Omecamtiv mecarbil lowers the contractile deficit in a mouse model of nebulin-based nemaline myopathy

Fig 1. Fiber type composition of Neb cKO muscle and dose-response curve of OM. (A) The proportion of type I myosin heavy chain (MHC) isoforms. (Top) Typical MHC-gel of soleus muscle of six months old CON and cKO mice. Control muscle (left lane) is a mixture of 40 days old TC and Sol muscle to reveal all MHC isoforms. In CON, MHC type I and IIa/x isoforms are present, while only the MHC type I isoform is clearly detectable in Neb cKO muscle. Bottom: The proportion of type I MHC is significantly greater (****: p<0.0001) in Neb cKO (n = 25) than CON (n = 20) mice. (B) Dose-response of OM on soleus muscle from Neb cKO mice (n = 6). Forces were measured at a 20Hz stimulation frequency. Relative increases in force from vehicle are shown. Relative increases in force beyond vehicle are shown. Results follow a dose-response curve with EC50 of 0.47±0.08 μM (based on fitting results from each individual muscle). Values are mean ± SEM.

Fig 2. Effect of OM on force-pCa relations of permeabilized fiber bundles from Neb cKO mice. A) Force-pCa curves in 0, 0.1, 0.3, 0.5, 1.0 and 10.0 μM OM. OM affects specific force in a dose-and activation-dependent manner. B) Force in OM relative to 0.0 OM at 5 pCa values. The two lowest OM concentrations (0.1 and 0.3 μM OM) increase force at low activation levels with minimal effects at high activation. At 0.5, 1.0, and 10 μM OM forces are minimally increased at low activation level and forces at high activation levels are depressed. C) Force-pCa curves normalized to their maximal force (pCa 4.0) are left shifted in OM (increased pCa50) and less steep (reduced Hill coefficient). D) Ktr values (pCa 6.75) at a range of OM concentrations. At high OM doses, Ktr is reduced. A and B shows mean ± SEM and C shows curve fits of data from 7 muscles except for the DMSO (vehicle) and 10 μM OM data that represent 6 muscles each. In D each data point represents one muscle. Asterisks in D denote significant differences between vehicle and OM (1W ANOVA with multiple testing correction); one symbol p<0.05 and three symbols p<0.001.

Fig 3. Effect of OM on active force of single soleus fibers from CON mice. A) Representative images of silver-stained gel with single fiber lysates loaded. Control is a mix of soleus and EDL lysates that shows the resolvable MHC isoforms (IIX and IIA integrate into a single band). Neb CON shows one type IIX/A fiber and one type I fiber while Neb cKO displays two type I fibers. (B and C) Relative force-pCa curves for slow type I (B) and fast type II (C) muscle fibers from CON soleus mice. Inset in B: Calcium-sensitivity (pCa50) of slow type I fibers with 0.1 μM OM was significantly greater than fibers with vehicle. Values are mean ± SEM. Multiple fibers per animal were studied (typically 5–6 fibers per animal) and their means were calculated. The shown values are the means of all studied animals: 5 mice with total 29 fibers (vehicle) and 5 mice with 22 fibers (OM) for type II fibers and 5 mice with 28 fibers (vehicle) and 5 mice with 25 fibers (OM) for type I fibers. (*p<0.05; **p<0.01 comparison vehicle vs. OM).

Fig 4. Effect of OM on active force of single type I fibers of Neb cKO and comparison with CON type I fibers. (A) Relative force-pCa relations of single type I fibers from Neb cKO soleus muscle. Relative forces were significantly greater at pCa 6.50, 6.25 and 6.00 with OM in Neb cKO fibers. Inset: Calcium sensitivity (pCa50) was significantly greater in fibers with OM than with vehicle. (B) Specific force-pCa relations in CON (solid lines) and cKO (dashed lines) in type I fibers with vehicle (Veh) or OM. Force-pCa curves were left shifted with OM relative to vehicle in both genotypes. Values are mean ± SEM. Multiple fibers per animal were studied and their means were calculated. The shown values are the means of 5 mice with total 28 fibers (CON + vehicle), 5 mice with 25 fibers (CON + OM) for type I fibers and 6 mice with 40 fibers (cKO+vehicle) and 5 mice with 28 fibers (cKO+OM). (*p<0.05; **p<0.01; ***p<0.001: ****p<0.0001; comparison CON vs. cKO, #p<0.0001 comparison vehicle vs. OM).

Fig 5. Effect of OM on force of intact soleus muscle. (A) Relative increase in force as a function of frequency in OM over vehicle. Neb cKO muscle (dark symbols); CON (light symbols). The relative increase in force with OM over vehicle was increased at all stimulation frequencies in Neb cKO muscles, while significance was found at only 10 and 20Hz in CON muscle. The OM effect was significantly greater in Neb cKO than CON at all frequencies except 10 Hz. The solid lines are the fits to the data and the broken lines normalize the response to the slow type I myosin content (50% in control muscle and 90% in Neb cKO muscle (Fig 1A)). (B-C) frequency for half-maximal force (half-frequency) in (B) and Hill-coefficient in (C) with vehicle or OM. A two-way repeated measures ANOVA revealed in B) a significant treatment effect with a significant interaction with genotype and in C) a significant treatment effect, a significant genotype effect and an interaction between treatment and genotype. Sidak multiple comparison tests revealed in B) a significant OM effect in Neb cKO muscle and in C) a significant OM effect in both CON and Neb cKO muscles. The number of muscles is 14 for each genotype. Values…

Fig 6. Effect of OM on power production in intact soleus muscle. A) Examples of force-velocity (left) and force-power curves (right) in CON (top) and Neb cKO (bottom) in vehicle and OM. (B) Maximal power with vehicle and OM solutions in both genotypes. A two-way repeated measures ANOVA revealed a significant treatment effect and a significant genotype effect. A Sidak multiple comparison test revealed a significant OM effect in Neb cKO muscle. Neb cKO mice have decreased power compared to CON. OM-treatment increased power in Neb cKO mice. Numbers of muscles are 6 for CON and 11 for cKO. Values are mean ± SEM. Comparison between vehicle and OM is marked as an asterisk(*). One symbol p<0.05.