Rationale Accurate knowledge of the cellular composition of the heart is essential to fully understand the changes that occur during pathogenesis and to devise strategies for tissue engineering and regeneration. Objective To examine the relative frequency of cardiac endothelial cells, hematopoietic-derived cells and fibroblasts in the mouse and human heart. Methods and Results Using a combination of genetic tools and cellular markers, we examined the occurrence of the most prominent cell types in the adult mouse heart. Immunohistochemistry revealed that endothelial cells constitute over 60%, hematopoietic-derived cells 5–10%, and fibroblasts under 20% of the non-myocytes in the heart. A refined cell isolation protocol and an improved flow cytometry approach provided an independent means of determining the relative abundance of non-myocytes. High dimensional analysis and unsupervised clustering of cell populations confirmed that endothelial cells are the most abundant cell population. Interestingly, fibroblast numbers are smaller than previously estimated, and two commonly assigned fibroblast markers, Sca-1 and CD90, underrepresent fibroblast numbers. We also describe an alternative fibroblast surface marker that more accurately identifies the resident cardiac fibroblast population. Conclusions This new perspective on the abundance of different cell types in the heart demonstrates that fibroblasts comprise a relatively minor population. By contrast, endothelial cells constitute the majority of non-cardiomyocytes and are likely to play a greater role in physiologic function and response to injury than previously appreciated.

In dramatic contrast to the poor repair outcomes for humans and rodent models such as mice, salamanders and some fish species are able to completely regenerate heart tissue following tissue injury, at any life stage. This capacity for complete cardiac repair provides a template for understanding the process of regeneration and for developing strategies to improve human cardiac repair outcomes. Using a cardiac cryo-injury model we show that heart regeneration is dependent on the innate immune system, as macrophage depletion during early time points post-injury results in regeneration failure. In contrast to the transient extracellular matrix that normally accompanies regeneration, this intervention resulted in a permanent, highly cross-linked extracellular matrix scar derived from alternative fibroblast activation and lysyl-oxidase enzyme synthesis. The activation of cardiomyocyte proliferation was not affected by macrophage depletion, indicating that cardiomyocyte replacement is an independent feature of the regenerative process, and is not sufficient to prevent fibrotic progression. These findings highlight the interplay between macrophages and fibroblasts as an important component of cardiac regeneration, and the prevention of fibrosis as a key therapeutic target in the promotion of cardiac repair in mammals.

Cardiac tissue macrophages (cTMs) are abundant in the murine heart but the extent to which the cTM phenotype changes with age is unknown. This study characterizes aging-dependent phenotypic changes in cTM subsets. Using the Cx3cr1GFP/+ mouse reporter line where GFP marks cTMs, and the tissue macrophage marker Mrc1, we show that two major cardiac tissue macrophage subsets, Mrc1−GFPhi and Mrc1+GFPhi cTMs, are present in the young (<10 week old) mouse heart, and a third subset, Mrc1+GFPlo, comprises ~50% of total Mrc1+ cTMs from 30 weeks of age. Immunostaining and functional assays show that Mrc1+ cTMs are the principal myeloid sentinels in the mouse heart and that they retain proliferative capacity throughout life. Gene expression profiles of the two Mrc1+ subsets also reveal that Mrc1+GFPlo cTMs have a decreased number of immune response genes (Cx3cr1, Lpar6, CD9, Cxcr4, Itga6 and Tgfβr1), and an increased number of fibrogenic genes (Ltc4s, Retnla, Fgfr1, Mmp9 and Ccl24), consistent with a potential role for cTMs in cardiac fibrosis. These findings identify early age-dependent gene expression changes in cTMs, with significant implications for cardiac tissue injury responses and aging-associated cardiac fibrosis.