Injury-mediated stiffening persistently activates muscle stem cells through YAP and TAZ mechanotransduction

Abstract

The skeletal muscle microenvironment transiently remodels and stiffens after exercise and injury, as muscle ages, and in myopathic muscle; however, how these changes in stiffness affect resident muscle stem cells (MuSCs) remains understudied. Following muscle injury, muscle stiffness remained elevated after morphological regeneration was complete, accompanied by activated and proliferative MuSCs. To isolate the role of stiffness on MuSC behavior and determine the underlying mechanotransduction pathways, we cultured MuSCs on strain-promoted azide-alkyne cycloaddition hydrogels capable of in situ stiffening by secondary photocrosslinking of excess cyclooctynes. Using pre- to post-injury stiffness hydrogels, we found that elevated stiffness enhances migration and MuSC proliferation by localizing yes-associated protein 1 (YAP) and WW domain-containing transcription regulator 1 (WWTR1; TAZ) to the nucleus. Ablating YAP and TAZ in vivo promotes MuSC quiescence in postinjury muscle and prevents myofiber hypertrophy, demonstrating that persistent exposure to elevated stiffness activates mechanotransduction signaling maintaining activated and proliferating MuSCs.

Fig. 1. Barium chloride injury induces muscle stiffening and persistently activates MuSCs.

(A) Myofibers isolated from EDL muscles before injury and at 14 and 28 days after injury (DPI). MuSCs are immunoreactive for Pax7 and for MyoD upon activation. Nuclei are 4′,6-diamidino-2-phenylindole–positive (DAPI⁺), and white arrowheads mark Pax7⁺ MuSCs. (B) Quantification of the percentage of MyoD⁺ expressing MuSCs. UI, uninjured. (C) TA muscle sections were assayed for EdU incorporation following 24-hour EdU pulse before harvest. Immunoreactivity to Pax7 identifies MuSCs, and laminin immunoreactivity demarcates the myofiber basement membrane; nuclei are detected by DAPI staining. White arrowheads identify EdU⁻ MuSCs, with EdU⁺ MuSCs in the insert. Scale bars, 10 μm. Quantification of density (D) and percentage of EdU⁺ (E) of Pax7⁺ MuSCs at 14 and 28 days after injury. (F) Spatial Young’s modulus maps of TA muscles measured by AFM. (G) Gaussian fits (dashed lines) of the frequency of the stiffness measurements and (H) their average values per muscle after injury. n = 3 biological replicates with three modulus maps per replicate. (I) TA muscle sections were stained with hematoxylin and eosin (H&E), Picrosirius red, and Masson’s trichrome to identify collagen and the ECM. Yellow arrowheads mark examples of centrally located nuclei in regenerated myofibers, and black arrowheads mark increased collagen deposition. n = 4 biological replicates. The collagen⁺ areas were quantified by Picrosirius (J) and Masson’s trichrome staining (K). Unless otherwise noted, n = 3 biological replicates. For MuSC quantification, >50 MuSCs scored per replicate. Error bars represent the SD, and *P < 0.05, **P < 0.01, and ***P < 0.001 in a one-way analysis of variance (ANOVA) test compared to uninjured controls.

Fig. 2. Secondary photostiffening of SPAAC hydrogels to model muscle stiffening.

(A) Schematic representation of hydrogel formation and the photocrosslinking reaction. Hydrogels are formed through a SPAAC reaction between cyclooctyne (PEG-DBCO)– and azide (PEG-N₃)–functionalized macromers, and the peptide N₃-KRGDS (1 mM) is incorporated to promote cell adhesion. Networks formed with an excess of DBCO functionalities can be further stiffened upon cytocompatible light irradiation in the presence of a photoinitiator (LAP). (B) Rheological traces of network evolution for different stoichiometric ratios of ─DBCO and ─N₃ functional groups. SPAAC network evolution is monitored for 600 s, followed by light exposure (365 nm, 10 mW/cm², 120 s; shaded region) to induce secondary photocrosslinking of pendant DBCO functionalities. (C) Quantification of Young’s modulus for hydrogels with different DBCO to ─N₃ stoichiometries before (striped) and after photocrosslinking (solid). n = 3 independent measurements, the plot displays the means ± SD, and ***P < 0.001 in a two-tailed Student’s t test. (D) Stepwise network stiffening performed through consecutive 30-s light exposures, demonstrating tunability of the magnitude of stiffening entirely with light irradiation.

Fig. 3. C2C12 cell proliferation increases in response to dynamic stiffening substrates.

(A and B) C2C12 cells cultured for 3 days on E′ = 2- and 32-kPa SPAAC hydrogels retained Pax7 and MyoD expression. (C) Representative confocal images EdU incorporation in C2C12 cells after 2-hour EdU incubation. (D) EdU⁺ cells were quantified as a function of substrate moduli between E′ = 2 to 32 kPa before and after photostiffening. n > 5 biological replicates with >100 cells analyzed per replicate. (E) C2C12 cells were tracked in real time with NucBlue staining using Imaris software. Images were acquired every 15 min over 12 hours. (F) Violin plots of the average C2C12 migration velocity for 12 hours of tracking. Solid lines indicate the mean, and dashed lines mark the 25th and 75th quartiles. n > 6 with >180 cells counted per hydrogel. For (B) and (D), plots display the means ± SD, and **P < 0.01 and ***P < 0.001 in a two-way ANOVA test.

