Characterizing Neonatal Heart Maturation, Regeneration, and Scar Resolution Using Spatial Transcriptomics

The neonatal mammalian heart exhibits a remarkable regenerative potential, which includes fibrotic scar resolution and the generation of new cardiomyocytes. To investigate the mechanisms facilitating heart repair after apical resection in neonatal mice, we conducted bulk and spatial transcriptomic analyses at regenerative and non-regenerative timepoints. Importantly, spatial transcriptomics provided near single-cell resolution, revealing distinct domains of atrial and ventricular myocardium that exhibit dynamic phenotypic alterations during postnatal heart maturation. Spatial transcriptomics also defined the cardiac scar, which transitions from a proliferative to secretory phenotype as the heart loses regenerative potential. The resolving scar is characterized by spatially and temporally restricted programs of inflammation, epicardium expansion and extracellular matrix production, metabolic reprogramming, lipogenic scar extrusion, and cardiomyocyte restoration. Finally, this study revealed the emergence of a regenerative border zone defined by immature cardiomyocyte markers and the robust expression of Sprr1a. Taken together, our study defines the spatially and temporally restricted gene programs that underlie neonatal heart regeneration and provides insight into cardio-restorative mechanisms supporting scar resolution.

Specific macrophage populations promote both cardiac scar deposition and subsequent resolution in adult zebrafish

Aims A robust inflammatory response to tissue injury is a necessary part of the repair process but the deposition of scar tissue is a direct downstream consequence of this response in many tissues including the heart. Adult zebrafish not only possess the capacity to regenerate lost cardiomyocytes but also to remodel and resolve an extracellular scar within tissues such as the heart, but this scar resolution process remains poorly understood. This study aims to characterize the scarring and inflammatory responses to cardiac damage in adult zebrafish in full and investigate the role of different inflammatory subsets specifically in scarring and scar removal. Methods and results Using stable transgenic lines, whole organ imaging and genetic and pharmacological interventions, we demonstrate that multiple inflammatory cell lineages respond to cardiac injury in adult zebrafish. In particular, macrophage subsets (tnfα+ and tnfα−) play prominent roles with manipulation of different phenotypes suggesting that pro-inflammatory (tnfα+) macrophages promote scar deposition following cardiac injury whereas tnfα− macrophages facilitate scar removal during regeneration. Detailed analysis of these specific macrophage subsets reveals crucial roles for Csf1ra in promoting pro-inflammatory macrophage-mediated scar deposition. Additionally, the multifunctional cytokine Osteopontin (Opn) (spp1) is important for initial scar deposition but also for resolution of the inflammatory response and in late-stage ventricular collagen remodelling. Conclusions This study demonstrates the importance of a correctly balanced inflammatory response to facilitate scar deposition during repair but also to allow subsequent scar resolution, and full cardiac regeneration, to occur. We have identified Opn as having both pro-fibrotic but also potentially pro-regenerative roles in the adult zebrafish heart, driving Collagen deposition but also controlling inflammatory cell resolution.

Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration

Zebrafish display a distinct ability to regenerate their heart following injury. However, this ability is not shared by another teleost, the medaka. In order to identify cellular and molecular bases for this difference, we performed comparative transcriptomic analyses following cardiac cryoinjury. This comparison points to major differences in immune cell dynamics between these models. Upon closer examination, we observed delayed and reduced macrophage recruitment in medaka, along with delayed neutrophil clearance. To investigate the role of immune responses in cardiac regeneration, we delayed macrophage recruitment in zebrafish and observed compromised neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. In contrast, stimulating Toll-like receptor signaling in medaka enhanced immune cell dynamics and promoted neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. Altogether, these data provide further insight into the complex role of the immune response during regeneration, and serve as a platform to identify and test additional regulators of cardiac repair.