Mg2+ influx mediated by TRPM7 triggers the initiation of muscle stem cell activation

Abstract

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 Mg²⁺-permeable ion channel, as a critical regulator of MuSC activation. Trpm7 deletion in MuSCs reduced Mg²⁺ 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 G₀ to G_Alert phase. In addition, Trpm7-deficient MuSCs exhibited impaired early responses, including quiescent projection retraction and AP-1 induction. Mg²⁺ supplementation rescued these defects, restoring normal MuSC activation. Our findings reveal a previously unrecognized mechanism where Mg²⁺ permeation through TRPM7 is essential for MuSC activation and efficient skeletal muscle regeneration, highlighting TRPM7 as a critical regulator of muscle repair.

Fig. 1.. Magnesium ion dynamics in MuSCs during skeletal muscle regeneration.

(A and B) Mg²⁺ measurements in MuSCs using Magnesium Green, AM. (A) Representative fluorescent images of MuSCs from uninjured (left) and injured (1 day after injury) (right) muscle. (B) Left: Histograms of FACS data. Gray: unstained MuSCs; light green: uninjured MuSCs; dark green: injured MuSCs 3 days post-CTX. Right: Quantification of mean fluorescence intensity at indicated time points. (C to E) Mg²⁺ measurements using Mag-Fura-2 (C) Left: Schematic of Mg²⁺ imaging. Right: Fluorescent images of MuSCs cultured for 0 to 4 days. Look-up tables (LUTs) indicate signal intensity. (D) Quantification of basal Mg²⁺ levels in MuSCs cultured for 0 to 4 days. (E) Maximum fluorescent intensity (Δratio) of Mag-Fura-2 after addition of 10 mM Mg²⁺ (final concentration) subtracted by the mean basal intensity. (F) Time course for induction of Trpm7 deficiency. Trpm7^+/+; Pax7^CreERT2/+ was used as control (con) and Trpm7^flox/flox; Pax7^CreERT2/+ as knockout (cKO). (G) Trpm7 mRNA expression in freshly isolated MuSCs. (H) Cytosolic Mg²⁺ measurements of MuSCs 3 days post-CTX injury. Left: Histogram of flow cytometric data using Magnesium green. Light gray: Unstained MuSCs; dark gray: con MuSCs; and red: cKO MuSCs. Right: Quantification of increase in cytosolic Mg²⁺. (I to K) Mg²⁺ imaging of MuSCs cultured for 3 days. (I) Fluorescent images of control and cKO MuSCs before (0 s) and after (65 s) adding 10 mM Mg²⁺. (J) Heatmap showing ΔF/F of Mag-Fura-2 transients in pseudo-color (30 cells per group). (K) Δratio of Mag-Fura-2 in con versus cKO MuSCs (control: n = 241 cells, cKO: n = 101 cells, three mice per condition). Scale bars, 10 μm in [(A), (C), and (I)]. a.u, arbitrary unit.

Fig. 2.. Impaired regeneration capacity of MuSC-specific Trpm7-deficient muscle after injury.

(A) Time course for induction of Trpm7 deficiency, injection of TA with CTX, and isolation of regenerating muscle samples for [(B) to (D)]. (B) Representative images of regenerating TA muscle samples isolated from con (left) and cKO mice (right). (C) Weight of TA muscle samples after CTX-induced muscle injury (N = 3 to 5 mice per condition). (D) Hematoxylin and eosin staining of cross sections from intact and CTX-injected TA muscle samples harvested at indicated time points. Top panels: con; bottom panels: cKO mice. (E) Detection of eMyHC in cross sections from control (top) and cKO muscle (bottom). The area of eMyHC per section was evaluated 5 days after CTX treatment. (N = 3 to 4 mice per condition). (F) Cross-section area (CSA) of intact and regenerating myofibers at 21 and 28 days post-CTX injection (N = 3 mice per condition). (G) Number of Pax7-positive cells in intact and regenerating myofibers at 28 days post-CTX injection. Representative images of intact and regenerating muscle sections. White arrows indicate Pax7-positive cells (N = 3 mice per condition). (H) Quantification of MuSCs on muscle section, 1 month following the first TMX injection. Representative immunohistochemical images of TA muscle sections are shown in fig S1I. Pax7⁺ cells per 1000 μm². Scale bars, 100 μm in [(D), (E), and (G)].

Fig. 3.. Characterization of Trpm7-deficient MuSCs based on RNA-seq analysis.

