Functional Characterization of the Intact Diaphragm in a Nebulin-Based Nemaline Myopathy (NM) Model-Effects of the Fast Skeletal Muscle Troponin Activator tirasemtiv

Figure 1. Force–frequency relations in control (ctrl) and of nebulin-based NM (Neb cKO) diaphragm muscle strips. (A) Force–frequency curves. Forces are shown as specific force (force normalized to the muscle’s cross-sectional area). Standard two-way ANOVA reveals that genotype significantly (p < 0.0001) affects specific force. A post-hoc test (Sidak) corrected for multiple comparisons reveals that at all stimulation frequencies, Neb cKO produces significantly less force than ctrl (two lowest frequencies p < 0.01, all other frequencies p < 0.0001). (B) Relative force–frequency curves. For each genotype, force is normalized to the maximal force at 200 Hz. The Neb cKO curve has a different shape than the ctrl curve. Standard two-way ANOVA reveals that genotype significantly (p < 0.0001) affects relative force. A post-hoc test (Sidak) corrected for multiple comparisons reveals that Neb cKO produces significantly less relative force than ctrl at 1–40 Hz (** p <0.01, **** p < 0.0001). (C) Force of Neb cKO muscles relative to that of ctrl diaphragm (this study) and EDL and soleus (data from Li et al. [13]), which uses the same mouse model (at 6 months old) and experimental protocols as the…

Figure 2. Effect of tirasemtiv on force production in control (ctrl) and Neb cKO diaphragm muscle strips. (A and B) Force–frequency curves (A) and maximal speed of specific force rise (dF/dt) (B) in ctrl and cKO muscle with vehicle or tirasemtiv. Two-way ANOVA with repeated measures reveals in both genotypes that tirasemtiv significantly (p < 0.0001) affects specific force. Post-hoc tests (Sidak) corrected for multiple comparisons reveal that tirasemtiv increases force at all stimulation frequencies except 1 Hz and maximal force rise at all stimulation frequencies, except 200 Hz for cKO (p < 0.01). (C and D) Effect of tirasemtiv on force (C) and speed of force rise (D) over vehicle at various stimulation frequencies. In both ctrl and cKO diaphragms, tirasemtiv increases force (C) and speed of force rise (D) with a maximal effect at ~20 Hz. A standard two-way ANOVA reveals that tirasemtiv has a significantly (p < 0.0001) different effect on force in Neb cKO diaphragm strips than in ctrl strips. Post-hoc tests (Sidak) corrected for multiple comparisons reveals that Neb cKO muscle has significantly greater enhancement with tirasemtiv than ctrl muscle in the mid-frequency range (** p < 0.01,…

Figure 3. Tirasemtiv reduces the force deficit of cKO diaphragm strips. Specific force of cKO in vehicle (circles) or tirasemtiv (squares) relative to ctrl (in vehicle). A standard two-way ANOVA reveals that tirasemtiv has a significant (p < 0.0001) effect on the force deficit. Post-hoc tests (Sidak) corrected for multiple comparisons reveal that tirasemtiv significantly reduces the force deficit of cKO diaphragm at all stimulation frequencies up to 100 Hz (p < 0.001) with the largest effect at intermediate stimulation frequencies (e.g., at 20 Hz the deficit goes from 76% to 29%). Mean ± SD are shown.

Figure 4. Effect of tirasemtiv on tension-velocity relation and power-velocity relations of diaphragm in control (ctrl) and Neb cKO mice. (A) Tension-velocity relationship measured in load-clamp experiments. At all load levels and in both genotypes, tirasemtiv significantly increases shortening velocity (p < 0.0001). (B) Tension-velocity relation with tension normalized to maximal tetanic tension. Tirasemtiv increases the shortening velocity at each tension level in both genotypes and statistical significance was found at Tension/Tensionmax of 0.1–0.7 for ctrl and 0.1–0.6 for cKO (p < 0.01). Shortening velocity in cKO with tirasemtiv overlaps with that of ctrl in vehicle. (C) Power–velocity relationship. In both genotypes, maximal power is significantly increased by tirasemtiv (p < 0.0001) and maximal power occurs at higher shortening speeds. (Shortening speed expressed as Lo (length at optimal force)/second). Results from 11 ctrl mice and 13 cKO mice. Mean ± SD are shown. Note that at many frequencies SD lines fall within the symbols.

Figure 5. Force–frequency relation of in vivo gastrocnemius muscle complex measured in vehicle or tirasemtiv in ctrl and Neb cKO mice. (A and B) Force in tirasemtiv (square) and in vehicle (circle) in ctrl (A) and cKO (B) mice. Two-way ANOVA with repeated measures reveals in both genotypes that tirasemtiv significantly (p < 0.0001) affects force in ctrl (A) and cKO (B) mice. Post-hoc tests (Sidak) corrected for multiple comparisons reveal that tirasemtiv increases force at low–intermediate frequencies. Insets show frequency that results in half-maximal force, F50 frequency (light color: vehicle, dark color-tirasemtiv). An unpaired two-tailed t-test reveals in both genotypes a significant reduction in F50. (C) Effect of tirasemtiv on force over vehicle at various stimulation frequencies. In both ctrl and cKO, tirasemtiv increases force with a maximal effect at ~20 Hz. A standard two-way ANOVA with post-hoc tests (Sidak) corrected for multiple comparisons reveals that tirasemtiv has a significantly larger effect on ctrl gastrocnemius than cKO at 30 and 40 Hz (*** p < 0.001, **** p < 0.0001). (D) Force deficit of cKO gastrocnemius complex. Force of cKO in vehicle (circles) or tirasemtiv…