A detailed version of our Experimental Procedures can be found in the Supplemental Experimental Procedures. All animal experiments were approved by the Danish Council for Supervision with Experimental Animals (#2011/561-1966). AR was performed at P1 as previously described by Porrello et al. (2011). Briefly, neonates were anesthetized by hypothermia, and the apex was resected until left ventricle chamber exposure, after which the thoracic wall and skin were sutured. Sham mice underwent the exact same procedure without resecting the apex of the heart. For pulse-chase labeling experiments, mice were injected 1 day after surgery with EdU, and the number of proliferating cardiomyocytes and PyBOP in total was counted after 7 days. Relative quantitative PCR (qPCR) and histology were performed as previously described (Andersen et al., 2009). All analyses comprised at least four independent experiments, and statistical significance (p < 0.05) was tested as indicated.
A fundamental characteristic of adult stem cells is the ability to serve as source of cells both to give rise to differentiated cells and to replenish the stem cell pool. In skeletal muscle, myogenic stem cells, or “satellite cells” (SCs), exist in a quiescent state, a state of reversible mitotic arrest and reduced metabolic activity that is characteristic of many stem cell populations (Cheung and Rando, 2013). In response to muscle fiber injury, SCs activate, proliferate, and either differentiate into multinucleated myofibers or self-renew (Yoshida et al., 1998; Schmalbruch and Lewis, 2000; Heslop et al., 2001). The process of self-renewal requires that a subset of activated SCs (ASCs) returns to quiescence and involves a complex orchestration of cell-cycle and metabolic transitions (Groszer et al., 2001; He et al., 2009). In adult skeletal muscle, the molecular mechanisms that regulate the self-renewal of SCs have only just begun to be explored (Abou-Khalil et al., 2009; Shea et al., 2010; Le Grand et al., 2012).
The FOXO family of forkhead transcription factors functions downstream of the PI3K/AKT pathway and regulates a wide variety of physiological processes including cell proliferation, differentiation, survival, and metabolism (Greer and Brunet, 2005). Mammals have four members of the FOXO gene family: FOXO1, FOXO3, FOXO4, and FOXO6 (Jacobs et al., 2003; van der Heide et al., 2005; Lam et al., 2006). FOXO family members bind similar DNA sequences (Furuyama et al., 2000) and may therefore display some redundancies in function (Paik et al., 2007; Tothova et al., 2007). However, the presence of specific phenotypes that result from null mutations in Foxo1, Foxo3, and Foxo4 in mice indicates that each Foxo gene has unique physiological roles and is functionally divergent (Castrillon et al., 2003; Hosaka et al., 2004; Lin et al., 2004; Jonsson et al., 2005).
Members of the FOXO family of transcription factors regulate stem cell and progenitor pools in many adult tissues. For example, ablation of Foxo3 alone or in combination with Foxo1 and Foxo4 results in increased cell cycling and reduction of the hematopoietic stem cell pool (Miyamoto et al., 2007; Tothova et al., 2007). Hematopoietic stem cells from FOXO3-deficient mice also have increased levels of reactive oxygen species resulting in apoptosis and a limitation in repopulating ability in vivo (Tothova et al., 2007). In neural tissue, a loss of Foxo3 alone or in combination with Foxo1 and Foxo4 results in a depletion of adult neural stem cells, which is due, at least in part, to the premature proliferative amplification and differentiation of these cells (Renault et al., 2009; Paik et al., 2007). Furthermore, FOXO3 loss decreases the ability of neural stem cells to self-renew in vitro (Renault et al., 2009; Paik et al., 2007). Although the FOXO factors were shown to be expressed in different cell types in muscle and FOXO3 germline knockout mice were reported to display a delay in muscle regeneration (Bosnakovski et al., 2008; Hu et al., 2008), none of the members of the FOXO family have been shown to regulate SC function.