(A) Time course for induction of Trpm7 deficiency, muscle injury, and sampling of MuSCs for RNA-seq. (B) Principal components analysis (PCA) of RNA-seq data. (C) Volcano plot of differentially expressed genes. Red and blue dots represent the genes with significantly increased (up-regulated) or decreased (down-regulated) expression, respectively (padj < 0.05). (D) GO enrichment analysis of significantly down-regulated genes in cKO. (E) Log₂fold changes (Log₂FCs) of representative down-regulated genes in cKO. (F) Time course for induction of Trpm7 deficiency, muscle injury, and sampling for [(G) to (K)]. (G) Detection of YFP-positive MuSCs and Cyclin D1 in cross sections from control (left) and cKO muscle (right). White arrows indicate YFP and Cyclin D1 double-positive cells. The ratio of Cyclin D1 per YFP-positive MuSCs was evaluated 3 days after CTX treatment (N = 4 mice per condition). (H) Detection of MyoD-positive cells in cross sections from con (top) and cKO muscle (bottom). MyoD-positive cells per 1000 μm² were evaluated 4 days after CTX treatment (N = 3 to 6 mice per condition). (I) Detection of Myogenin-positive cells in cross sections from control (top) and cKO muscle (bottom). Myogenin-positive cells per 1000 μm² were evaluated 4 days after CTX treatment (N = 3 to 6 mice per condition). (J) Ratio of EdU-positive cells per Pax7-positive cells were evaluated 4 days after CTX treatment (N = 3 mice per condition). (K) Representative FACS profiles of MuSCs isolated at 3 days post-injury, stained with MitoTracker Deep Red. Left: Histograms representing the mean fluorescent intensity of MitoTracker. Light gray: Unstained MuSCs; dark gray: con MuSCs; and red histogram: cKO MuSCs isolated from injured muscle 3 days post-CTX injection. Right: Quantification of mean fluorescence intensity. Scale bars, 100 μm in [(G) to (I)] and 10 μm (L).

Fig. 4.. Trpm7 deficiency inhibits cell cycle entry and proliferation of MuSC.

(A) Time course for induction of Trpm7 deficiency, harvesting of myofibers, and EdU incorporation. (B and C) TRPM7 and Pax7 expression in freshly isolated myofibers. (B) TRPM7 (red) detection in MuSCs; nuclei (DAPI, blue). Top: con MuSCs; bottom: cKO MuSCs. Right: Quantification of TRPM7-positive MuSCs (>50 MuSCs per condition, N = 3 mice). (C) Pax7-positive MuSCs per myofiber (>15 myofibers, N = 3 mice). (D to F) MuSC analysis after 30-hour culture. (D) Pax7 and MyoD expression (>15 myofibers, N = 3 mice). (E) Pax7 and Ki67 expression (>15 myofibers, N = 4 mice). (F) EdU incorporation assay in Pax7-positive MuSCs (>15 myofibers, N = 3 to 4 mice). (G) Immunofluorescent analysis after 72-hour culture. Left: Pax7 (green), MyoD (red), and nuclei (blue) in con (top) and cKO (bottom) MuSCs. Middle: Pax7- and MyoD-positive nuclei per myofiber. Right: Percent of positive nuclei (>15 myofibers, N = 5 mice). (H to J) Detection of CDK4-positive, Cyclin D1-positive, and phosphorylated Rb (pRb)–positive MuSCs cultured in growth medium for 48 hours. Left panels: CDK4 (H), Cyclin D1 (I), or pRb (J) were detected in con (top panels) and cKO (bottom panels). Right graphs: Quantification of CDK4 (H)–, Cyclin D1 (I)–, or pRb (J)–positive MuSCs. (>300 MuSCs per condition from N = 4 mice). (K) qPCR of MuSCs cultured 72 hours. (L and M) TRPM7-KR MuSCs. (L) Pax7-positive cells per myofiber (>15 myofibers, N = 3 mice). (M) Ki67 expression and EdU incorporation after 30 hours (>15 myofibers, N = 3 to 4 mice). Scale bars, 10 μm in [(B), (E), (F), (G), (H), (I), and (J)]. h, hours.

Fig. 5.. Loss of TRPM7 impairs activation of the mTOR signaling and induction of G_Alert in MuSCs.

