration forms the cornerstone of the reparative process in the injured zebrafish heart, recent studies have shown that this is dependent on prior neovascularization and lymphangiogenesis, which in turn require epicardial, endocardial, and inflammatory cell signalling within an extracellular milieu that is optimized for regeneration. Indeed, it is the amalgamation of multiple regenerative systems and gene regulatory patterns that drives the much-heralded success of the adult zebrafish response to cardiac injury. Increasing evidence supports the emerging paradigm that developmental transcriptional programmes are re-activated during adult tissue regeneration, including in the heart, and the zebrafish represents an optimal model organism to explore this concept. In this review, we summarize recent advances from the zebrafish cardiovascular research community with novel insight into the mechanisms associated with endogenous cardiovascular repair and regeneration, which may be of benefit to inform future strategies for patients with cardiovascular disease.

Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration.

Cardiac injury leads to the loss of cardiomyocytes, which are rapidly replaced by the proliferation of the surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronized live imaging, RNA sequencing, and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterize macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium, which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signaling. The identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair.