Mood, Cognitive Function, and Plasma Kynurenine Metabolites Responses Following Severe Changes in Physical Activity.

PURPOSE: To monitor changes in mood, cognitive function, brain electrical activity, and circulating kynurenine pathway metabolites in response to a three-week severe physical activity restriction, followed by three weeks of resumed activity adding resistance and high-intensity interval exercise training. METHODS: Twenty healthy participants (14 males, six females; 25.4 ± 5.2 years) underwent three weeks of limited physical activity using forearm crutches with one leg suspended (INACT) and then three weeks of resumed activity plus supervised resistance and high-intensity interval training sessions (ACT, three to six sessions per week). At baseline, after INACT, and then after ACT, venous blood was sampled for analysis of major kynurenine pathway metabolites, a short version of the International Physical Activity Questionnaire (IPAQ-SF), Hospital Anxiety and Depression Scale (HADS) and Profile of Mood States (POMS) questionnaires were completed, and cognitive tests with EEG were performed. RESULTS: During INACT, the depression score on the HADS scale tended to increase (3.5 to 6.8; p = 0.065), while it was reduced with ACT compared with after INACT (2.8; p = 0.022). On the POMS scale, depression, fatigue, and confusion increased within INACT (p < 0.05). Notably, subjects exhibited considerable variability, and those experiencing depression symptoms recorded by the HADS scale (n = 4) displayed distinct mood disturbances on POMS. All HADS and POMS scores were fully restored to baseline with ACT. Neither INACT nor ACT induced significant changes in cognition, brain electrical activity, or kynurenine pathway metabolites (p > 0.05). CONCLUSIONS: While young healthy individuals with three weeks of severely restricted physical activity do not undergo changes in circulating kynurenine pathway metabolites, cognitive performance, and brain electrical activity, their mood response is quite variable, and depression develops in some. Three weeks of resuming mobility plus exercise training reversed the mood profile.

Ca2+ and force during dynamic contractions in mouse intact skeletal muscle fibers.

Muscle fiber force production is determined by the excitation frequency of motor nerves, which induce transient increases in cytoplasmic free Ca2+ concentration ([Ca2+]i) and the force-generating capacity of the actomyosin cross-bridges. Previous studies suggest that, in addition to altered cross-bridge properties, force changes during dynamic (concentric or eccentric) contraction might be affected by Ca2+-dependent components. Here we investigated this by measuring [Ca2+]i and force in mouse muscle fibers undergoing isometric, concentric, and eccentric contractions. Intact single muscle fibers were dissected from the flexor digitorum brevis muscle of mice. Fibers were electrically activated isometrically at 30-100 Hz and after reaching the isometric force plateau, they were actively shortened or stretched. We calculated the ratio (relative changes) in force and [Ca2+]i attained in submaximal (30 Hz) and near-maximal (100 Hz) contractions under isometric or dynamic conditions. Tetanic [Ca2+]i was similar during isometric, concentric and eccentric phases of contraction at given stimulation frequencies while the forces were clearly different depending on the contraction types. The 30/100 Hz force ratio was significantly lower in the concentric (44.1 ± 20.3%) than in the isometric (50.3 ± 20.4%) condition (p = 0.005), whereas this ratio did not differ between eccentric and isometric conditions (p = 0.186). We conclude that the larger force decrease by decreasing the stimulation frequency during concentric than during isometric contraction is caused by decreased myofibrillar Ca2+ sensitivity, not by the decreased [Ca2+]i.

Zfp697 is an RNA-binding protein that regulates skeletal muscle inflammation and regeneration.

Muscular atrophy is a mortality risk factor that happens with disuse, chronic disease, and aging. Recovery from atrophy requires changes in several cell types including muscle fibers, and satellite and immune cells. Here we show that Zfp697/ZNF697 is a damage-induced regulator of muscle regeneration, during which its expression is transiently elevated. Conversely, sustained Zfp697 expression in mouse muscle leads to a gene expression signature of chemokine secretion, immune cell recruitment, and extracellular matrix remodeling. Myofiber-specific Zfp697 ablation hinders the inflammatory and regenerative response to muscle injury, compromising functional recovery. We uncover Zfp697 as an essential interferon gamma mediator in muscle cells, interacting primarily with ncRNAs such as the pro-regenerative miR-206. In sum, we identify Zfp697 as an integrator of cell-cell communication necessary for tissue regeneration.

