Detyrosinated microtubules modulate mechanotransduction in heart and skeletal muscle

Abstract

In striated muscle, X-ROS is the mechanotransduction pathway by which mechanical stress transduced by the microtubule network elicits reactive oxygen species. X-ROS tunes Ca(2+) signalling in healthy muscle, but in diseases such as Duchenne muscular dystrophy (DMD), microtubule alterations drive elevated X-ROS, disrupting Ca(2+) homeostasis and impairing function. Here we show that detyrosination, a post-translational modification of α-tubulin, influences X-ROS signalling, contraction speed and cytoskeletal mechanics. In the mdx mouse model of DMD, the pharmacological reduction of detyrosination in vitro ablates aberrant X-ROS and Ca(2+) signalling, and in vivo it protects against hallmarks of DMD, including workload-induced arrhythmias and contraction-induced injury in skeletal muscle. We conclude that detyrosinated microtubules increase cytoskeletal stiffness and mechanotransduction in striated muscle and that targeting this post-translational modification may have broad therapeutic potential in muscular dystrophies.

Figure 1. Detyrosinated α-tubulin is decreased following parthenolide treatment.

(a) Skeletal FDB fibres isolated from WT rats were treated with PTL, taxol, or vehicle (control) before fixation, and immunostained for α-tubulin (green) and detyrosinated α-tubulin (red). (scale bar, 10 μm). (b) The density of the fraction of α-tubulin or detyrosinated α-tubulin was quantified (see Methods) following PTL (red) or taxol (blue) treatment and expressed as a per cent change from control (DMSO (dimethylsulphoxide)). PTL and taxol significantly decreased and increased detyrosinated tubulin, respectively. No significant differences in α-tubulin were identified with either treatment (n=7 fibres per group; *P<0.05). (c) Western blots of isolated FDB fibres showing that PTL significantly decreased, and taxol increased, the amount of detyrosinated tubulin, with neither treatment affecting the total α-tubulin content (n=5 mice; *P<0.05). Each treatment was normalized to DMSO control condition. (d,e) Cardiomyocytes isolated from WT rats treated with PTL, taxol, or vehicle control as above in A (scale bar, 10 μm) and quantified as in B. The detyrosinated MT fraction was significantly decreased with PTL and increased following taxol treatment, while the density of the total α-tubulin network was unchanged by PTL treatment and taxol treatment (n=8 cells per group; *P<0.05). (f) Western blots of cardiomyocytes revealed that PTL significantly decreased detyrosinated α-tubulin while taxol increased the amount of detyrosination. Neither treatment had an effect on total α-tubulin content (n=6 mice; *P<0.05). Blots quantitated as per cent change from controls. Differences between groups were determined by using a one-way analysis of variance followed by Dunnett's post hoc test. All values are mean±s.e.m.

Figure 2. Contractile mechanics in cardiomyocytes are regulated by detyrosinated microtubules.

(a) Sarcomere shortening was assayed in single cardiomyocytes under single action potential stimulation (1 Hz; field stimulation), contracting under no-load conditions (see Methods). SL measures of a control (black) and PTL (red) treated fiber are shown. Neither taxol nor PTL treatment significantly altered resting SL compared with the control (ctrl) condition. The per cent change (contraction %) in SL during contraction was significantly increased with PTL treatment but not by taxol. *P<0.05. (b) First derivative calculations of the SL shortening of a control (black) and PTL (red) treated cell expressed as a % from control. PTL significantly increased the velocity of SL shortening and relaxation while taxol decreased the velocity of each. *P<0.05 (n=36 control cells, 30 PTL cells, 30 taxol cells). (c) Ca²⁺ transients were assayed simultaneously with sarcomere mechanics following PTL treatment in single cardiomyocytes (n=5 cells per treatment). PTL (red) did not affect peak amplitude (measured by Fluo-3; see Methods) or the rate of decay of the global Ca²⁺ transient, and slightly slowed the rate of rise compared with the control condition (black/grey). *P<0.05 (n=38 control cells, 27 PTL cells). Statistical significance determined by one-way analysis of variance followed by Tukey post hoc test. All values are mean±s.e.m.

Figure 3. Mechanical properties of skeletal muscle are regulated by detyrosinated MT filaments.

