Abstract
Muscle satellite cells are myogenic stem cells whose quiescence, activation, self-renewal, and differentiation are influenced by oxygen supply, an environmental regulator of stem cell activity. Accordingly, stem cell-specific oxygen signaling pathways precisely control the balance between muscle growth and regeneration in response to oxygen fluctuations, and hypoxia-inducible factors (HIFs) are central mediators of these cellular responses. However, the in vivo roles of HIFs in quiescent satellite cells and activated satellite cells (myoblasts) are poorly understood. Using transgenic mouse models for cell-specific HIF expression, we show here that HIF1α and HIF2α are preferentially expressed in pre- and post-differentiation myoblasts, respectively. Interestingly, double knockouts of HIF1α and HIF2α (HIF1α/2α dKO) generated with the MyoDCre system in embryonic myoblasts resulted in apparently normal muscle development and growth. However, HIF1α/2α dKO produced with the tamoxifen-inducible, satellite cell-specific Pax7CreER system in postnatal satellite cells delayed injury-induced muscle repair due to a reduced number of myoblasts during regeneration. Analysis of satellite cell dynamics on myofibers confirmed that HIF1α/2α dKO myoblasts exhibit reduced self-renewal but more pronounced differentiation under hypoxic conditions. Mechanistically, the HIF1α/2α dKO blunted hypoxia-induced activation of Notch signaling, a key determinant of satellite cell self-renewal. We conclude that HIF1α and HIF2α are dispensable for muscle stem cell function under normoxia but are required for maintaining satellite cell self-renewal in hypoxic environments. Our insights into a critical mechanism in satellite cell homeostasis during muscle regeneration could help inform research efforts to treat muscle diseases or improve muscle function.
Highlights
Oxygen is absolutely critical for life and is one of the most important microenvironmental cues that regulates cellular energy metabolism and survival
We measured myofiber number and size of extensor digitorum longus (EDL) muscles and did not find any differences between control and MyoD-HIFdKO mice (Fig. 1D). These results suggest that HIF1␣ and HIF2␣ are dispensable for skeletal muscle development and postnatal growth under normal oxygen conditions
Given the important role of Hypoxia-inducible factors (HIFs) in mediating hypoxia signaling, it is plausible to assume that their absence should affect muscle progenitor cell function during development
Summary
MyoDCre-mediated double knock-out of HIF1␣ and HIF2␣ did not affect muscle development. There were significantly fewer numbers of Pax7ϩ cells per TA cross-sectional areas in Pax7CreER-HIFdKO mice compared with control mice at 7 DPI (Fig. 4B) To confirm this observation, we counted Pax7ϩ/CD34ϩ satellite cells in fresh-isolated EDL myofibers (Fig. 4C). Consistent with the TA muscle cross-section results, the numbers of satellite cells per myofiber were identical between control and Pax7CreER-HIFdKO mice in non-injured muscles, but the Pax7CreER-HIFdKO mice had significantly fewer satellite cells after CTX injury at both 7 and 21 DPI (Fig. 4D; see Fig. 5D) These results indicate that co-deletion of HIF1␣ and HIF2␣ does not reduce satellite cells in noninjured muscles, it impairs injury-induced satellite cell proliferation or self-renewal, leading to a reduced number of satellite cells during and after muscle regeneration. These results indicate that activation of Notch signaling is sufficient to rescue the self-renewal defect of HIF1␣/HIF2␣ dKO myoblasts, confirming that HIF1␣/HIF2␣ promote self-renewal of satellite cells through activating Notch signaling
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