Calcium measurements in enzymatically dissociated or mechanically microdissected mouse primary skeletal muscle fibers.

Here, we provide a protocol for isolation of mouse primary skeletal muscle fibers using two alternative approaches-enzymatic dissociation or mechanical microdissection. We describe the procedures for surgical removal of muscle of interest and isolation of intact single-muscle fibers by either collagenase digestion or mechanical microdissection. We then detail intracellular calcium measurements by microinjecting or loading the isolated muscle fibers with membrane permeable calcium dyes. Finally, we outline steps for intracellular calcium quantification by fluorescent measurement. For complete details on the use and execution of this protocol, please refer to Gineste et al.1.

Increased tetanic calcium in early fatigue of mammalian muscle fibers is accompanied by accelerated force development despite a decreased force.

During the initial phase of fatigue induced by repeated contractions in fast-twitch muscle fibers, tetanic force decreases despite increasing tetanic free cytosolic [Ca2+ ] ([Ca2+ ]cyt ). Here, we hypothesized that the increase in tetanic [Ca2+ ]cyt nevertheless has positive effects on force in early fatigue. Experiments on enzymatically isolated mouse flexor digitorum brevis (FDB) fibers showed that an increase in tetanic [Ca2+ ]cyt during ten 350 ms contractions required trains of electrical pulses to be elicited at short intervals (≤2 s) and at high frequencies (≥70 Hz). Mechanically dissected mouse FDB fibers showed greater decrease in tetanic force when the stimulation frequency during contractions was gradually reduced to prevent the increase in tetanic [Ca2+ ]cyt . Novel analyses of data from previous studies revealed an increased rate of force development in the tenth fatiguing contraction in mouse FDB fibers, as well as in rat FDB and human intercostal fibers. Mouse FDB fibers deficient in creatine kinase showed no increase in tetanic [Ca2+ ]cyt and slowed force development in the tenth contraction; after injection of creatine kinase to enable phosphocreatine breakdown, these fibers showed an increase in tetanic [Ca2+ ]cyt and accelerated force development. Mouse FDB fibers exposed to ten short contractions (43 ms) produced at short intervals (142 ms) showed increased tetanic [Ca2+ ]cyt accompanied by a marked (~16%) increase in the developed force. In conclusion, the increase in tetanic [Ca2+ ]cyt in early fatigue is accompanied by accelerated force development, which under some circumstances can counteract the decline in physical performance caused by the concomitant decrease in maximum force.

Enzymatically dissociated muscle fibers display rapid dedifferentiation and impaired mitochondrial calcium control.

Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.

Citrullination is linked to reduced Ca2+ sensitivity in hearts of a murine model of rheumatoid arthritis.

AIMS: Cardiac contractile dysfunction is prevalent in rheumatoid arthritis (RA), with an increased risk for heart failure. A hallmark of RA has increased levels of peptidyl arginine deaminases (PAD) that convert arginine to citrulline leading to ubiquitous citrullination, including in the heart. We aimed to investigate whether PAD-dependent citrullination in the heart was linked to contractile function in a mouse model of RA during the acute inflammatory phase. METHODS: We used hearts from the collagen-induced arthritis (CIA) mice, with overt arthritis, and control mice to analyze cardiomyocyte Ca2+ handling and fractional shortening, the force-Ca2+ relationship in isolated myofibrils, the levels of PAD, protein post-translational modifications, and Ca2+ handling protein. Then, we used an in vitro model to investigate the role of TNF-α in the PAD-mediated citrullination of proteins in cardiomyocytes. RESULTS: Cardiomyocytes from CIA mice displayed larger Ca2+ transients than controls, whereas cell shortening was similar in the two groups. Myofibrils from CIA hearts required higher [Ca2+ ] to reach 50% of maximum shortening, ie Ca2+ sensitivity was lower. This was associated with increased PAD2 expression and α-actin citrullination. TNF-α increased PAD-mediated citrullination which was blocked by pre-treatment with the PAD inhibitor 2-chloroacetamide. CONCLUSION: Using a mouse RA model we found evidence of impaired cardiac contractile function linked to reduced Ca2+ sensitivity, increased expression of PAD2, and citrullination of α-actin, which was triggered by TNF-α. This provides molecular and physiological evidence for acquired cardiomyopathy and a potential mechanism for RA-associated heart failure.