Fig. 4. Stiffness induces MuSC proliferation on encapsulated myofibers.

(A) Myofibers from Pax7^CreERT;ROSA26-stop-lox-stop^NLS-tdTOM mice were isolated from an EDL muscle and cultured floating in media supplemented with 4-hydroxytamoxifen for 1 day to induce nuclear MuSC tdTomato expression (Tom⁺MuSCs). Myofibers were encapsulated after 1 day of culture in either Matrigel or SPAAC hydrogels with stiffness values ranging between E′ = 300 and 4600 Pa. Myofibers were imaged on three consecutive days. (B) Representative images of the encapsulated myofibers where Tom⁺MuSCs are pseudo-colored on the basis of the imaging day and overlaid with the day 3 brightfield images. (C) Data are plotted for MuSCs cultured in Matrigel compared to stiffness-controlled SPAAC hydrogels (E′ = 300 to 4600 Pa). n > 6 myofibers analyzed from three independent experiments, means ± SD, and *P < 0.05 and ***P < 0.001 in a two-way ANOVA test comparing the means of other conditions to E′ = 300-Pa SPAAC hydrogels.

Fig. 5. YAP and TAZ mediate mechanosensitive C2C12 behavior.

(A) Representative images of C2C12 cells cultured for 3 days on different stiffness hydrogels assayed for immunoreactivity to YAP and TAZ. Nuclear and cytoplasmic borders are demarcated by DAPI and phalloidin staining, respectively. (B) YAP/TAZ nuclear-to-cytoplasmic (Nuc:Cyto) ratio quantified as a function of substrate stiffness. Error bars represent 95% confidence intervals (CIs). (C) YAP/TAZ Nuc:Cyto ratio quantified upon in situ photostiffening and assayed at 1 and 2 days after stiffening (DPS). Error bars represent 95% CI. (D) Representative images of C2C12s cells treated with 5 μM verteporfin or vehicle control [dimethyl sulfoxide (DMSO)] for 24 hours. Verteporfin decreased YAP/TAZ Nuc:Cyto ratio in C2C12 cells. (E) Normalized YAP/TAZ Nuc:Cyto ratio of C2C12 cells with or without verteporfin treatment. (F) Quantification of EdU incorporation in C2C12 cells after 24 hours of 5 μM verteporfin treatment beginning on day 2 of culture. (G) Quantification of the mRNA expression and (H) protein levels of YAP and TAZ 48 hours after knockdown in C2C12 cells. YAP was knocked down with a short hairpin RNA (shRNA), and TAZ was knocked down with a siRNA. Each were compared to respective scrambled control vectors. shNT, non-targeted shRNA; siSCR, scrambled siRNA; w.r.t., with respect to. (I) Representative images for a 2-hour EdU incorporation after YAP and/or TAZ knockdown of C2C12 cells on 32-kPa hydrogels and their quantification (J). Unless noted elsewhere, n > 3 per experiment and >100 cells per hydrogel were quantified, means ± SD, and *P < 0.05, **P < 0.01, and ***P < 0.001 in a two-way ANOVA test.

Fig. 6. YAP and TAZ signaling restores MuSC fate in stiff muscle.

(A) Images of wild-type MuSCs cultured on hydrogels for 72 or 48 hours after in situ stiffening. MuSCs were identified by Pax7 immunoreactivity, assessed for subcellular YAP/TAZ localization with phalloidin and DAPI demarcating the cytoplasm and nucleus, respectively. (B) YAP/TAZ mean nuclear intensity was quantified on static and dynamically stiffened hydrogels. n = 3 with >15 cells scored per replicate. a.u., arbitrary units. (C) mRNA quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after 72 hours of culture. Images (D) and quantification (E) of EdU⁺ MuSCs following an EdU treatment after 3 days after seeding or 2 days after in situ stiffening. (F) MuSCs from Pax7^CreERT;YAP^fl/fl;TAZ^fl/fl mice were cultured on hydrogels. The medium was supplemented with 4-hydroxytamoxifen (4-OHT), and proliferation was assessed 48 hours later. Images of MuSCs immunoreactive for Pax7 and MyoD and assayed for EdU incorporation and quantification (G) of proliferating MuSCs. (H) Schematic for YAP/TAZ knockout and EdU treatment after injury in YAP^fl/fl;TAZ^fl/fl;Pax7^CreERT (dKO) mice or YAP^fl/fl;TAZ^fl/fl;Pax7^+/+ (control) mice. Images of 28–day after injury TA muscle sections were assayed for EdU incorporation and immunoreactivity with laminin and Pax7 to identify MuSCs (white arrowheads). DAPI detected nuclei. Quantification of Pax7⁺ (I) and EdU⁺ (J) MuSCs from 28–day after injury or contralateral (CL) TA muscle sections. (K) Quantification of the myofiber minimum Feret diameter identified via laminin immunoreactivity. Unless noted elsewhere, n = 3 biological replicates. More than 50 MuSCs and >250 myofibers scored per replicate. Graphs display means ± SD, and *P < 0.05, **P < 0.01, and ***P < 0.001 in a one-way ANOVA test.

Fig. 7. Muscle stiffening induces mechanosensitive behavior through YAP and TAZ localization.

A model for persistent injury-mediated mechanical stiffening of skeletal muscle promoting MuSC activation and proliferation. YAP and TAZ localization transduces the mechanical signals into proliferative MuSCs and enhances their migration.