(a) Sarcomere shortening was assayed in single FDB fibres contracting with single action potential stimulation (1 Hz; field stimulation) under no-load conditions (see Methods). Representative raw SL measures of control (black) and PTL (red) treated fibres are shown. Neither taxol nor PTL treatment significantly altered resting SL or the per cent change (% change) in SL compared with the control condition. (b) PTL significantly increased the maximal velocity of contraction, although relaxation kinetics were unaltered. Taxol treatment did not significantly alter either contraction or relaxation velocity. However, PTL significantly decreased, while taxol significantly increased, the time to peak SL shortening. *P<0.05 (n=10 control, 18 PTL, 14 taxol). Statistical significance determined by using a Kruskal–Wallis with Dunn's post hoc test. (c) Following PTL treatment, Ca²⁺ transients (displayed as the resulting ratio, F(a.u.)) were assayed using the ratiometric dye, Indo1-PE during unloaded shortening of single FDB fibres (n=5 cells per treatment). PTL did not affect peak calcium or the kinetics of rise or decay. Statistical significance determined by Student's t-test. All values are mean±s.e.m.

Figure 4. Detyrosinated tubulin affects cytoskeletal stiffness

(a) Differentiated C2C12 cells were treated with either DMSO (dimethylsulphoxide), taxol (10 μM), or PTL (10 μM) before extraction for western blotting. Following PTL or taxol treatment, no change in α-tubulin was noted. In contrast, PTL significantly decreased and taxol significantly increased detyrosinated tubulin (normalized to control, as in (b)). *P<0.05 (n=6 per treatment) Statistical significance was determined by using a one-way analysis of variance (ANOVA) with a Tukey's post hoc test. (c) Differentiated C2C12 cells treated with DMSO, taxol, or PTL were fixed and immunostained for α-tubulin and detyrosinated tubulin. PTL decreased the amount of detyrosinated tubulin detected, while taxol increased detyrosinated MTs. Scale bar, 10 μm. (d) α-actinin staining confirming sarcomere development in differentiated C2C12 cells used for AFM measurements (Scale bar, 10 μm.) (e) Phase contrast showing AFM tip and C2C12 cells (Scale bar, 10 μm). (f) PTL treatment significantly decreased while taxol treatment significantly increased cytoskeletal stiffness. Unhashed bars indicate pre-treatment measures; hashed bars indicate measurements taken post-treatment. Statistical significance determined by repeated measures ANOVA with post hoc Tukey, *P<0.05 (n=12 cells per treatment) All values are mean±s.e.m.

Figure 5. Mechano-activated ROS production and Ca²⁺ influx are regulated by detyrosinated tubulin.

(a) Apparatus for the mechanical manipulation of single skeletal muscle fibres mounted over an inverted fluorescence microscope (left panel). Middle panel is close up view of myofibre holders mounted to the piezo length controller (left) and force transducer (right). (b) Microfabricated glass myofibre holders (kind gift of Ionoptix, Inc.; see Methods) have an etched channel (30 μm diameter; side view on top, bottom view below), that when coated with Myotak adhesive, are designed to receive and hold a single muscle fibre. The far right panel shows an isolated myofibre (pseudocolored pink) held by the microfabricated glass holders. (c) Single FDB myofibres were challenged with a protocol of dynamic sinusoidal stretch. Representative experimental records of piezo actuator length output (top) and SL(bottom) recorded in single control fibres. (d) Averaged fluorescence records of single FDB fibres loaded with the ROS indicator DFF at resting length (SL ∼1.83), during 20 s of dynamic sinusoidal stretch (8 μM length excursion at 1 Hz; from resting SL to ∼1.95 μm SL, approximately 8% SL excursion) and post stretch. (e) Dynamic stretch of control fibres (grey) elicited a significant increase in DFF fluorescence that returned to pre-stretch rates with the cessation of stretch (hashed bars). The Nox2 inhibitor peptide gp91ds-TAT ameliorated the mechano-activated ROS signal indicating X-ROS as the mechanism of ROS generation. Pre-treatment with PTL or overexpression of TTL significantly reduced the mechano-activated ROS signal. *P<0.05 (n=12 cells per treatment). (f) FDB fibres loaded with near-membrane Indo-1 were assayed for sarcolemmal Ca²⁺ permeability during mechanical stretch. The rate of decline in Indo1 fluorescence due to internalization of extracellular Mn and subsequent quenching of Indo1 fluorescence was taken as sarcolemmal Ca²⁺ permeability. (g) Compared with pre-stretch rates, dynamic sinusoidal stretch elicited a significant increase in Mn quench that was abrogated by PTL. The stretch-dependent Mn influx was abrogated by the mechanosensitive channel inhibitor GsMTx4. *P<0.05 (n=10 cells per treatment). Statistical significance determined by one-way analysis of variance with Tukey's post hoc test. All values are mean±s.e.m.

Figure 6. Mechanical stress-induced ROS production and Ca²⁺ sparks in heart.

