Yuji Hara

Muscle satellite cells (MuSCs) are muscle-resident stem cells responsible for myofibre regeneration. Although the importance of calcium ions (Ca2+) in muscle physiology has been well established, the mechanism by which Ca2+ mobilisation governs MuSC function is not well understood. In this study, we aimed to systematically characterize Ca2+ dynamics in MuSCs and to define the mechanisms regulating these signals during muscle regeneration. By employing modified protocols for mouse MuSC isolation and Ca2+ measurement, we observed spontaneous Ca2+ fluctuations in MuSCs isolated from regenerating muscle after cardiotoxin-induced myofibre injury. Our detailed analysis using chemical Ca2+ indicators and a genetically encoded Ca2+ indicator revealed that the frequency and amplitude of Ca2+ fluctuations increased significantly during the activation and proliferative stages of MuSCs. This effect was more pronounced in MuSCs isolated from dystrophic and aged mice. Mechanistically, these Ca2+ fluctuations were partially mediated by mechanosensitive ion channels including Piezo-type mechanosensitive ion channel component 1 (PIEZO1) and Transient Receptor Potential Melastatin 7 (TRPM7) which promote MuSC migration. Collectively, our findings demonstrate that Ca2+ fluctuations through mechanosensitive ion channels act as a key regulator of MuSC activation during muscle regeneration and may provide new insights into the role of Ca2+ influx in muscle biology and the pathogenesis of muscle diseases.

: Phospholipid asymmetry is a hallmark of mammalian cell membranes and reflects the selective distribution of distinct phospholipid species between the two leaflets of the lipid bilayer. Although this asymmetry is tightly maintained, the membrane proteins whose functions depend on it remain largely unknown. To perturb phospholipid asymmetry experimentally, we expressed a constitutively active phospholipid scramblase and thereby identified transient receptor potential melastatin 8 (TRPM8) as an ion channel regulated by this membrane property. Activation of TRPM8 by both l -menthol and innocuous cold was markedly suppressed following disruption of phospholipid asymmetry. Likewise, selective depletion of phosphatidylserine (PS), a phospholipid enriched in the cytoplasmic leaflet, using a cytosolically targeted PS decarboxylase attenuated TRPM8 activation, indicating that cytoplasmic PS is required for proper TRPM8 function. Mechanistically, our findings suggest that cytoplasmic PS supports efficient TRPM8 activation by maintaining the biochemical state of the channel. Together, these findings identify TRPM8 as a phospholipid asymmetry-dependent ion channel and establish an experimental strategy for systematically identifying membrane proteins regulated by phospholipid asymmetry. This work provides a foundation for future studies investigating the biological significance of this fundamental membrane property.

Muscle satellite cells (MuSCs) are muscle-resident stem cells that are responsible for myofiber regeneration. Although the importance of calcium ions (Ca ²⁺ ) in muscle physiology has been well established, the mechanism by which Ca ²⁺ mobilization governs MuSC function remains poorly understood. In this study, we aimed to systematically characterize Ca ²⁺ dynamics in MuSCs and to define the mechanisms regulating these signals during muscle regeneration. By employing modified protocols for mouse MuSC isolation and Ca ²⁺ measurement, we observed spontaneous Ca ²⁺ fluctuations in MuSCs isolated from regenerating muscle after cardiotoxin-induced myofiber injury. Our detailed analysis using chemical Ca ²⁺ indicators and a genetically encoded Ca ²⁺ indicator revealed that the frequency and amplitude of Ca ²⁺ fluctuations increased significantly during the activated and proliferative stages of MuSCs in muscle regeneration. This effect was more pronounced in MuSCs isolated from dystrophic and aged mice. Mechanistically, these Ca ²⁺ fluctuations were at least partially mediated by mechanosensitive ion channels, including PIEZO1 and TRPM7, which promote MuSC migration. Collectively, our findings demonstrate that Ca ²⁺ fluctuations through mechanosensitive ion channels act as a key regulator of MuSC activation during muscle regeneration and may provide new insights into the role of Ca ²⁺ influx in muscle biology and the pathogenesis of muscle diseases.

Primary tissue stem cells are useful not only for basic cell biological research but also for therapeutic applications. However, their broader utility is often limited by technical challenges, such as a low efficiency of exogenous gene expression, particularly when introducing large genes. PIEZO1 is a large mechanosensitive ion channel that plays an important role in muscle-resident stem cells, known as muscle satellite cells (MuSCs), during muscle regeneration. In this study, we developed a method for the ectopic expression of PIEZO1 in isolated MuSCs. Using a baculovirus vector system, we expressed PIEZO1 in myoblast C2C12 cells. Following optimization of the infection condition, we achieved robust PIEZO1 expression in isolated MuSCs during activated and differentiated states, with appropriate subcellular localization and ion channel activity. Importantly, the baculovirus-mediated PIEZO1 expression restored the defective cell functions in Piezo1-deficient MuSCs to a level comparable to that in wild-type cells, indicating that the exogenously expressed PIEZO1 is functionally equivalent to the endogenous protein. Overall, we established an efficient method for the transfer of the Piezo1 gene into isolated MuSCs, which should provide a versatile platform to study other large proteins in MuSCs.

