Skeletal muscle contraction occurs following motor neuron firing and transmission of this impulse across the neuromuscular junction (NMJ). The ensuing action potential triggers the release of calcium from the sarcoplasmic reticulum (SR). As the frequency of nerve firing increases, the amount of calcium released from the SR increases, causing the force of muscle contraction to ramp proportionally from a twitch up to a maximal tetanic contraction2
. The relationship of the frequency of motor neuron firing to muscle force is termed rate coding
and is one of the means by which muscle strength is controlled. Under normal conditions, skeletal muscle operates in a range of forces between 10% and 65% of maximum4
In many diseases, muscle weakness is the result of limited neuromuscular input and can lead to significant disability and increased mortality. Peripheral motor neuropathies such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and Charcot-Marie-Tooth disease (CMT) cause motor neuron damage and death, straining the ability of surviving motor neurons to stimulate muscle effectively to generate force5-8
. In myasthenia gravis (MG), weakness and fatigue result from failure of signal transmission at the NMJ which limits calcium release and force production9
; treatment consists of acetylcholinesterase inhibitors and immunosuppression although weakness and fatigue are still common in these patients10
. Therapeutic options for other neuropathies are limited or non-existent.
We hypothesized that amplifying the response of the sarcomere, the fundamental contractile unit in skeletal muscle, to inadequate motor neuron input would improve muscle force generation and physical function in patients with neuromuscular diseases. One means to do so is to increase the calcium sensitivity of the troponin-tropomyosin regulatory complex, the calcium sensor within the sarcomere that regulates the actin-myosin force-generating interaction. We identified a structural class of fast skeletal troponin activators following high-throughput screens of fast (Type II) and slow (Type I) skeletal myofibrils. This compound class was subsequently optimized for potency, physiochemical properties, and pharmacokinetics leading to the synthesis of CK-2017357 (molecular weight = 230.3, ).
CK-2017357 is a selective calcium sensitizer of the fast skeletal troponin complex
CK-2017357 selectively sensitized fast skeletal muscle to calcium by binding to its troponin complex. We prepared nearly pure populations of fast, slow, and cardiac myofibrils from well characterized sources11-14
: rabbit psoas (fast), bovine masseter (slow), and bovine cardiac muscle (muscle composition confirmed by myosin heavy chain analysis, Supplementary Fig. 1a
). Addition of CK-2017357 to fast skeletal myofibrils resulted in a leftward shift in the calcium-dependent myosin adenosine triphosphatase (ATPase) relationship () with the pCa producing half-maximal force (pCa50
) shifting from 5.61 ± 0.01 (control) to 6.52 ± 0.02 (CK-2017357, 5.0 μM). We characterized potency by measuring an increase in the ATPase rate at a fixed calcium concentration (pCa = 6.0) resulting in an EC50
of 390 ± 17 nM (). There was little or no effect in myofibrils from slow skeletal and cardiac muscle (), demonstrating its selectivity for fast skeletal muscle. Activation of myofibrils from bovine rectus abdominis muscle (EC50
= 770 ± 100 nM, Supplementary Fig. 1b
), a mixed fiber type muscle that contains in part fast skeletal muscle, confirmed its selectivity profile in muscle derived from the same species.
We leveraged this selectivity to determine the target of CK-2017357 using heterologous reconstituted versions of troponin-tropomyosin–regulated actin-myosin15
. CK-2017357 activated only those reconstitutions containing the fast skeletal troponin complex (). Isothermal titration calorimetry further confirmed a direct interaction of CK-2017357 with fast skeletal troponin (Kd
= 40 ± 6 nM). Addition of CK-2017357 to purified fast skeletal troponin complex resulted in an exothermic reaction that fit well to a single site binding model. Consistent with its myofibril selectivity, there was modest affinity for slow skeletal troponin (Kd
= 3800 ± 700 nM) and none measureable for cardiac troponin, (). This selectivity is not surprising given the substantially different amino acid composition (~50%) of the troponin complex in each muscle type.
CK-2017357 binds to the skeletal troponin complex and slows calcium release
The troponin complex contains three subunits, troponin C, I, and T. Troponin C is a calcium sensor with four binding sites, two high affinity (Kd
~50 nM) and two low affinity (Kd
~5 μM) 16
. Following calcium release from the SR, binding to the low affinity sites results in tropomyosin movement and allows the actin-myosin interaction to proceed17
. As calcium is pumped back into the SR, calcium levels fall, the low affinity sites release calcium, and the muscle contraction ends. We measured calcium release from the low affinity sites of fast skeletal troponin using a fluorescent calcium indicator in a stopped flow apparatus. CK-2017357 (20 μM) slowed the rate of Ca2+
release from 14.7 s-1
[95% CI 14.5-14.8] to 4.0 s-1
[95% CI 3.9-4.1], consistent with an increase in the affinity of troponin for calcium (). Similar experiments with isolated fast skeletal troponin C failed to demonstrate either a change in calcium release rate () or a binding interaction (), suggesting the CK-2017357 binding site lies at an interface between two or more troponin subunits.
