hapln1Defines an Epicardial Cell Subpopulation Required for Cardiomyocyte Expansion During Heart Morphogenesis and Regeneration

Abstract

Background:Certain nonmammalian species such as zebrafish have an elevated capacity for innate heart regeneration. Understanding how heart regeneration occurs in these contexts can help illuminate cellular and molecular events that can be targets for heart failure prevention or treatment. The epicardium, a mesothelial tissue layer that encompasses the heart, is a dynamic structure that is essential for cardiac regeneration in zebrafish. The extent to which different cell subpopulations or states facilitate heart regeneration requires research attention.Methods:To dissect epicardial cell states and associated proregenerative functions, we performed single-cell RNA sequencing and identified 7 epicardial cell clusters in adult zebrafish, 3 of which displayed enhanced cell numbers during regeneration. We identified paralogs ofhapln1as factors associated with the extracellular matrix and largely expressed in cluster 1. We assessedHAPLN1expression in published single-cell RNA sequencing data sets from different stages and injury states of murine and human hearts, and we performed molecular genetics to determine the requirements forhapln1-expressing cells and functions of eachhapln1paralog.Results:A particular cluster of epicardial cells had the strongest association with regeneration and was marked by expression ofhapln1aandhapln1b. Thehapln1paralogs are expressed in epicardial cells that enclose dedifferentiated and proliferating cardiomyocytes during regeneration. Induced genetic depletion ofhapln1-expressing cells or genetic inactivation ofhapln1baltered deposition of the key extracellular matrix component hyaluronic acid, disrupted cardiomyocyte proliferation, and inhibited heart regeneration. We also found thathapln1-expressing epicardial cells first emerge at the juvenile stage, when they associate with and are required for focused cardiomyocyte expansion events that direct maturation of the ventricular wall.Conclusions:Our findings identify a subset of epicardial cells that emerge in postembryonic zebrafish and sponsor regions of active cardiomyogenesis during cardiac growth and regeneration. We provide evidence that, as the heart achieves its mature structure, these cells facilitate hyaluronic acid deposition to support formation of the compact muscle layer of the ventricle. They are also required, along with the function ofhapln1bparalog, in the production and organization of hyaluronic acid–containing matrix in cardiac injury sites, enabling normal cardiomyocyte proliferation and muscle regeneration.