Sphingomyelinase activity promotes atrophy and attenuates force in human muscle fibres and is elevated in heart failure patients.

BACKGROUND: Activation of sphingomyelinase (SMase) as a result of a general inflammatory response has been implicated as a mechanism underlying disease-related loss of skeletal muscle mass and function in several clinical conditions including heart failure. Here, for the first time, we characterize the effects of SMase activity on human muscle fibre contractile function and assess skeletal muscle SMase activity in heart failure patients. METHODS: The effects of SMase on force production and intracellular Ca2+ handling were investigated in single intact human muscle fibres. Additional mechanistic studies were performed in single mouse toe muscle fibres. RNA sequencing was performed in human muscle bundles exposed to SMase. Intramuscular SMase activity was measured from heart failure patients (n = 61, age 69 ± 0.8 years, NYHA III-IV, ejection fraction 25 ± 1.0%, peak VO2 14.4 ± 0.6 mL × kg × min) and healthy age-matched control subjects (n = 10, age 71 ± 2.2 years, ejection fraction 60 ± 1.2%, peak VO2 25.8 ± 1.1 mL × kg × min). SMase activity was related to circulatory factors known to be associated with progression and disease severity in heart failure. RESULTS: Sphingomyelinase reduced muscle fibre force production (-30%, P < 0.05) by impairing sarcoplasmic reticulum (SR) Ca2+ release (P < 0.05) and reducing myofibrillar Ca2+ sensitivity. In human muscle bundles exposed to SMase, RNA sequencing analysis revealed 180 and 291 genes as up-regulated and down-regulated, respectively, at a FDR of 1%. Gene-set enrichment analysis identified 'proteasome degradation' as an up-regulated pathway (average fold-change 1.1, P = 0.008), while the pathway 'cytoplasmic ribosomal proteins' (average fold-change 0.8, P < 0.0001) and factors involving proliferation of muscle cells (average fold-change 0.8, P = 0.0002) where identified as down-regulated. Intramuscular SMase activity was ~20% higher (P < 0.05) in human heart failure patients than in age-matched healthy controls and was positively correlated with markers of disease severity and progression, and with several circulating inflammatory proteins, including TNF-receptor 1 and 2. In a longitudinal cohort of heart failure patients (n = 6, mean follow-up time 2.5 ± 0.2 years), SMase activity was demonstrated to increase by 30% (P < 0.05) with duration of disease. CONCLUSIONS: The present findings implicate activation of skeletal muscle SMase as a mechanism underlying human heart failure-related loss of muscle mass and function. Moreover, our findings strengthen the idea that SMase activation may underpin disease-related loss of muscle mass and function in other clinical conditions, acting as a common patophysiological mechanism for the myopathy often reported in diseases associated with a systemic inflammatory response.

Improved skeletal muscle fatigue resistance in experimental autoimmune myositis mice following high-intensity interval training.

BACKGROUND: Muscle weakness and decreased fatigue resistance are key manifestations of systemic autoimmune myopathies (SAMs). We here examined whether high-intensity interval training (HIIT) improves fatigue resistance in the skeletal muscle of experimental autoimmune myositis (EAM) mice, a widely used animal model for SAM. METHODS: Female BALB/c mice were randomly assigned to control (CNT) or EAM groups (n = 28 in each group). EAM was induced by immunization with three injections of myosin emulsified in complete Freund's adjuvant. The plantar flexor (PF) muscles of mice with EAM were exposed to either an acute bout or 4 weeks of HIIT (a total of 14 sessions). RESULTS: The fatigue resistance of PF muscles was lower in the EAM than in the CNT group (P < 0.05). These changes were associated with decreased activities of citrate synthase and cytochrome c oxidase and increased expression levels of the endoplasmic reticulum stress proteins (glucose-regulated protein 78 and 94, and PKR-like ER kinase) (P < 0.05). HIIT restored all these alterations and increased the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and the mitochondrial electron transport chain complexes (I, III, and IV) in the muscles of EAM mice (P < 0.05). CONCLUSIONS: HIIT improves fatigue resistance in a SAM mouse model, and this can be explained by the restoration of mitochondria oxidative capacity via inhibition of the ER stress pathway and PGC-1α-mediated mitochondrial biogenesis.

Increasing the resting time between drop jumps lessens delayed-onset muscle soreness and limits the extent of prolonged low-frequency force depression in human knee extensor muscles.

