39 | Exploring the role of the chemokine receptor CXCR2 in muscle stem cell heterogeneity

Angela Gambardella, G. Andolfi, G. Minchiotti, O. Guardiola | Stem Cell Fate Laboratory, Institute of Genetics and Biophysics "A. Buzzati-Traverso", CNR, Naples, Italy.

Skeletal muscle regeneration is driven by a heterogeneous population of muscle stem cells (MuSCs). This heterogeneity, particularly the non-genetic cell-to-cell variability arising from intrinsic and extrinsic factors, plays a critical role in shaping the diverse regenerative responses of this cell population. Our previous work identified surface CRIPTO protein as a key factor influencing MuSC plasticity and adaptation to the regenerative microenvironment, suggesting its involvement in generating this heterogeneity. Transcriptomic analysis of distinct CRIPTO-expressing MuSC fractions revealed a significant upregulation of the chemokine receptor CXCR2 in CRIPTO-positive cell fraction, prompting us to investigate its involvement in MuSC plasticity. CRIPTO-mediated heterogeneity involves phospholipase C (PLC) activity, which in turn regulates intracellular calcium levels. Therefore, as CXCR2 is a G-protein coupled receptor known to modulate PLC, we hypothesized the involvement of CXCR2/PLC/calcium axis in driving non-genetic heterogeneity within the MuSC population. Our results revealed variable surface expression of CXCR2 in both activated primary MuSCs and C2C12 myoblasts. Notably, isolated CXCR2positive and CXCR2negative cells displayed a dynamic interconversion, suggesting a potential role in MuSC cell plasticity. Additionally, inhibiting PLC activity in C2C12 cultures resulted in the generation of different cell populations, over time, exhibiting distinct surface CXCR2 levels as well as distinct intracellular calcium levels. Moreover, pharmacological manipulation of CXCR2 in C2C12 myoblasts resulted in altered myogenic markers profiles. These preliminary findings indicate that the dynamic expression of CXCR2 and modulation of PLC and calcium signaling may define distinct functional states within the MuSC population, suggesting their involvement in cellular metastability.