In contrast to lower vertebrates, the mammalian heart has a very limited regenerative capacity. Cardiomyocytes, lost after ischemia, are replaced by fibroblasts. Although the human heart is able to form new cardiomyocytes throughout its lifespan, the efficiency of this phenomenon is not enough to substitute sufficient myocardial mass after an infarction. In contrast, zebrafish hearts regenerate through epicardial activation and initiation of myocardial proliferation. With this study we obtain insights into the activation and cellular contribution of the mammalian epicardium in response to ischemia. In a mouse myocardial infarction model we analyzed the spatio-temporal changes in expression of embryonic epicardial, EMT, and stem cell markers and the contribution of cells of the Wt1-lineage to the infarcted area. Though the integrity of the epicardial layer overlaying the infarct is lost immediately after the induction of the ischemia, it was found to be regenerated at three days post infarction. In this regenerated epicardium, the embryonic gene program is transiently re-expressed as well as proliferation. Concomitant with this activation, Wt1-lineage positive subepicardial mesenchyme is formed until two weeks post-infarction. These mesenchymal cells replace the cardiomyocytes lost due to the ischemia and contribute to the fibroblast population, myofibroblasts and coronary endothelium in the infarct, and later also to the cardiomyocyte population. We show that in mice, as in lower vertebrates, an endogenous, epicardium-dependent regenerative response to injury is induced. Although this regenerative response leads to the formation of new cardiomyocytes, their number is insufficient in mice but sufficient in lower vertebrates to replace lost cardiomyocytes. These molecular and cellular analyses provide basic knowledge essential for investigations on the regeneration of the mammalian heart aiming at epicardium-derived cells.

The endothermic state of mammals and birds requires high heart rates to accommodate the high rates of oxygen consumption. These high heart rates are driven by very similar conduction systems consisting of an atrioventricular node that slows the electrical impulse and a His-Purkinje system that efficiently activates the ventricular chambers. While ectothermic vertebrates have similar contraction patterns, they do not possess anatomical evidence for a conduction system. This lack amongst extant ectotherms is surprising because mammals and birds evolved independently from reptile-like ancestors. Using conserved genetic markers, we found that the conduction system design of lizard (Anolis carolinensis and A. sagrei), frog (Xenopus laevis) and zebrafish (Danio rerio) adults is strikingly similar to that of embryos of mammals (mouse Mus musculus, and man) and chicken (Gallus gallus). Thus, in ectothermic adults, the slow conducting atrioventricular canal muscle is present, no fibrous insulating plane is formed, and the spongy ventricle serves the dual purpose of conduction and contraction. Optical mapping showed base-to-apex activation of the ventricles of the ectothermic animals, similar to the activation pattern of mammalian and avian embryonic ventricles and to the His-Purkinje systems of the formed hearts. Mammalian and avian ventricles uniquely develop thick compact walls and septum and, hence, form a discrete ventricular conduction system from the embryonic spongy ventricle. Our study uncovers the evolutionary building plan of heart and indicates that the building blocks of the conduction system of adult ectothermic vertebrates and embryos of endotherms are similar.

SignificanceThe adult mammalian heart does not functionally repair itself after injury. Therefore, identification of cardiac stem cell (CSC) populations is of great interest for regenerative intervention. However, the significance of such CSC populations remains heavily debated. Using single-cell mRNA sequencing and genetic lineage tracing, we interrogate the existence of CSCs with unbiased mouse models of proliferation. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium consisted mainly of various noncardiomyocyte cell types. Injury-activated cardiac fibroblasts that acquire a gene expression profile similar to that of neonatal cardiac fibroblasts signal—in an autocrine fashion—to prevent cardiac rupture. We find no evidence for the existence of a quiescent CSC population.