PURPOSE: Unaccustomed eccentric contractions generally result in a long-lasting contractile impairment, referred to as prolonged low-frequency force depression (PLFFD), and delayed-onset muscle soreness (DOMS). We here used repeated drop jumps (DJs) as an eccentric contraction model and studied the effects of increasing the time between DJs from 20 s to 5 min. We hypothesized that both PLFFD and DOMS would be less marked at the longer DJ interval due to the longer time to restore structural elements between DJs. METHODS: Young men (n = 12) randomly performed 50 DJs with either 20-s (DJ-20 s) or 5-min (DJ-5 min) rest between DJs. Voluntary, 20 Hz and 100 Hz electrically stimulated isometric knee extension torques and muscle soreness were monitored before and for 7 days after DJs; serum CK activity was measured to assess muscle fibre protein leakage. In additional experiments, changes in mRNA levels were assessed in muscle biopsies collected before and 1 h after exercise. RESULTS: A marked PLFFD was observed with both protocols and the extent of 20 Hz torque depression was smaller immediately and 1 day after DJ-5 min than after DJ-20 s (p < 0.05), whereas the MVC and 100 Hz torques were similarly decreased with the two protocols. Markedly larger differences between the two protocols were observed for the muscle soreness score, which 1-4 days after exercise was about two times larger with DJ-20 s than with DJ-5 min (p < 0.01). CONCLUSIONS: The larger protective effect of the longer DJ interval against DOMS than against PLFFD indicates that their underlying mechanisms involve different structural elements.

Adding High-Intensity Interval Training to Classical Resistance Training Does Not Impede the Recovery from Inactivity-Induced Leg Muscle Weakness.

Inactivity is known to induce muscle weakness, and chronically increased levels of reactive oxygen species (ROS) are proposed to have a central causative role in this process. Intriguingly, high-intensity interval training (HIIT), which involves bursts of high ROS production, can have positive effects in pathological conditions with chronically increased ROS. Here, young male volunteers were exposed to 3 weeks of unloading of the dominant leg followed by 3 weeks of resistance training without (Ctrl group) or with the addition of all-out cycling HIIT. Changes in muscle thickness were assessed by ultrasonography, and contractile function was studied by measuring the torque during maximal voluntary contractions (MVC). The results show an ~6% decrease in vastus lateralis thickness after the unloading period, which was fully restored after the subsequent training period in both the Ctrl and HIIT groups. MVC torque was decreased by ~11% after the unloading period and recovered fully during the subsequent training period in both groups. All-out cycling performance was improved by the 3 weeks of HIIT. In conclusion, the decline in muscle size and function after 3 weeks of unloading was restored by 3 weeks of resistance training regardless of whether it was combined with HIIT.

Larger improvements in fatigue resistance and mitochondrial function with high- than with low-intensity contractions during interval training of mouse skeletal muscle.

Interval training (IT) results in improved fatigue resistance in skeletal muscle mainly due to an increased aerobic capacity, which involves increased muscle mitochondrial content and/or improved mitochondrial function. We hypothesized that IT with high-intensity contractions is more effective in increasing mitochondrial function, and hence fatigue resistance, than low-intensity contractions. To study this hypothesis without interference from differences in muscle fiber recruitment obliged to occur during voluntary contractions, IT was performed with in situ supramaximal electrical stimulation where all muscle fibers are recruited. We compared the effect of IT with repeated low-intensity (20 Hz stimulation, IT20) and high-intensity (100 Hz stimulation, IT100) contractions on fatigue resistance and mitochondrial content and function in mouse plantar flexor muscles. Muscles were stimulated every other day for 4 weeks. The averaged peak torque during IT bouts was 4.2-fold higher with IT100 than with IT20. Both stimulation protocols markedly improved in situ fatigue resistance, although the improvement was larger with IT100. The citrate synthase activity, a biomarker of mitochondrial content, was similarly increased with IT20 and IT100. Conversely, increased expression of mitochondrial respiratory chain (MRC) complexes I, III, and IV was only observed with IT100 and this was accompanied by increases in MRC supercomplex formation and pyruvate-malate-driven state 3 respiration in isolated mitochondria. In conclusion, the IT-induced increase in fatigue resistance is larger with high-intensity than with low-intensity contractions and this is linked to improved mitochondrial function due to increased expression of MRC complexes and assembly of MRC supercomplexes.

