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Nemaline myopathies are the most common form of congenital myopathies. Variants in ACTA1 (NEM3) comprise 15-25% of all nemaline myopathy cases. Patients harboring variants in ACTA1 present with a heterogeneous disease course characterized by stable or progressive muscle weakness and, in severe cases, respiratory failure and death. To date, no specific treatments are available. Since NEM3 is an actin-based thin filament disease, we tested the ability of tirasemtiv, a fast skeletal muscle troponin activator, to improve skeletal muscle function in a mouse model of NEM3, harboring the patient-based p.Asp286Gly variant in Acta1. Acute and long-term tirasemtiv treatment significantly increased muscle contractile capacity at submaximal stimulation frequencies in both fast-twitch extensor digitorum longus and gastrocnemius muscle, and intermediate-twitch diaphragm muscle in vitro and in vivo. Additionally, long-term tirasemtiv treatment in NEM3 mice resulted in a decreased respiratory rate with preserved minute volume, suggesting more efficient respiration. Altogether, our data support the therapeutic potential of fast skeletal muscle troponin activators in alleviating skeletal muscle weakness in a mouse model of NEM3 caused by the Acta1:p.Asp286Gly variant., (© 2024 Galli et al.)

Respiratory failure due to diaphragm dysfunction is considered a main cause of death in nemaline myopathy (NM) and we studied both isometric force and isotonic shortening of diaphragm muscle in a mouse model of nebulin-based NM (Neb cKO). A large contractile deficit was found in nebulin-deficient intact muscle that is frequency dependent, with the largest deficits at low-intermediate stimulation frequencies (e.g., a deficit of 72% at a stimulation frequency of 20 Hz). The effect of the fast skeletal muscle troponin activator (FSTA) tirasemtiv on force was examined. Tirasemtiv had a negligible effect at maximal stimulation frequencies, but greatly reduced the force deficit of the diaphragm at sub-maximal stimulation levels with an effect that was largest in Neb cKO diaphragm. As a result, the force deficit of Neb cKO diaphragm fell (from 72% to 29% at 20 Hz). Similar effects were found in in vivo experiments on the nerve-stimulated gastrocnemius muscle complex. Load-clamp experiments on diaphragm muscle showed that tirasemtiv increased the shortening velocity, and reduced the deficit in mechanical power by 33%. Thus, tirasemtiv significantly improves muscle function in a mouse model of nebulin-based nemaline myopathy.

Objective: To assess the efficacy of tirasemtiv, a fast skeletal muscle troponin activator, vs. placebo in patients with amyotrophic lateral sclerosis. Methods: VITALITY-ALS (NCT02496767) was a multinational, double-blind, randomized, placebo-controlled clinical trial. Participants tolerating 2 weeks of open-label tirasemtiv (125 mg twice daily) were randomized 3:2:2:2 to placebo or one of three target tirasemtiv dose levels, using an escalating dosage protocol lasting 28 days. The primary outcome measure was changed in slow vital capacity (SVC) at 24 weeks. Secondary endpoints included a change in muscle strength and time to respiratory milestones of disease progression., Results: Of 744 participants, 565 tolerated open-label tirasemtiv and received randomized treatment. By 24 weeks, 23 (12.2%) placebo-treated participants discontinued study treatment vs. 129 (34.2%) randomized to tirasemtiv. SVC declined by 14.4% (95% CI: −16.8, −11.9) in the placebo group and 13.4% (95% CI: −15.3, −11.6) in the tirasemtiv group (p = 0.56). Secondary endpoints did not show significant differences. However, participants who tolerated tirasemtiv at their randomized dose showed a numeric trend toward a dose-related slowing of decline in SVC (p = 0.11). Dizziness, fatigue, nausea, weight loss, and insomnia occurred more frequently on tirasemtiv. Serious adverse events were similar across groups., Conclusions: Tirasemtiv did not alter the decline of SVC or significantly impact secondary outcome measures. Poor tolerability of tirasemtiv may have contributed to this result. However, participants tolerating their intended dose exhibited a trend toward treatment benefit on SVC, suggesting the underlying mechanism of action may still hold promise, as is being tested with a different fast skeletal muscle troponin activator (NCT03160898).

