Tubulin acetylation increases cytoskeletal stiffness to regulate mechanotransduction in striated muscle

Abstract

Microtubules tune cytoskeletal stiffness, which affects cytoskeletal mechanics and mechanotransduction of striated muscle. While recent evidence suggests that microtubules enriched in detyrosinated α-tubulin regulate these processes in healthy muscle and increase them in disease, the possible contribution from several other α-tubulin modifications has not been investigated. Here, we used genetic and pharmacologic strategies in isolated cardiomyocytes and skeletal myofibers to increase the level of acetylated α-tubulin without altering the level of detyrosinated α-tubulin. We show that microtubules enriched in acetylated α-tubulin increase cytoskeletal stiffness and viscoelastic resistance. These changes slow rates of contraction and relaxation during unloaded contraction and increased activation of NADPH oxidase 2 (Nox2) by mechanotransduction. Together, these findings add to growing evidence that microtubules contribute to the mechanobiology of striated muscle in health and disease.

Figure 1.

HDAC6 inhibition increases α-tubulin acetylation independently of altering the density of microtubules and increases cell viscoelastic resistance. (A and B) Western blot of isolated cardiomyocytes and FDB fibers treated with HDAC6 inhibitor Tubacin (10 µM, 2 h, n = 4). Quantification of α-tubulin and acetylated and detyrosinated tubulin normalized to Ponceau and expressed as fold-change from control conditions (vehicle only). (C and D) Immunofluorescence and quantification of isolated cardiomyocytes and FDB fibers, respectively. Tubulin abundance was determined from a binary quantification normalized to cell area and expressed as fold change from control. FDB, n = 12; cardiomyocytes, n = 10. (E–H) Young’s modulus of cardiomyocytes and FDB fibers treated with Tubacin (red) from nanoindentation at different speeds (E and G, respectively). The derived viscoelastic resistance from experiments shown in E and G is represented in F (n = 4; control, n = 25; Tubacin, n = 27) and H (n = 4; control, n = 29; Tubacin, n = 29), respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001; scale bars, 10 µm.

Figure 2.

Increased MT acetylation slows contractile kinetics and modulates mechanosensitive ROS production. (A–C and E–G) Representative sarcomere length and velocity during contraction (A and E) treated with Tubacin (red) and respective controls (gray); measured fractional shortening (B and F) and contractile kinetics (C and G) from cardiomyocytes and FDB fibers, respectively. (D) Quantification of the change in ROS production in cardiac cells during 2-Hz stimulated contractions measured by the change in dihydrofluorescein fluorescence increase (n = 7; control, n = 30; Tubacin, n = 31). (H) Quantification of the change in ROS production during 2-Hz cyclic stretch in FDB fibers (n = 3; control, n = 16; Tubacin, n = 18). *, P < 0.05; ***, P < 0.001.

Figure 3.

Overexpression of αTAT increases viscoelastic resistance and slows contractile kinetics. (A) Immunofluorescent staining and quantification of acetylated and detyrosinated tubulin in FDBs overexpressing αTAT (fold-change from GFP-only controls). (B and C) Young’s modulus of FDB fibers overexpressing αTAT (blue) and GFP control (gray) from nanoindentation at different speeds (detyrosinated tubulin, n = 10; acetylated tubulin, n = 20; B) and their derived viscoelastic resistance (n = 4; GFP, n = 15; αTAT, n = 17; C). (D and E) Peak contraction, relaxation velocities (D), and fractional shortening (E) measured from FDB fibers overexpressing αTAT (blue) and GFP-only controls (gray). Unloaded shortening parameters (n = 4; GFP, n = 27; αTAT, n = 45). n.s, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; scale bars, 10 µm.

Figure 4.

Microtubule acetylation has diminished impact on contractile mechanics in intact muscle. (A) Western blot of contralateral EDL from animals treated with Tubastatin A or vehicle control (n = 8). (B) Quantification of α-tubulin, acetylated tubulin, and detyrosinated tubulin protein expression. (C) Representative traces of successive stretches from intact EDL muscle in a bath. (D) Fold-change from control of Tubastatin A–treated animals. Quantification of peak force and decay time to 80% of peak for each successive stretch. (E) Force frequency curve from intact EDL muscle in a bath. (F) Contraction and relaxation rate from intact EDL stimulated at increasing frequencies; n = 8. *, P < 0.05; ***, P < 0.001.