Vitamin C and E Treatment Blocks Changes in Kynurenine Metabolism Triggered by Three Weeks of Sprint Interval Training in Recreationally Active Elderly Humans.

The kynurenine pathway (KP) is gaining attention in several clinical fields. Recent studies show that physical exercise offers a therapeutic way to improve ratios of neurotoxic to neuroprotective KP metabolites. Antioxidant supplementation can blunt beneficial responses to physical exercise. We here studied the effects of endurance training in the form of sprint interval training (SIT; three sessions of 4-6 × 30 s cycling sprints per week for three weeks) in elderly (~65 years) men exposed to either placebo (n = 9) or the antioxidants vitamin C (1 g/day) and E (235 mg/day) (n = 11). Blood samples and muscle biopsies were taken under resting conditions in association with the first (untrained state) and last (trained state) SIT sessions. In the placebo group, the blood plasma level of the neurotoxic quinolinic acid was lower (~30%) and the neuroprotective kynurenic acid to quinolinic acid ratio was higher (~50%) in the trained than in the untrained state. Moreover, muscle biopsies showed a training-induced increase in kynurenine aminotransferase (KAT) III in the placebo group. All these training effects were absent in the vitamin-treated group. In conclusion, KP metabolism was shifted towards neuroprotection after three weeks of SIT in elderly men and this shift was blocked by antioxidant treatment.

Impaired aerobic capacity and premature fatigue preceding muscle weakness in the skeletal muscle Tfam-knockout mouse model.

Mitochondrial diseases are genetic disorders that lead to impaired mitochondrial function, resulting in exercise intolerance and muscle weakness. In patients, muscle fatigue due to defects in mitochondrial oxidative capacities commonly precedes muscle weakness. In mice, deletion of the fast-twitch skeletal muscle-specific Tfam gene (Tfam KO) leads to a deficit in respiratory chain activity, severe muscle weakness and early death. Here, we performed a time-course study of mitochondrial and muscular dysfunctions in 11- and 14-week-old Tfam KO mice, i.e. before and when mice are about to enter the terminal stage, respectively. Although force in the unfatigued state was reduced in Tfam KO mice compared to control littermates (wild type) only at 14 weeks, during repeated submaximal contractions fatigue was faster at both ages. During fatiguing stimulation, total phosphocreatine breakdown was larger in Tfam KO muscle than in wild-type muscle at both ages, whereas phosphocreatine consumption was faster only at 14 weeks. In conclusion, the Tfam KO mouse model represents a reliable model of lethal mitochondrial myopathy in which impaired mitochondrial energy production and premature fatigue occur before muscle weakness and early death.

Loss of α-actinin-3 during human evolution provides superior cold resilience and muscle heat generation.

The protein α-actinin-3 expressed in fast-twitch skeletal muscle fiber is absent in 1.5 billion people worldwide due to homozygosity for a nonsense polymorphism in ACTN3 (R577X). The prevalence of the 577X allele increased as modern humans moved to colder climates, suggesting a link between α-actinin-3 deficiency and improved cold tolerance. Here, we show that humans lacking α-actinin-3 (XX) are superior in maintaining core body temperature during cold-water immersion due to changes in skeletal muscle thermogenesis. Muscles of XX individuals displayed a shift toward more slow-twitch isoforms of myosin heavy chain (MyHC) and sarcoplasmic reticulum (SR) proteins, accompanied by altered neuronal muscle activation resulting in increased tone rather than overt shivering. Experiments on Actn3 knockout mice showed no alterations in brown adipose tissue (BAT) properties that could explain the improved cold tolerance in XX individuals. Thus, this study provides a mechanism for the positive selection of the ACTN3 X-allele in cold climates and supports a key thermogenic role of skeletal muscle during cold exposure in humans.

Eccentric Resistance Training Ameliorates Muscle Weakness in a Mouse Model of Idiopathic Inflammatory Myopathies.

