Macrophages and cardiac lesion in zebrafish: what can single-cell RNA sequencing reveal?

Abstract

Unlike mammals, zebrafish can regenerate their heart after cardiac insult. There are several ways to perform cardiac injury in zebrafish, but cryoinjury most closely resembles human myocardial infarction (MI). Studies demonstrated that macrophages are essential cells from the beginning to later stages of cardiac injury throughout the regenerative process in zebrafish. These cells have phenotypic plasticity; hence, overly sensitive techniques, such as single-cell RNA sequencing (scRNAseq), are essential for uncovering the phenotype needed for zebrafish cardiac injury regeneration, from inflammatory profile initiation to scar resolution. This technique enables the RNA sequencing of individual cells, thus generating clusters of cells with similar gene expression and allowing the study of a particular cell population. Therefore, in this review, we focused on discussing data obtained by scRNAseq of macrophages in the context of cardiac injury. We found that from 1 to 7 days post-injury (dpi), macrophages are present with inflammatory and reparative functions in either cryoinjury or ventricular resection. At 14 dpi, there were differences between the injury models, especially in the expression profile of inflammatory cytokines, and studies with later time points are needed to understand the gene expression that enrolls the collagen scar resorption dynamic.

Keywords: cardiac resection; cryoinjury; fibrosis; macrophages; regeneration; repair; single-cell RNA sequencing; zebrafish.

Figure 1

The most commonly used means of mimicking human heart disease are hypoxia, where the animal's habitat is deprived of oxygen, leading to lesions; explant culture, where the animal's organ is kept in culture, and it is possible to observe cell-extracellular matrix interactions in regeneration; ventricular resection, to mark the mechanisms and expression of genes in the regeneration process; cryoinjury, which uses a cryo-cooled probe pressed on the myocardium to mimic an infarction; laser injury, which generates selective damage in the region of interest; and genetic ablation, in which cardiomyocytes are genetically modified to generate cytotoxic metabolites when activated via a prodrug, causing mosaic cell death in a temporally controlled manner.

Figure 2

Schema of scRNAseq workflow. Red boxes are exclusive to scRNAseq, while blue boxes represent the workflow for any RNAseq technique. For scRNAseq, single cell separation and sample digestion/preparation is performed in nano wells (first box), this is done after sample processing (second box), and then, after cDNA extraction and PCR indexing, the cDNA is sequenced, aligned and analyzed.

Figure 3

Schematic showing the relationship between studies that performed scRNAseq of macrophages in cardiac cryoinjury and cardiac resection at other time points. DPI, days post-injury.