Objective: To assess the efficacy of tirasemtiv, a fast skeletal muscle troponin activator, vs. placebo on respiratory function and other functional measures in patients with amyotrophic lateral sclerosis (ALS). This study was designed to confirm and extend results from a large phase IIb trial and maximize tolerability with a slower dose escalation., Methods: VITALITY-ALS (NCT02496767) was a multinational, double-blind, randomized, placebo-controlled, parallel-group study in ALS patients. Participants who tolerated two weeks of open-label tirasemtiv (125 mg twice a day) were randomized 3:2:2:2 to placebo or one of three target total daily dose levels of tirasemtiv (250, 375, or 500 mg). Participants randomized to tirasemtiv escalated their dose every two weeks to their target dose level or maximum tolerated dose. The primary outcome measure was change in slow vital capacity from baseline to 24 weeks. Secondary endpoints assessed the effect of tirasemtiv on muscle strength and certain respiratory milestones of disease progression. A four-week randomized withdrawal phase followed 48 weeks of treatment to evaluate the possibility of sustained benefit or rebound decline., Results: Data collection will be complete in the fourth quarter of 2017., Conclusions: VITALITY-ALS was a phase III trial designed to evaluate the efficacy, safety, and tolerability of tirasemtiv in ALS.

Key Points: We report that the small molecule CK-2066260 selectively slows the off-rate of Ca 2 + from fast skeletal muscle troponin, leading to increased myofibrillar Ca 2 + sensitivity in fast skeletal muscle. Rodents dosed with CK-2066260 show increased hindlimb muscle force and power in response to submaximal rates of nerve stimulation in situ. CK-2066260 has no effect on free cytosolic [Ca 2 + ] during contractions of isolated muscle fibres. We conclude that fast skeletal muscle troponin sensitizers constitute a potential therapy to address an unmet need of improving muscle function in conditions of weakness and premature muscle fatigue., Abstract: Skeletal muscle dysfunction occurs in many diseases and can lead to muscle weakness and premature muscle fatigue. Here we show that the fast skeletal troponin activator, CK-2066260, counteracts muscle weakness by increasing troponin Ca 2+ affinity, thereby increasing myofibrillar Ca 2+ sensitivity. Exposure to CK-2066260 resulted in a concentration-dependent increase in the Ca 2+ sensitivity of ATPase activity in isolated myofibrils and reconstituted hybrid sarcomeres containing fast skeletal muscle troponin C. Stopped-flow experiments revealed a ∼2.7-fold decrease in the Ca 2+ off-rate of isolated troponin complexes in the presence of CK-2066260 (6 vs. 17 s -1 under control conditions). Isolated mouse flexor digitorum brevis fibres showed a rapidly developing, reversible and concentration-dependent force increase at submaximal stimulation frequencies. This force increase was not accompanied by any changes in the free cytosolic [Ca 2+ ] or its kinetics. CK-2066260 induced a slowing of relaxation, which was markedly larger at 26°C than at 31°C and could be linked to the decreased Ca 2+ off-rate of troponin C. Rats dosed with CK-2066260 showed increased hindlimb isometric and isokinetic force in response to submaximal rates of nerve stimulation in situ producing significantly higher absolute forces at low isokinetic velocities, whereas there was no difference in force at the highest velocities. Overall muscle power was increased and the findings are consistent with a lack of effect on crossbridge kinetics. In conclusion, CK-2066260 acts as a fast skeletal troponin activator that may be used to increase muscle force and power in conditions of muscle weakness., (© 2016 Cytokinetics, Inc. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Tirasemtiv is a fast skeletal troponin activator that sensitizes the sarcomere to calcium and increases muscle force following subtetanic nerve input. In an animal model of myasthenia gravis (MG), single oral doses of tirasemtiv improved muscle force and reduced fatigability. The purpose of this study was to determine the effect of single doses of tirasemtiv on skeletal muscle function and fatigability in patients with generalized MG. Thirty-two patients with acetylcholine receptor-antibody positive MG and muscle weakness received single doses of tirasemtiv (250 mg or 500 mg) or placebo in a double-blind, randomized treatment sequence with each treatment separated by at least 1 week. Outcome measures included the Quantitative MG Score (QMG), MG Composite, Manual Muscle Testing, and forced vital capacity. At 6 h after dosing, tirasemtiv produced dose-related improvements from baseline in the QMG score (slope: -0.49 QMG point per 250 mg; p = 0.02) and in percent predicted forced vital capacity (slope: 2.2% per 250 mg; p = 0.04). QMG improved >3 points in twice as many patients after 500 mg tirasemtiv than after placebo. Both doses of tirasemtiv were well tolerated; there were no premature terminations or serious adverse events. The results of this study suggest that tirasemtiv may improve muscle function in MG and will be used to support further development of tirasemtiv in neuromuscular diseases.

