Fast skeletal muscle troponin activator CK-2066260 increases fatigue resistance by reducing the energetic cost of muscle contraction.

KEY POINTS: Skeletal muscle fatigue limits performance in various physical activities, with exercise intolerance being a key symptom in a broad spectrum of diseases. We investigated whether a small molecule fast skeletal troponin activator (FSTA), CK-2066260, can mitigate muscle fatigue by reducing the cytosolic free [Ca2+ ] required to produce a given submaximal force and hence decreasing the energy requirement. Isolated intact single mouse muscle fibres and rat muscles in-situ treated with CK-2066260 showed improved muscle endurance., which was accompanied by decreased ATP demand and reduced glycogen usage. CK-2066260 treatment improved in-vivo exercise capacity in healthy rats and in a rat model of peripheral artery insufficiency. In conclusion, we show that the FSTA CK-2066260 effectively counteracts muscle fatigue in rodent skeletal muscle in vitro, in situ, and in vivo. This may translate to humans and provide a promising pharmacological treatment to patients suffering from severe muscle weakness and exercise intolerance. ABSTRACT: Skeletal muscle fatigue limits performance during physical exercise and exacerbated muscle fatigue is a prominent symptom among a broad spectrum of diseases. The present study investigated whether skeletal muscle fatigue is affected by the fast skeletal muscle troponin activator (FSTA) CK-2066260, which increases myofibrillar Ca2+ sensitivity and amplifies the submaximal force response. Because more force is produced for a given Ca2+ , we hypothesized that CK-2066260 could mitigate muscle fatigue by reducing the energetic cost of muscle activation. Isolated single mouse muscle fibres were fatigued by 100 repeated 350 ms contractions while measuring force and the cytosolic free [Ca2+ ] or [Mg2+ ] ([Mg2+ ]i ). When starting fatiguing stimulation at matching forces (i.e. lower stimulation frequency with CK-2066260): force was decreased by ∼50% with and by ∼75% without CK-2066260; [Mg2+ ]i was increased by ∼10% with and ∼32% without CK-2066260, reflecting a larger decrease in [ATP] in the latter. The glycogen content in in situ stimulated rat muscles fatigued by repeated contractions at matching forces was about two times higher with than without CK-2066260. Voluntary exercise capacity, assessed by rats performing rotarod exercise and treadmill running, was improved in the presence of CK-2066260. CK-2066260 treatment also increased skeletal muscle fatigue resistance and exercise performance in a rat model of peripheral artery insufficiency. In conclusion, we demonstrate that the FSTA CK-2066260 mitigates skeletal muscle fatigue by reducing the metabolic cost of force generation.

DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers.

A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow or fast fibers were hypomethylated, which was supported by the RNA-sequencing analysis. These are the first fiber type-specific methylation data from human skeletal muscle and provide a unique platform for future research.NEW & NOTEWORTHY This study validates a low-DNA input reduced representation bisulfite sequencing method for human muscle biopsy samples to investigate the methylation patterns at a fiber type-specific level. These are the first fiber type-specific methylation data reported from human skeletal muscle and thus provide initial insight into basal state differences in myosin heavy chain I and IIa muscle fibers among young, healthy men.

The ACTN3 R577X genotype is associated with muscle function in a Japanese population.

Homozygosity for the common nonsense polymorphism R577X in the α-actinin-3 gene (ACTN3) causes complete α-actinin-3 deficiency in fast-twitch skeletal muscle fibers. This study investigated whether the ACTN3 R577X polymorphism affects fitness status using a battery of tests in a large Japanese cohort. In the present study, 1227 subjects (age: 25-85 years) were genotyped for the ACTN3 R577X polymorphism (rs1815739) using a TaqMan SNP genotyping assay (Applied Biosystems). All subjects were divided into 2 groups based on their age (<55 years and ≥55 years). All subjects completed a questionnaire about exercise habits and were subjected to a battery of tests to assess their fitness status (including grip strength test, chair stand test, and 8-foot walking test). A significant association between the ACTN3 R577X genotype and chair stand test performance was observed in the group of men ≥55 using ANCOVA adjusted for age and exercise habits (p = 0.036). The ACTN3 R577X genotype accounted for 2.5% of the variability in the results of the chair stand test among men in the ≥55 age group. Moreover, for the ≥55 age group, performance in the chair stand test was lower among those with the XX genotype than among those with the RR genotype (p = 0.024) or RX genotype (p = 0.005), unlike results for the <55 age group. No significant difference was noted for hand grip strength or 8-foot walking time. Thus, our results suggest that the ACTN3 R577X genotype is associated with lower-extremity muscle function in the Japanese population.

[Muscle activity and energy expenditure]. / Activité musculaire et dépense d'énergie.

Most of increases in energy metabolism are induced by exercise. They are related with power and efficiency. In cycle ergometer exercise efficiency is positively correlated with exercise power and negatively correlated with pedaling rate. Ramp exercises are associated with an apparent increase in efficiency. Movements of daily life activity are too complex to make evaluation of power or efficiency possible. Energy expenditure assessment is based on direct measurement of the energy metabolism increase. The energy cost of movement or economy is calculated. Daily activity recording provides an assessment of the energy metabolism ability of patients. Muscle contractile activity is linked with ATP splitting. The pathways to resynthesize ATP include anaerobic glycogenolysis and the aerobic breakdown of substrates. Type I fibres have a higher oxidative capacity than type II fibres. Type II fibres demonstrate a higher glycolytic capacity, contract faster, and are more fatigable. Information relative to energy expenditure during daily life activity allows clinicians to better assess the clinical implications of the stress tests results.