We sought to understand how an increase in troponin calcium affinity would translate to changes in muscle force. Chemically “skinned” human muscle fibers devoid of the plasma membrane and sarcoplasmic reticulum were prepared from muscle biopsies of the vastus lateralis (VL), a mixed fast (type IIa, IIx) and slow (type I) muscle. These fibers contract when exogenous calcium is added to the muscle fiber. As expected, treatment of fast fibers with CK-2017357 shifted the force-calcium relationship leftwards without increasing maximum force or the shape of the curve; in contrast, slow fibers were approximately ten fold less responsive to CK-2017357 (). Skinned skeletal muscle fibers from animal sources demonstrated similar selectivity and notably skinned cardiac muscle fibers obtained from rat heart were unresponsive to CK-2017357 (60 μM) (). These experiments confirmed the selectivity of CK-2107357 for fast skeletal muscle in an intact muscle system.
CK-2017357 shifts the force-calcium relationship in fast skeletal muscle leftwards and amplifies the response of muscle to nervous input
Given its effects on calcium sensitivity, CK-2017357 should result in an increase in the response of muscle to neuromuscular input. To test this hypothesis, we measured the response of living skeletal muscle to treatment with CK-2017357 using an in situ
preparation of the extensor digitorum longus (EDL) muscle in the rat, where nerve and blood supply are intact18
. In this fast fiber skeletal muscle, systemic infusion of CK-2017357 resulted in rapid, dose-dependent increases in muscle force (Supplementary Fig. 2
). Examination of the force-frequency relationship demonstrated a substantial increase in muscle force at sub-maximal stimulation rates without increasing maximum tension at maximal tetanic stimulation rates (). Tension in the absence of nerve stimulation did not change following treatment (data not shown). Thus, the change in the force-frequency relationship was consistent with the effect of CK-2017357 on the force-calcium relationship in skinned muscle fibers.
The leftward shift in the force-frequency relationship of fast skeletal muscle led us to test whether CK-2017357 could increase muscle force in a model of neuromuscular disease. Passive transfer experimental autoimmune myasthenia gravis (PT-EAMG) is a rat model of myasthenia gravis in which treatment with an inhibitory nicotinic acetylcholine receptor (nAChR) antibody leads to muscle weakness and fatigue19
. Antibody treatment led to a substantial decline in grip strength (~50%), reaching a plateau 72 hours post-injection () that remained stable to 96 hours. Underlying this weakness was lower muscle force measured in situ
across a wide range of nerve stimulation frequencies (). In this model, administration of CK-2017357 rapidly increased muscle force in situ
() and eliminated the decline in force produced by prolonged stimulation, so-called use dependent fatigue or sag ().
CK-2017357 improves muscle and physical function in a model of neuromuscular disease
We next tested whether these increases in the performance of individual, intact muscles could translate to enhanced physical performance in the same PT-EAMG model. Following randomization of the animals to two groups, CK-2017357 or vehicle was dosed orally in a blinded fashion at 72 hours followed by crossover to the opposite treatment at 96 hours with grip strength assessments at each time point. CK-2017357 treatment increased grip strength by up to 0.97 kg kg-1 body weight relative to placebo 60 minutes after dosing, representing more than a 50% increase in grip strength relative to baseline (). Of note, control animals (no nAChR injection) did not show a change in grip strength with CK-2017357 () suggesting that increases in function in this setting required weakness produced by neuromuscular blockade.
CK-2017357 is a selective sensitizer of the fast skeletal troponin complex and a direct activator of skeletal muscle function. CK-2017357 may represent a therapeutic option for a range of serious neuromuscular disorders, by increasing muscle strength and reducing fatigability. Evidence also points to reduced efficiency of motorneuron excitation-contraction coupling in the sarcopenia of old age20-22
, suggesting potentially broader applicability of this mechanism of action. Direct activators of the skeletal sarcomere, like the fast skeletal troponin activator, CK-2017357, may therefore hold promise in an array of patient conditions marked by muscle weakness.