(I-330) Regulation of Muscle Stem Cell Structure and Function by Piezo1

Introduction:: Our daily lives require constant flexion and extension of our skeletal muscles. The mechanical strain from these movements can damage the multinucleated cells within a myofiber. Thus, muscle regeneration is vital to a higher quality of life. Regeneration of myofibers is achieved by muscle stem cells (MuSCs), a heterogeneous population residing between the basal lamina and the sarcolemma. Until recently, MuSCs’ morphology had never been characterized in their endogenous environment. The Mourkioti lab generated Pax7EGFP mice that fluorescently labeled MuSCs in situ and reported novel presence of cellular protrusions [1]. Upon injury, MuSCs retract their protrusions and increase their circularity. We hypothesize that MuSCs may use these protrusions to mechanically sense their environment. It is becoming increasingly evident that mechanosensing plays a significant role in the development and regulation of stem cells. Piezo1, a calcium cation membrane channel, is a stretch activated mechanosensor that activates numerous intracellular calcium-dependent pathways. This project aims to clarify how Piezo1 pathways regulate MuSC subtype function.

Materials and Methods:: We generated and examined the behavior of Piezo1 MuSC-specific knockout (KO) mice. We first verified the genotypes by PCR and gel electrophoresis. Then, two-photon microscopy was used for in situ observation of MuSC morphological protrusions. Fiji software was used to measure the length and number of protrusions per cell from uninjured and injured muscles. To look deeper into Piezo1 regulated molecular mechanisms, MuSCs were isolated by fluorescence-activated cell sorting. Subsequent RNA sequencing compared genes with significant changes in expression level between control and KO MuSCs.

Results, Conclusions, and Discussions:: By observing MuSCs in their endogenous environment, we see that upon injury Piezo1 KO MuSCs lose the ability to retract their protrusions. Thus, they exhibit elongated, aberrant protrusions and are unable to activate or proliferate. These data demonstrate that the absence of Piezo1 in MuSCs leads to a lower rate of activation and proliferation upon injury and failure to adequately enter the myogenic program, which eventually contributes to an overall impairment of muscle regeneration over time. Altogether, we concluded that Piezo1 is sufficient to prime MuSCs and is necessary for the proper retraction of cellular protrusions and the shift of MuSCs toward more activated states that restore muscle regeneration. While our data demonstrate that Piezo1 drives MuSC morphological heterogeneity and activation, the molecular signals responsible for this behavior remain unresolved. To further assess the mechanistic basis of the cytoskeletal effects we observe in Piezo1 KO injured mice, we performed RNAseq analysis and found that genes involved in early activation and cell cycle arrest are upregulated in KOs compared to control MuSCs. Thus, Piezo1 may regulate MuSC cell cycle progression by influencing signaling pathways responsible for the switch from the early activation state to proliferation. Now, we have a more in depth understanding of how Piezo1 functions in MuSCs. Collectively, these studies provided a starting point to delve deeper into the molecular signals associated with different MuSC subtypes as well as the mechanisms by which Piezo1 regulates cytoskeletal protrusions and mechanosensing signatures, resulting in altered MuSC morphological and functional properties. As MuSCs are a useful model for studying stem cell behavior, these mechanisms may be applicable to a variety of stem cell types.

Acknowledgements (Optional): : This project was funded by the NSF-funded Science and Technology Center, Center for Engineering Mechanobiology (CEMB), award number CMMI-1548571 (to MM) and startup funds from Perelman School of Medicine, the NASA (80NSSC19K1602), and the NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01HL146662) (to FM).

References (Optional): : [1] Nuoying Ma et al., Piezo1 regulates the regenerative capacity of skeletal muscles via orchestration of stem cell morphological states. Sci. Adv. 8, 2022. DOI:10.1126/sciadv.abn0485