Modulation of energy expenditure independent of contraction: a novel paradigm in muscle

Skeletal muscle is known as a major site of energy expenditure mainly due to its intense contraction and relaxation in daily life. Recent studies have identified some critical molecular mechanisms involved in skeletal muscle function and maintenance of energy expenditure. This has ignited research into strategies to target these mechanisms to modulate energy expenditure and improve functionality of skeletal muscle. While it is proposed that increasing energy expenditure can counter metabolic diseases like obesity and type 2 diabetes, lowering energy expenditure may ameliorate disorders of muscle impairment including muscular dystrophy and myasthenia gravis; skeletal muscle fatigue is one of the major causes of reduced mobility and exercise intolerance in the older population. Inability of the muscle to supply the required ATP in older people leads to fatigue, and this can be improved if ATP demand can be lowered. Cellular Ca2+ fluxes during activation and relaxation of skeletal muscle are mediated by the ryanodine receptor (RyR) and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), respectively. SERCA is the only transporter of Ca2+ from the cytosol to the sarcoplasmic reticulum (SR), and SERCA Ca2+ uptake is essential for the replenishment of SR Ca2+ stores and in mediating muscle relaxation. SERCA, however, is one of the major consumers of energy in muscle. The SERCA-based ATP utilization load can be modulated by pharmacological agents to manipulate muscle energy expenditure as well as myofibrillar contractility. In a study recently published in The Journal of Physiology, Cheng et al. (2019) have tried to unravel the molecular basis of the action of a small molecule, CK-2066260, a fast skeletal troponin activator that is known to improve contractile force kinetics in weak or fatigued muscles. Cheng et al. demonstrated that CK-2066260 restores contractility to muscle fatigued with mid-frequency stimulation. It also increases submaximal contraction. They showed that CK-2066260 alleviates skeletal muscle fatigue by increasing the Ca2+ sensitivity of the Ca2+–troponin complex in contractile fibres (Hwee et al. 2017). In order to examine the role of CK-2066260 in the Ca2+ dynamics of electro-mechanical coupling, they administered it or caffeine in isolated flexor digitorum brevis fibres. Caffeine (a RyR activator) is a muscle stimulant which potentiates [Ca2+] release from the SR thereby increasing availability of Ca2+ for troponin binding. The effect of caffeine in fatigued muscle is to produce a transient increase in ‘peak force’ and [Ca2+]i followed by a rapid decrease in both, whereas CK-2066260 produces an increased submaximal and sustained force response in repetitive stimulations without significant reduction in [Ca2+]i. In the interpretation of the authors, caffeine-induced SR Ca2+ release causes increased SERCA [Ca2+]i uptake leading to enhanced ATP utilization in the fibre, whereas with CK-2066260 less Ca2+ is needed for the same submaximal force production and therefore less Ca2+ has to be pumped back by SERCA, relieving the ATP requirement. However, when high-frequency stimulation (120 Hz) was applied, the disparity of force response was not the same for caffeine- and CK-2066260-treated fibres upon reaching fatigue. Cheng et al. tested whether CK-2066260 improved fatigue resistance vs. vehicle. In pre-fatigued muscle CK-2066260 slowed the decline in force and maintained tetanic [Ca2+]i levels during repetitive contractions vs. vehicle-mitigated fatigue. As myosin-ATPase and SERCA are two major ATP utilizing sites, they analysed ATP consumption by detecting cytosolic [Mg2+]i levels with the indicator mag-indo-1, which inversely correlates with [ATP] levels. Lower frequency repetitive stimulation revealed a lower ATP consumption in CK-2066260-treated fibres than in control. This shows that in drug-facilitated contraction, the contribution of SERCA-mediated ATP hydrolysis is lowered. To evaluate the substrate utilization rate, quantification of glycogen content by periodic acid–Schiff staining was performed in medial gastrocnemius muscle. At equivalent force, treatment with CK-2066260 gave higher glycogen levels vs. treatment with vehicle. Whole body in vivo muscle performance of rats treated with CK-2066260 showed twice the enhancement in a rota rod and treadmill exercise experiment than in rats treated with vehicle. CK-2066260 administration in rats with experimental femoral artery ligation (FAL), which mimics peripheral arterial insufficiency (PAI), also improved muscle performance and increased tension. This implies that CK-2066260 has the dual benefit of increasing muscle performance while reducing energy expenditure. Muscle fatigue is a major impediment in prolonged repetitive muscle contraction and causes exercise intolerance. Various molecular mechanisms have been proposed in muscle fatigue, such as acidosis, high Pi levels and limiting ATP concentration around the contractile machinery (Westerblad et al. 2002). To counter muscle fatigue, many pharmacological strategies have been designed to target the myofibrils. CK-2066260, a structural analogue of tirasemtiv, is a troponin-binding Ca2+ sensitizer that acts specifically at fast skeletal myofibrils. The dynamics of muscle contraction and relaxation in skeletal muscle are intricately associated with the flow of myoplasmic and SR Ca2+. In contrast to caffeine treatment, which leads to a peak transient force response due to increased SR Ca2+ release, CK-2066260 (a myofibrillar Ca2+ sensitizer) showed a sustained force response. This implies that CK-2066260 maintains a lower but sustained flow of RyR Ca2+ release and SERCA re-uptake. The Ca2+ sensitization action of CK-2066260 on muscle fibres has an effective range at mid frequency stimulation, since high frequency (∼120 Hz) stimulation gives high [Ca2+]i which saturates the Ca2+–troponin complex nullifying the drug's effect in the muscle. This implies that the action of CK-2066260 is dependent on RYR Ca2+ release. Role of Ca2+ myofibrillar sensitizers is still poorly defined during muscle fatigue. We hypothesize that CK-2066260 may also act via 3 contiguous ways to mitigate fatigue. CK-2066260 might counter low pH induced acidosis by enhancing the affinity of troponin for Ca2+ over other ions like H+ (arisen due to fatigue). Increased troponin Ca2+ sensitivity decreases rate of myofibrillar Ca2+ dispersion and SR Ca2+-uptake. Lesser metabolic requirement (ATP demand) on SERCA uptake thus lowers energy utilization. Lowered ATP breakdown reduces cellular Pi accumulation during repetitive contractions which can be a factor in lessening fatigue. Altogether, it indicates CK-2066260 increases contraction/ATP consumption ratio thereby providing fatigue resistance. In vivo administration of CK-2066260 has a positive ionotropic effect on skeletal muscle function. CK-2066260 lowers energy expenditure and improves muscle performance, which explains the increased time to failure in rats under treadmill and rota rod exercise. PAI is a blood circulatory problem in which reduced flow causes muscle ischaemia. FAL models have been shown to have impaired muscle contractility in lower limbs due to low oxygen supply. CK-2066260's action on muscle increases treadmill performance in both acute and chronic FAL models. SERCA function is intricately regulated by transcriptional and post-transcriptional modifications in many disease states including muscle disorders (Sahu et al. 2019). In skeletal muscle, RyR Ca2+ release from the SR and cytosolic removal by SERCA have been considered to be highly coupled to force generation, i.e. actin–myosin crossbridge cycling. This study provides a novel paradigm in which these can be uncoupled raising the possibility that SERCA ATP utilization can be manipulated independent of the recruitment status of the contractile fibres. None declared. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. None. We thank Dr Naresh C. Bal for his helpful insights and critical discussion on the manuscript.