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Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases.

ABSTRACT: Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal-troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal-troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards, as does the force-frequency relationship of a nerve-muscle pair, so that CK-2017357 increases the production of muscle force in situ at sub-maximal nerve stimulation rates. Notably, we show that sensitization of the fast-skeletal-troponin complex to calcium improves muscle force and grip strength immediately after administration of single doses of CK-2017357 in a model of the neuromuscular disease myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.

SUBMITTER: Russell AJ

PROVIDER: S-EPMC3296825 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases.

Russell Alan J AJ Hartman James J JJ Hinken Aaron C AC Muci Alexander R AR Kawas Raja R Driscoll Lena L Godinez Guillermo G Lee Kenneth H KH Marquez David D Browne William F WF Chen Michael M MM Clarke David D Collibee Scott E SE Garard Marc M Hansen Richard R Jia Zhiheng Z Lu Pu-Ping PP Rodriguez Hector H Saikali Khalil G KG Schaletzky Julia J Vijayakumar Vipin V Albertus Daniel L DL Claflin Dennis R DR Morgans David J DJ Morgan Bradley P BP Malik Fady I FI

Nature medicine 20120219 3

Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal-troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal-tropo ...[more]

PMID: 22344294

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Project description:BackgroundAgeing skeletal muscle undergoes chronic denervation, and the neuromuscular junction (NMJ), the key structure that connects motor neuron nerves with muscle cells, shows increased defects with ageing. Previous studies in various species have shown that with ageing, type II fast-twitch skeletal muscle fibres show more atrophy and NMJ deterioration than type I slow-twitch fibres. However, how this process is regulated is largely unknown. A better understanding of the mechanisms regulating skeletal muscle fibre-type specific denervation at the NMJ could be critical to identifying novel treatments for sarcopenia. Cardiac troponin T (cTnT), the heart muscle-specific isoform of TnT, is a key component of the mechanisms of muscle contraction. It is expressed in skeletal muscle during early development, after acute sciatic nerve denervation, in various neuromuscular diseases and possibly in ageing muscle. Yet the subcellular localization and function of cTnT in skeletal muscle is largely unknown.MethodsStudies were carried out on isolated skeletal muscles from mice, vervet monkeys, and humans. Immunoblotting, immunoprecipitation, and mass spectrometry were used to analyse protein expression, real-time reverse transcription polymerase chain reaction was used to measure gene expression, immunofluorescence staining was performed for subcellular distribution assay of proteins, and electromyographic recording was used to analyse neurotransmission at the NMJ.ResultsLevels of cTnT expression in skeletal muscle increased with ageing in mice. In addition, cTnT was highly enriched at the NMJ region-but mainly in the fast-twitch, not the slow-twitch, muscle of old mice. We further found that the protein kinase A (PKA) RIα subunit was largely removed from, while PKA RIIα and RIIβ are enriched at, the NMJ-again, preferentially in fast-twitch but not slow-twitch muscle in old mice. Knocking down cTnT in fast skeletal muscle of old mice: (i) increased PKA RIα and reduced PKA RIIα at the NMJ; (ii) decreased the levels of gene expression of muscle denervation markers; and (iii) enhanced neurotransmission efficiency at NMJ.ConclusionsCardiac troponin T at the NMJ region contributes to NMJ functional decline with ageing mainly in the fast-twitch skeletal muscle through interfering with PKA signalling. This knowledge could inform useful targets for prevention and therapy of age-related decline in muscle function.