Increased resistance to fatigue in creatine kinase deficient muscle is not due to improved contractile economy.

There has been speculation on the origin of the increased endurance of skeletal muscles in creatine kinase (CK)-deficient mice. Important factors that have been raised include the documented increased mitochondrial capacity and alterations in myosin heavy chain (MyHC) isoform composition in CK-deficient muscle. More recently, the absence of inorganic phosphate release from phosphocreatine hydrolysis in exercising CK-deficient muscle has been postulated to contribute to the lower fatigueability in skeletal muscle. In this study, we tested the hypothesis that the reported shift in MyHC composition to slower isoforms in CK-deficient muscle leads to a decrease in oxygen cost of twitch performance. To that aim, extensor digitorum longus (EDL) and soleus (SOL) muscles were isolated from wild-type (WT) and knock-out mice deficient in the cytoplasmic muscle-type and sarcomeric mitochondrial isoenzymes of CK, and oxygen consumption per twitch time-tension-integral (TTI) was measured. The results show that the adaptive response to loss of CK function does not involve any major change to contractile economy of skeletal muscle.

Thermogenesis and energy expenditure: control of heat production by the Ca(2+)-ATPase of fast and slow muscle.

This report measured the amount of heat released during Ca(2+) transport and ATP hydrolysis by vesicles derived from the sarcoplasmic reticulum of rabbit slow (SM) and fast (FM) muscle. During ATP hydrolysis, part of the chemical energy released is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles), the heat produced per mol of ATP cleaved by SM and FM vesicles was the same and varied between 7.7-9.1 kcal. In the presence of the gradient the heat produced by SM and FM vesicles differed, 13.4 kcal/mol and 23.0 kcal/mol ATP cleaved, respectively. After formation of the gradient, part of the ATPase activity was not coupled to Ca(2+) transport. The difference of heat produced by FM and SM vesicles during ATP hydrolysis was related to the rate of uncoupled ATPase activity. When extended to the living cell, the data described indicate that the amount of heat produced by the Ca(2+)-ATPase of SM muscle is 36 times smaller than that produced by the FM. Thyroid hormone 3,5,3'-triiodo L-thyronine (T3) regulate both thermogenesis and the transcription of the sarcoplasmic reticulum Ca(2+)-ATPase isoforms found in SM and FM. The findings described in this report raise the possibility that one of the mechanisms of thermogenesis control may be related to the regulation of Ca(2+)-ATPase isoforms expression by T3.

Post-tetanic potentiation increases energy cost to a higher extent than work in rat fast skeletal muscle.

We studied the effects of (post-tetanic) potentiation on myosin light chain (MLC-2) phosphorylation, work and energy cost in skeletal muscle. Experiments were performed using in situ medial gastrocnemius muscles of male Wistar rats, which were electrically stimulated through the severed sciatic nerve. One group of muscles was first potentiated with an isometric tetanus before a series of 10 concentric contractions (PRC). A second group performed the same series of contractions without previous potentiation (RC). Following the last contraction the muscles were rapidly frozen and excised after which the high-energy phosphate content, lactate concentration and the level of MLC-2 phosphorylation were measured. The results indicate that PRC muscles had a higher (P < 0.05) total work output 144.5 +/- 17.0 (SD) (n = 6) vs. 121.6 +/- 11.4 (SD) (n = 6) mJ and level of MLC-2 phosphorylation (49.2 +/- 7.3 vs. 40.8 +/- 3.6%) than RC muscles. The energy cost of the series of concentric contractions in the PRC muscles (9.8 +/- 1.9 micromol approximately P/muscle) was significantly higher (P < 0.05) than the energy cost in the RC muscles (6.2 +/- 0.97 micromol approximately P/muscle). It was shown that the relative increase in energy cost of PRC muscles was higher (P < 0.05) than in total work output. It is proposed that the relative high increase in energy cost is the direct result of the increase in muscle performance rather than a property of potentiation.

Acidosis has no effect on the ATP cost of contraction in cat fast- and slow-twitch skeletal muscles.

Studies of skinned fibers suggest that the rate of ATP turnover in skeletal muscle is depressed by acidosis. To examine whether this occurs in intact muscles, the ATP cost of isometric contractions was measured in ex vivo, arterially perfused cat biceps (predominantly fast-twitch) and soleus (slow-twitch) muscles under normocapnic (5% CO2) and hypercapnic (70% CO2) conditions. Hypercapnia decreased extracellular pH from 7.4 to 6.7 and intracellular pH from 7.1 to 6.5 (soleus) or 6.6 (biceps) but had no significant effect on the phosphocreatine (PCr)-to-ATP ratio in muscles at rest. The ATP cost of contraction was estimated from PCr changes, measured by gating the acquisition of 31P-nuclear magnetic resonance spectra to times before and after brief tetani (1 s at 100 Hz and 2 s at 25 Hz for biceps and soleus, respectively) or 10-s trains of twitches (2 and 1 Hz, respectively). Peak isometric force and the ATP cost of tetanic contraction (PCr/force x time integral) were not significantly different under hypercapnic compared with normocapnic conditions in either muscle (mean: 7.97 and 2.44 micromol x kg(-1) x s(-1) for biceps and soleus, respectively). Twitch force and the ATP cost per twitch decreased by nearly 50% during hypercapnic perfusion in both muscle types. The results indicate that hypercapnic acidosis has no significant effect on the ATPase rate per active myosin head in intact mammalian skeletal muscle.