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 >60%, hematopoietic-derived cells 5% to 10%, and fibroblasts <20% of the nonmyocytes in the heart. A refined cell isolation protocol and an improved flow cytometry approach provided an independent means of determining the relative abundance of nonmyocytes. 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 2 commonly assigned fibroblast markers, Sca-1 and CD90, under-represent 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 noncardiomyocytes and are likely to play a greater role in physiological function and response to injury than previously appreciated.

The failure to replace damaged body parts in adult mammals results from a muted growth response and fibrotic scarring. Although infiltrating immune cells play a major role in determining the variable outcome of mammalian wound repair, little is known about the modulation of immune cell signaling in efficiently regenerating species such as the salamander, which can regrow complete body structures as adults. Here we present a comprehensive analysis of immune signaling during limb regeneration in axolotl, an aquatic salamander, and reveal a temporally defined requirement for macrophage infiltration in the regenerative process. Although many features of mammalian cytokine/chemokine signaling are retained in the axolotl, they are more dynamically deployed, with simultaneous induction of inflammatory and anti-inflammatory markers within the first 24 h after limb amputation. Systemic macrophage depletion during this period resulted in wound closure but permanent failure of limb regeneration, associated with extensive fibrosis and disregulation of extracellular matrix component gene expression. Full limb regenerative capacity of failed stumps was restored by reamputation once endogenous macrophage populations had been replenished. Promotion of a regeneration-permissive environment by identification of macrophage-derived therapeutic molecules may therefore aid in the regeneration of damaged body parts in adult mammals.

Macrophages play an essential role in the resolution of tissue damage through removal of necrotic cells, thus paving the way for tissue regeneration. Macrophages also directly support the formation of new tissue to replace the injury, through their acquisition of an anti-inflammatory, or M2, phenotype, characterized by a gene expression program that includes IL-10, the IL-13 receptor, and arginase 1. We report that deletion of two CREB-binding sites from theCebpbpromoter abrogatesCebpbinduction upon macrophage activation. This blocks the downstream induction of M2-specificMsr1,Il10,II13ra, andArg-1genes, whereas the inflammatory (M1) genesIl1,Il6,Tnfa, andIl12are not affected. Mice carrying the mutatedCebpbpromoter (βΔCre) remove necrotic tissue from injured muscle, but exhibit severe defects in muscle fiber regeneration. Conditional deletion of theCebpbgene in muscle cells does not affect regeneration, showing that the C/EBPβ cascade leading to muscle repair is muscle-extrinsic. While βΔCre macrophages efficiently infiltrate injured muscle they fail to upregulateCebpb, leading to decreasedArg-1expression. CREB-mediated induction ofCebpbexpression is therefore required in infiltrating macrophages for upregulation of M2-specific genes and muscle regeneration, providing a direct genetic link between these two processes.

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.