Introduction: In this study we tested the hypothesis that tirasemtiv, a selective fast skeletal muscle troponin activator that sensitizes the sarcomere to calcium, could amplify the response of muscle to neuromuscular input in humans., Methods: Healthy men received tirasemtiv and placebo in a randomized, double-blind, 4-period, crossover design. The deep fibular nerve was stimulated transcutaneously to activate the tibialis anterior muscle and produce dorsiflexion of the foot. The force-frequency relationship of tibialis anterior dorsiflexion was assessed after dosing., Results: Tirasemtiv increased force produced by the tibialis anterior in a dose-, concentration-, and frequency-dependent manner with the largest increases [up to 24.5% (SE 3.1), P < 0.0001] produced at subtetanic nerve stimulation frequencies (10 Hz)., Conclusions: The data confirm that tirasemtiv amplifies the response of skeletal muscle to nerve input in humans. This outcome provides support for further studies of tirasemtiv as a potential therapy in conditions marked by diminished neuromuscular input., (© 2014 The Authors. Muscle Nerve published by Wiley Periodicals, Inc.)

Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease characterized by progressive motor neuron loss resulting in muscle atrophy, declining muscle function, and eventual paralysis. Patients typically die from respiratory failure 3 to 5 years from the onset of symptoms. Tirasemtiv is a fast skeletal troponin activator that sensitizes the sarcomere to calcium; this mechanism of action amplifies the response of muscle to neuromuscular input producing greater force when nerve input is reduced. Here, we demonstrate that a single dose of tirasemtiv significantly increases submaximal isometric force, forelimb grip strength, grid hang time, and rotarod performance in a female transgenic mouse model (B6SJL-SOD1 G93A) of ALS with functional deficits. Additionally, diaphragm force and tidal volume are significantly higher in tirasemtiv-treated female B6SJL-SOD1 G93A mice. These results support the potential of fast skeletal troponin activators to improve muscle function in neuromuscular diseases.

Introduction: Tirasemtiv is a novel small molecule activator of the fast skeletal muscle troponin complex that produces sensitization of the sarcomere to calcium. Tirasemtiv is currently in Phase II clinical trials for neuromuscular disease., Methods: We conducted a blinded, randomized, placebo-controlled preclinical study of the effect of tirasemtiv on forearm grip strength, endurance, respiratory physiology, and muscle pathology in adequate sample sizes of the Lama2(dy-2J) mouse model of congenital muscular dystrophy., Results: Mice receiving a high dose of tirasemtiv had significantly higher muscle fiber cross-sectional area and respiratory response to CO₂ stimulation at 16 weeks than mice on low dose or placebo. There were no changes in muscle pathology, serum creatine kinase, strength, endurance, or respiration following long-term treatment., Conclusions: We conclude that tirasemtiv influences the structure of the skeletal muscle fiber in this model of muscular dystrophy but does not impact muscle function, as evaluated in this study., (© 2013 Wiley Periodicals, Inc.)

Background: Nemaline myopathy-the most common non-dystrophic congenital myopathy-is caused by mutations in thin filament genes, of which the nebulin gene is the most frequently affected one. The nebulin gene codes for the giant sarcomeric protein nebulin, which plays a crucial role in skeletal muscle contractile performance. Muscle weakness is a hallmark feature of nemaline myopathy patients with nebulin mutations, and is caused by changes in contractile protein function, including a lower calcium-sensitivity of force generation. To date no therapy exists to treat muscle weakness in nemaline myopathy. Here, we studied the ability of the novel fast skeletal muscle troponin activator, CK-2066260, to augment force generation at submaximal calcium levels in muscle cells from nemaline myopathy patients with nebulin mutations., Methods: Contractile protein function was determined in permeabilised muscle cells isolated from frozen patient biopsies. The effect of 5 μM CK-2066260 on force production was assessed., Results: Nebulin protein concentrations were severely reduced in muscle cells from these patients compared to controls, while myofibrillar ultrastructure was largely preserved. Both maximal active tension and the calcium-sensitivity of force generation were lower in patients compared to controls. Importantly, CK-2066260 greatly increased the calcium-sensitivity of force generation-without affecting the cooperativity of activation-in patients to levels that exceed those observed in untreated control muscle., Conclusions: Fast skeletal troponin activation is a therapeutic mechanism to augment contractile protein function in nemaline myopathy patients with nebulin mutations and with other neuromuscular diseases.