Primary tissue stem cells are useful not only for basic cell biological research but also for therapeutic applications: however, their broader utility is often limited by technical challenges, such as a low efficiency of exogenous gene expression. PIEZO1 is a large mechanosensitive ion channel that plays an important role in muscle-resident stem cells, known as muscle satellite cells (MuSCs), during muscle regeneration. In this study, we developed a method for the ectopic expression of PIEZO1 in isolated MuSCs. Using a baculovirus vector system, we expressed PIEZO1 in myoblast C2C12 cells. Following optimization of the infection condition, we achieved robust PIEZO1 expression in isolated MuSCs during activated and differentiated states, with appropriate subcellular localization and ion channel activity. Importantly, the baculovirus-mediated PIEZO1 expression restored the reduced proliferative capacity of Piezo1 -deficient MuSCs to a level comparable to wild-type cells, indicating that the exogenously expressed PIEZO1 is functionally equivalent to the endogenous protein. Overall, we established an efficient method for the transfer of the Piezo1 gene into isolated MuSCs, which should provide a versatile platform to study other large proteins in MuSCs. Summary Statement: A baculovirus-based method was developed, enabling robust and functional PIEZO1 expression in isolated muscle satellite cells and providing a platform to study large proteins in isolated stem cells.

Muscle satellite cells (MuSCs) respond immediately to environmental cues upon skeletal muscle injuries. Despite decades of research into muscle regeneration, the specific molecular factors that trigger the transition of MuSCs from a quiescent to an active state remain largely unidentified. Here, we identify transient receptor potential melastatin 7 (TRPM7), an Mg ²⁺ -permeable ion channel, as a critical regulator of MuSC activation. Trpm7 deletion in MuSCs reduced Mg ²⁺ influx, impairing myofiber regeneration and leading to decreased MuSC numbers and cell cycle arrest during regeneration. These changes were linked to disrupted mTOR signaling, which drives the transition of MuSCs from G ₀ to G _Alert phase. In addition, Trpm7 -deficient MuSCs exhibited impaired early responses, including quiescent projection retraction and AP-1 induction. Mg ²⁺ supplementation rescued these defects, restoring normal MuSC activation. Our findings reveal a previously unrecognized mechanism where Mg ²⁺ permeation through TRPM7 is essential for MuSC activation and efficient skeletal muscle regeneration, highlighting TRPM7 as a critical regulator of muscle repair.

Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscles, play an essential role in muscle regeneration. After skeletal muscle injury, quiescent MuSCs are activated to enter the cell cycle and proliferate, thereby initiating regeneration; however, the mechanisms that ensure successful MuSC division, including chromosome segregation, remain unclear. Here, we show that PIEZO1, a calcium ion (Ca²⁺)-permeable cation channel activated by membrane tension, mediates spontaneous Ca²⁺influx to control the regenerative function of MuSCs. Our genetic engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs and thatPiezo1deletion in these cells delays myofibre regeneration after injury. These results are, at least in part, due to a mitotic defect in MuSCs. Mechanistically, this phenotype is caused by impaired PIEZO1-Rho signalling during myogenesis. Thus, we provide the first concrete evidence that PIEZO1, a bona fide mechanosensitive ion channel, promotes proliferation and regenerative functions of MuSCs through precise control of cell division.

Dystroglycan (DG) requires extensive post-translational processing to function as a receptor for extracellular matrix proteins containing laminin-G-like (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose and glucuronic acid that is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1) and binds with high affinity to matrix proteins like laminin. Defects in the post-translational processing of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. However, little is known regarding mechanisms that generate full-length matriglycan on α-DG (~150-250 kDa). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in a ~100-125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force induced by eccentric contractions and abnormalities in neuromuscular junctions. Collectively, our study demonstrates that α-DGN is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.

Although immobility is a common cause of muscle atrophy, the mechanism underlying this causality is unclear. We here show that Krüppel-like factor 15 (KLF15) and IL-6 are upregulated in skeletal muscle of limb-immobilized mice and that mice with KLF15 deficiency in skeletal muscle or with systemic IL-6 deficiency are protected from immobility-induced muscle atrophy. A newly developed Ca²⁺ bioimaging revealed that the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) of skeletal muscle is reduced to below the basal level by immobilization, which is associated with the downregulation of Piezo1. Acute disruption of Piezo1 in skeletal muscle induced Klf15 and Il6 expression as well as muscle atrophy, which was prevented by antibodies against IL-6. A role for the Piezo1/KLF15/IL-6 axis in immobility-induced muscle atrophy was validated in human samples. Our results thus uncover a paradigm for Ca²⁺ signaling in that a decrease in [Ca²⁺]_i from the basal level triggers a defined biological event.