Pre-existing fibroblasts of epicardial origin are the primary source of pathological fibrosis in cardiac ischemia and aging.

Serum response factor (SRF) and the SRF co-activators myocardin-related transcription factors (MRTFs) are essential for epicardium-derived progenitor cell (EPDC)-mobilization during heart development; however, the impact of developmental EPDC deficiencies on adult cardiac physiology has not been evaluated. Here, we utilize the Wilms Tumor-1 (Wt1)-Cre to delete Mrtfs or Srf in the epicardium, which reduced the number of EPDCs in the adult cardiac interstitium. Deficiencies in Wt1-lineage EPDCs prevented the development of cardiac fibrosis and diastolic dysfunction in aged mice. Mice lacking MRTF or SRF in EPDCs also displayed preservation of cardiac function following myocardial infarction partially due to the depletion of Wt1 lineage-derived cells in the infarct. Interestingly, depletion of Wt1-lineage EPDCs allows for the population of the infarct with a Wt1-negative cell lineage with a reduced fibrotic profile. Taken together, our study conclusively demonstrates the contribution of EPDCs to both ischemic cardiac remodeling and the development of diastolic dysfunction in old age, and reveals the existence of an alternative Wt1-negative source of resident fibroblasts that can populate the infarct.

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.

Coordination of endothelial cell positioning and fate specification by the epicardium.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.