(a) Average traces (baseline subtracted, see Methods) of contraction-induced ROS production in isolated cardiomyocytes. (b) Parthenolide (PTL) reduces contraction-induced ROS production, quantified as per cent change in ROS production from baseline during (solid bars) and after (striped bars) 2 Hz field stimulation of isolated cardiomyocytes. *P<0.05 (n=49 control cells, 48 PTL cells). (c) PTL blocks stretch-dependent ROS production, quantified as per cent change in ROS production from baseline during (solid bars) and after (striped bars) 2 Hz 10% cyclic stretch of isolated cardiomyocytes. *P<0.05 (n=11 control cells, 12 PTL cells). (d) Representative fluorescence surface plots of calcium sparks in cyclically stretched myocytes with or without PTL treatment. PTL abrogated the stretch-dependent increase in calcium sparks seen in control cells. (e) Quantification of Ca²⁺ spark rate on first 10 s of initial cyclic stretch, after 1 min of sustained cyclical stretch, immediately following the release of stretch, and 30 s after the release of stretch, normalized to the pre-stretch rate *P<0.05 (n=22 control, 16 PTL cells). Statistical significance determined by repeated measures analysis of variance with post hoc Tukey. All values are mean±s.e.m.

Figure 7. Targeting detyrosinated tubulin in vivo reduces contraction-induced force decline.

Mdx mice (12 +/− 3.2 months) were treated with the PTL prodrug, LC-1 (100 mg kg⁻¹) or vehicle control by oral gavage for 3 days. (a) On the fourth day, a protocol of 20 eccentric contractions revealed a decline in isometric force before stretch in both vehicle and LC-1 treated mdx mice. (b) Repetitive eccentric contractions of the gastrocnemius muscle were elicited in vivo. The characteristic decline in isometric force was significantly inhibited with LC-1 treatment. *P<0.05 (n=5). (c) Western blot of gastrocnemius muscles following LC-1 treatment show significant decrease in detyrosinated α-tubulin, but no change in Nox2 (gp91^phox) or total α-tubulin content. Blots quantitated as per cent change from vehicle treatment. *P<0.05 (n=5) (d–f) FDB fibres from vehicle control or LC-1 treated mice were enzymatically isolated, loaded with calcium indicator dye (Fluo-4-AM). (d) Aggregate normalized Fluo4 traces during a sustained train of action potential pulses (20 Hz for 3 min). Ten sec before the cessation of stimulation, FDB myofibres from LC-1 treated mdx mice exhibited a significantly reduced peak cytosolic Ca²⁺. *P<0.05 (n=15 cells per treatment). (e) Quantitation of D. (f) Fibres from LC-1 treated mdx mice exhibited a significant increase in the decay of cytosolic Ca²⁺ following stimulation, as the post-stimulation fluorescence (F_post) declined lower than pre-stimulation levels (F_o) which yielded a negative F_post-F₀. In contrast, the F_post in vehicle-treated FDBs were significantly greater than pre-stimulation values 60 s after the end of the stimulation. *P<0.05 (n=15 cells per treatment). Statistical significance determined by Student's t-test. All values are mean±s.e.m.

Figure 8. In vivo targeting of detyrosination in mdx heart rescues isoproterenol induced arrhythmia and lethality.

(a) Representative ECG recordings from mdx mice given vehicle treatment or the parthenolide prodrug, LC-1, and exposed to an isoproterenol challenge to increase cardiac work. Off-scale readings in mdx trace represent agonal breathing shortly preceding death. (LC-1: n=6; vehicle: n=13) Scale bar, 1 s. (b) Per cent survival of mice following isoproterenol challenge. All 6 LC-1 treated mice survived (100% survival rate); 12 of 13 vehicle-treated mice were anesthetized due to fatal arrhythmia (7.6% survival rate). (c) Western blotting of isolated hearts following LC-1 or vehicle treatment shows a significant decrease in detyrosinated tubulin without significantly altering α-tubulin or Nox2 content. Blots quantitated as per cent change from vehicle-treated. Statistical significance determined by Student's t-test. *P<0.05 (n=3 per group). All values are mean±s.e.m.

Figure 9. Parthenolide treatment ablates stress-induced Ca²⁺ waves and leak in mdx cardiomyocytes.

(a) Representative traces of calcium transients and calcium waves during and after high-frequency (3 Hz) stimulation of mdx myocytes. (b) Parthenolide treatment significantly reduced the frequency of Ca²⁺ waves following high-frequency stimulation of mdx myocytes. (c) Parthenolide treatment prevented an elevation in resting calcium concentration following high-frequency stimulation of mdx myocytes *P< 0.01 versus mdx untreated, #P<0.05 versus mdx immediately post stim. (n=36 control cells, 36 PTL cells). Statistical significance determined by Student's t-test. All values are mean±s.e.m.