Macrophages promote both injury and repair following myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping and single-cell transcriptomics to demonstrate that at steady state, TIMD4 + LYVE1 + MHC-II lo CCR2 − resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4 − LYVE1 − MHC-II hi CCR2 − macrophages and fully replaced TIMD4 − LYVE1 − MHC-II hi CCR2 + macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4 + and TIMD4 − resident macrophage abundance within infarcted tissue while recruited, CCR2 + monocyte-derived macrophages adopted multiple cell fates, including those nearly indistinguishable from resident macrophages. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1 -based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, highlighting a non-redundant, cardioprotective role of resident cardiac macrophages. Lastly, we demonstrate the ability of TIMD4 to be used as a durable lineage marker of a subset of resident cardiac macrophages.

High-dimensional analysis reveals three macrophage subsets with common transcriptional and biological properties across organs.