Project description:BackgroundMuscle weakness is a common symptom in numerous diseases and a regularly occurring problem associated with ageing. Prolonged low-frequency force depression (PLFFD) is a form of exercise-induced skeletal muscle weakness observed after exercise. Three different intramuscular mechanisms underlying PLFFD have been identified: decreased sarcoplasmic reticulum Ca2+ release, decreased myofibrillar Ca2+ sensitivity, and myofibrillar dysfunction. We here used these three forms of PLFFD as models to study the effectiveness of a fast skeletal muscle troponin activator, CK-2066260, to mitigate muscle weakness.MethodsExperiments were performed on intact single muscle fibres or fibre bundles from mouse flexor digitorum brevis, which were stimulated with electrical current pulses, while force and the free cytosolic [Ca2+ ] ([Ca2+ ]i ) were measured. PLFFD was induced by three different stimulation protocols: (i) repeated isometric contractions at low intensity (350 ms tetani given every 5 s for 100 contractions); (ii) repeated isometric contractions at high intensity (250 ms tetani given every 0.5 s for 300 contractions); and (iii) repeated eccentric contractions (350 ms tetani with 20% length increase given every 20 s for 10 contractions). The extent and cause of PLFFD were assessed by comparing the force-[Ca2+ ]i relationship at low (30 Hz) and high (120 Hz) stimulation frequencies before (control) and 30 min after induction of PLFFD, and after an additional 5 min of rest in the presence of CK-2066260 (10 μM).ResultsProlonged low-frequency force depression following low-intensity and high-intensity fatiguing contractions was predominantly due to decreased sarcoplasmic reticulum Ca2+ release and decreased myofibrillar Ca2+ sensitivity, respectively. CK-2066260 exposure resulted in marked increases in 30 Hz force from 52 ± 16% to 151 ± 13% and from 6 ± 4% to 98 ± 40% of controls with low-intensity and high-intensity contractions, respectively. Following repeated eccentric contractions, PLFFD was mainly due to myofibrillar dysfunction, and it was not fully reversed by CK-2066260 with 30 Hz force increasing from 48 ± 8% to 76 ± 6% of the control.ConclusionsThe fast skeletal muscle troponin activator CK-2066260 effectively mitigates muscle weakness, especially when it is caused by impaired activation of the myofibrillar contractile machinery due to either decreased sarcoplasmic reticulum Ca2+ release or reduced myofibrillar Ca2+ sensitivity.

Project description:The contractile properties of fast-twitch and slow-twitch skeletal muscles are primarily determined by the myosin isoform content and modulated by a variety of sarcomere proteins. X-ray diffraction studies of regulatory mechanisms in muscle contraction have focused predominately on fast- or mixed-fibre muscle with slow muscle being much less studied. Here, we used time-resolved X-ray diffraction to investigate the dynamic behaviour of the myofilament proteins in relatively pure slow-twitch-fibre rat soleus (SOL) and pure fast-twitch-fibre rat extensor digitorum longus (EDL) muscle during twitch and tetanic contractions at optimal length. During twitch contractions the diffraction signatures indicating a transition in the myosin heads from ordered OFF states, where heads are held close to the thick filament backbone, to disordered ON states, where heads are free to bind to thin filaments, were found in EDL and not in SOL muscle. During tetanic contraction, changes in the disposition of myosin heads as active tension develops is a quasi-stepwise process in EDL muscle whereas in SOL muscle this relationship appears to be linear. The observed reduced extensibility of the thick filaments in SOL muscle as compared to EDL muscles indicates a molecular basis for this behaviour. These data indicate that for the EDL, thick filament activation is a cooperative strain-induced mechano-sensing mechanism, whereas for the SOL, thick filament activation has a more graded response. These different approaches to thick filament regulation in fast- and slow-twitch muscles may be adaptations for short-duration, strong contractions versus sustained, finely controlled contractions, respectively. KEY POINTS: Fast-twitch muscle and slow-twitch muscle are optimized for strong, short-duration contractions and for tonic postural activity, respectively. Structural events (OFF to ON transitions) in the myosin-containing thick filaments in fast muscle help determine the timing and strength of contractions, but these have not been studied in slow-twitch muscle. The X-ray diffraction signatures of structural OFF to ON transitions are different in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle, being completely absent during twitches in soleus muscle and blunted during tetanic contractions SOL as compared to EDL Quasi-stepwise thick filament structural OFF to ON transitions in fast twitch muscle may be an adaptation for rapid, ballistic movements, whereas more graded OFF to ON structural transitions in slow-twitch muscle may be an adaptation for slower, finer motions.

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Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases.

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Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases.

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