(A) Time course for induction of Trpm7 deficiency and isolation of myofibers. (B and C) Analysis of MuSCs on myofibers cultured for 30 h. (B) MuSC size: Representative YFP-positive MuSC images and size distribution (>100 cells per condition). (C) pS6: Representative fluorescent images and quantification of pS6 intensity (>50 cells per condition, N = 3 mice). (D) Time course for induction of Trpm7 deficiency, injection of CTX, and isolation of myofibers. MuSCs on myofibers without injury was defined as quiescent satellite cells (QSCs). MuSCs on myofibers isolated from the contralateral leg of injury was defined as contralateral satellite cells (CSCs). Myofibers were isolated 4 days after CTX injection for G_Alert induction. (E) Morphological evaluation of MuSCs after induction of G_Alert. Phase contrast images of con (top) and cKO (bottom) MuSCs. Size distribution of QSCs and CSCs (>100 MuSCs per condition; data are individually plotted.). (F) pS6 in QSCs and CSCs: Representative images and quantification in con and cKO MuSCs (>50 cells per condition, N > 3 mice). (G) FACS histograms of MitoTracker and PyroninY staining. Gray: Unstained MuSCs; blue: con QSCs; green: con CSCs; red: cKO CSCs. (H) Time course for induction of Trpm7 deficiency, injection of HGFA, and isolation of myofibers. MuSCs from HGFA-treated mice were defined as HGFA for G_Alert induction. PBS was used for control. (I) HGFA effect on pS6 in cKO MuSCs: Representative images and quantification of pS6 in con and cKO MuSCs after G_Alert induction with HGFA (>50 cells per condition, N = 3 mice). Scale bars, 10 μm in [(B), (C), (E), (F), and (I)].

Fig. 6.. Trpm7 deficiency impairs early responses required for MuSCs activation.

(A) Evaluation of MuSCs with quiescent projection on myofibers from Pax7^CreERT2/+; Rosa26^YFP. TRPM7 expression (white) and YFP (magenta) were detected on myofibers freshly isolated (T0), or cultured for 1 hour (T1) and 2 hours (T2). MuSC morphology was visualized by detection of YFP (magenta). (B) MuSCs morphology cultured for 1 hour in DMEM with or without Mg²⁺. MuSC morphology was evaluated by detection of YFP (yellow). Nuclei were detected by DAPI (blue) (>50 MuSCs per condition from N = 3 mice). (C) MuSCs morphology in T0, T1, and T2 MuSCs. Left: Representative images of MuSCs. Top panels: con; bottom panels: cKO. YFP: yellow; nuclei (DAPI): blue. Right: Quantification of MuSCs with or without projections (>50 MuSCs per condition from N = 3 mice). (D) Time course for induction of Trpm7-deficiency, isolation, and culture of myofibers. (E) Expression of AP-1 transcription factor in T2 MuSCs. Left panels: Representative fluorescent images of AP-1 transcription factor (Fos and c-Jun). Quantification of AP-1 expression in T2 MuSCs (>50 MuSCs per condition from N = 3 mice). (F) Detection of pS6 (left) and phosphorylated ERK (pERK1/2; right) expression in T0, T2, and T4 MuSCs. Top: Representative images of con MuSCs. Bottom: Quantification of fluorescence intensity of pS6 and pERK1/2 normalized to con T0 MuSCs intensity. (G) MyoD expression in T8 MuSCs. Left: Representative fluorescent images of MyoD. Top: con; bottom: cKO. Right: Quantification of MyoD intensity (>50 MuSCs per condition from N = 3 mice). Scale bars: 10 μm in [(A), (B), (C), (E), (F), and (G)]. h, hours.

Fig. 7.. Mg²⁺ supplementation rescues defects of Trpm7-deficient MuSCs.

(A) Experimental procedures for myofiber isolation and culture in plating medium (PM) supplemented with 10 mM Mg²⁺. (B to E) Evaluation of con and cKO MuSCs on myofibers cultured in PM supplemented with 10 mM Mg²⁺ for 30 hours. con or cKO mice harboring the Rosa26-YFP were used for these experiments. (B) EdU incorporation assay on YFP-positive MuSCs in con and cKO MuSC on myofibers. (>15 myofibers per group were investigated from N > 4 mice). (C) MuSCs cell size (>100 MuSCs per group were investigated from N = 3 mice). (D) pS6 expression in MuSCs on myofibers (>50 MuSCs per group were investigated from N = 6 mice). (E) MyoD expression in MuSCs on myofibers (>50 MuSCs per group were investigated from N = 3 mice). (F) Quantification of Cyclin D1–positive MuSCs of con and cKO MuSCs cultured in GM supplemented with 10 mM Mg²⁺ for 48 hours (>300 MuSCs per group were investigated from N = 3 mice). (G) EdU assay on MuSCs cultured in growth medium supplemented with 10 mM Mg²⁺ for 3 days. EdU was incorporated into MuSCs for 3 hours before fixation. Left panels: Representative images of EdU assays. Right: Quantification of EdU-positive MuSCs (>500 cells from N = 3 mice per condition). Scale bars, 10 μm in [(B), (D), and (E)] and 100 μm in (H). h, hours.