OBJECTIVE: High-force eccentric contractions (ECCs) have traditionally been excluded from rehabilitation programs that include patients with idiopathic inflammatory myopathies (IIMs) due to unverified fear of causing muscle damage and inflammation. In an IIM animal model that used mice with experimental autoimmune myositis (EAM), we undertook this study to investigate whether ECC training can safely and effectively be used to counteract muscle weakness in IIM. METHODS: EAM was induced in BALB/c mice by immunization with 3 injections of myosin emulsified in Freund's complete adjuvant. Controls (n = 12) and mice with EAM (n = 12) were exposed to either an acute bout of 100 ECCs or 4 weeks of ECC training (20 ECCs every other day). To induce ECCs, plantar flexor muscles were electrically stimulated while the ankle was forcibly dorsiflexed. RESULTS: Less cell damage, as assessed by Evans blue dye uptake, was observed in the muscles of mice with EAM, compared to controls, after an acute bout of 100 ECCs (P < 0.05). Maximum Ca2+ -activated force was decreased in skinned gastrocnemius muscle fibers from mice with EAM, and this was accompanied by increased expression of endoplasmic reticulum (ER) stress proteins, including Gsp78 and Gsp94 (P < 0.05). ECC training prevented the decrease in force and the increase in ER stress proteins and also enhanced the expression and myofibrillar binding of small heat-shock proteins (HSPs) (P < 0.05), which can stabilize myofibrillar structure and function. CONCLUSION: ECC training protected against the reduction in myofibrillar force-generating capacity in an IIM mouse model, and this occurred via inhibition of ER stress responses and small HSP-mediated myofibrillar stabilization.

Quantification of Plasma Kynurenine Metabolites Following One Bout of Sprint Interval Exercise.

The kynurenine pathway of tryptophan degradation produces several neuroactive metabolites suggested to be involved in a wide variety of diseases and disorders, however, technical challenges in reliably detecting these metabolites hampers cross-comparisons. The main objective of this study was to develop an accurate, robust and precise bioanalytical method for simultaneous quantification of ten plasma kynurenine metabolites. As a secondary aim, we applied this method on blood samples taken from healthy subjects conducting 1 session of sprint interval exercise (SIE). It is well accepted that physical exercise is associated with health benefits and reduces risks of psychiatric illness, diabetes, cancer and cardiovascular disease, but also influences the peripheral and central concentrations of kynurenines. In line with this, we found that in healthy old adults (n = 10; mean age 64 years), levels of kynurenine increased 1 hour (P = .03) after SIE, while kynurenic acid (KYNA) concentrations were elevated after 24 hours (P = .02). In contrast, no significant changes after exercise were seen in young adults (n = 10; mean age 24 years). In conclusion, the described method performs well in reliably detecting all the analyzed metabolites in plasma samples. Furthermore, we also detected an age-dependent effect on the degree by which a single intense training session affects kynurenine metabolite levels.

Vitamin C and E Treatment Blunts Sprint Interval Training-Induced Changes in Inflammatory Mediator-, Calcium-, and Mitochondria-Related Signaling in Recreationally Active Elderly Humans.

Sprint interval training (SIT) has emerged as a time-efficient training regimen for young individuals. Here, we studied whether SIT is effective also in elderly individuals and whether the training response was affected by treatment with the antioxidants vitamin C and E. Recreationally active elderly (mean age 65) men received either vitamin C (1 g/day) and vitamin E (235 mg/day) or placebo. Training consisted of nine SIT sessions (three sessions/week for three weeks of 4-6 repetitions of 30-s all-out cycling sprints) interposed by 4 min rest. Vastus lateralis muscle biopsies were taken before, 1 h after, and 24 h after the first and last SIT sessions. At the end of the three weeks of training, SIT-induced changes in relative mRNA expression of reactive oxygen/nitrogen species (ROS)- and mitochondria-related proteins, inflammatory mediators, and the sarcoplasmic reticulum Ca2+ channel, the ryanodine receptor 1 (RyR1), were blunted in the vitamin treated group. Western blots frequently showed a major (>50%) decrease in the full-length expression of RyR1 24 h after SIT sessions; in the trained state, vitamin treatment seemed to provide protection against this severe RyR1 modification. Power at exhaustion during an incremental cycling test was increased by ~5% at the end of the training period, whereas maximal oxygen uptake remained unchanged; vitamin treatment did not affect these measures. In conclusion, treatment with the antioxidants vitamin C and E blunts SIT-induced cellular signaling in skeletal muscle of elderly individuals, while the present training regimen was too short or too intense for the changes in signaling to be translated into a clear-cut change in physical performance.

Fast skeletal muscle troponin activator CK-2066260 mitigates skeletal muscle weakness independently of the underlying cause.

BACKGROUND: Muscle 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. METHODS: Experiments 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). RESULTS: Prolonged 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. CONCLUSIONS: The 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.