Piperine enhances contractile force in slow- and fast-twitch muscle.

Piperine has been shown to bind to myosin and shift the distribution of conformational states of myosin molecules from the super-relaxed state to the disordered relaxed state. However, little is known about the implications for muscle force production and potential underlying mechanisms. Muscle contractility experiments were performed using isolated muscles and single fibres from rats and mice. The dose-response effect of piperine on muscle force was assessed at several stimulation frequencies. The potentiation of muscle force was also tested in muscles fatigued by eccentric contractions. Potential mechanisms of force potentiation were assessed by measuring Ca2+ levels during stimulation in enzymatically dissociated muscle fibres, while myofibrillar Ca2+ sensitivity was assessed in chemically skinned muscle fibres. Piperine caused a dose-dependent increase in low-frequency force with no effect on high-frequency force in both slow- and fast-twitch muscle, with similar relative increases in twitch force, rate of force development and relaxation rate. The potentiating effect of piperine on low-frequency force was reversible, and piperine partially recovered low-frequency force in fatigued muscle. Piperine had no effect on myoplasmic free [Ca2+] levels in mouse muscle fibres, whereas piperine substantially augmented the force response to submaximal levels of [Ca2+] in rat MyHCII fibres and MyHCI fibres along with a minor increase in maximum Ca2+-activated force. Piperine enhances low-frequency force production in both fast- and slow-twitch muscle. The effects are reversible and can counteract muscle fatigue. The primary underlying mechanism appears to be an increase in Ca2+ sensitivity. KEY POINTS: Piperine is a plant alkaloid derived from black pepper. It is known to bind to skeletal muscle myosin and enhance resting ATP turnover but its effects on contractility are not well known. We showed for the first time a piperine-induced force potentiation that was pronounced during low-frequency electrical stimulation of isolated muscles. The effect of piperine was observed in both slow and fast muscle types, was reversible, and could counteract the force decrements observed after fatiguing muscle contractions. Piperine treatment caused an increase in myofibrillar Ca2+ sensitivity in chemically skinned muscle fibres, while we observed no effect on intracellular Ca2+ concentrations during electrical stimulation in enzymatically dissociated muscle fibres.

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.

Time course changes in in vivo muscle mechanical function and Ca2+ regulation of force following experimentally induced gradual ovarian failure in mice.

The abrupt cessation of ovarian hormone release is associated with declines in muscle contractile function, yet the impact of gradual ovarian failure on muscle contractility across peri-, early- and late-stage menopause remains unclear. In this study, a 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure mouse model was used to examine time course changes in muscle mechanical function. Plantar flexors of female mice (VCD: n = 10; CON: n = 8) were assessed at 40 (early perimenopause), 80 (late perimenopause), 120 (menopause onset) and 176 (late menopause) days post-initial VCD injection. A torque-frequency relationship was established across a range of frequencies (10-200 Hz). Isotonic dynamic contractions were elicited against relative loads (10-80% maximal isometric torque) to determine the torque-velocity-power relationship. Mice then performed a fatigue task using intermittent 100 Hz isometric contractions until torque dropped by 60%. Recovery of twitch, 10 Hz and 100 Hz torque were tracked for 10 min post-task failure. Additionally, intact muscle fibres from the flexor digitorum brevis underwent a fatigue task (50 repetitions at 70 Hz), and 10 and 100 Hz tetanic [Ca2+] were monitored for 10 min afterward. VCD mice exhibited 16% lower twitch torque than controls across all time points. Apart from twitch torque, 10 Hz torque and 10 Hz tetanic [Ca2+], where VCD showed greater values relative to pre-fatigue during recovery, no significant differences were observed between control and VCD mice during recovery. These results indicate that gradual ovarian failure has minimal detriments to in vivo muscle mechanical function, with minor alterations observed primarily for low-frequency stimulation during recovery from fatigue.

Intrinsic contractile dysfunction due to impaired sarcoplasmic reticulum Ca2+ release in compensatory hypertrophied muscle fibers following synergist ablation.

Synergist ablation (SA) is an experimental procedure for the induction of hypertrophy. However, SA causes a decrease in specific force (i.e., force per cross-sectional area), likely due to excessive muscle use. Here, we investigated the mechanisms behind the SA-induced intrinsic contractile dysfunction, especially focusing on the excitation-contraction (EC) coupling. Male Wistar rats had unilateral surgical ablation of gastrocnemius and soleus muscles to induce compensatory hypertrophy in the plantaris muscles. Two weeks after SA, plantaris muscle was dissected from each animal and used for later analyses. SA significantly increased the mean fiber cross-sectional area (+18%). On the other hand, the ratio of depolarization-induced force to the maximum Ca2+-activated specific force, an indicator of sarcoplasmic reticulum (SR) Ca2+ release, was markedly reduced in mechanically skinned fibers from the SA group (-51%). These functional defects were accompanied by an extensive fragmentation of the SR Ca2+ release channel, the ryanodine receptor 1 (RyR1), and a decrease in the amount of other triad proteins (i.e., DHPR, STAC3, and junctophilin1). SA treatment also caused activation of calpain-1 and increased the amount of NADPH oxidase 2, endoplasmic reticulum (ER) stress proteins (i.e., Grp78, Grp94, PDI, and Ero1), and lipid peroxidation [i.e., 4-hydroxynonenal (4-HNE)] in SA-treated muscles. Our findings show that SA causes skeletal muscle weakness due to impaired EC coupling. This is likely to be induced by Ca2+-dependent degradation of triad proteins, which may result from Ca2+ leak from fragmented RyR1 triggered by increased oxidative stress.NEW & NOTEWORTHY Synergist ablation (SA) has widely been used to understand the mechanisms behind skeletal muscle hypertrophy. However, compensatory hypertrophied muscles display intrinsic contractile dysfunction, i.e., a hallmark of overuse. Here, we demonstrate that SA-induced compensatory hypertrophy is accompanied by muscle weakness due to impaired sarcoplasmic reticulum Ca2+ release. This dysfunction may be caused by the degradation of triad proteins due to the reciprocal amplification of reactive oxygen species and Ca2+ signaling at the junctional space microdomain.

Mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force.

The hypoxia-inducible nuclear-encoded mitochondrial protein NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) has been demonstrated to decrease oxidative phosphorylation and production of reactive oxygen species in neonatal cardiomyocytes, brain tissue and hypoxic domains of cancer cells. Prolonged local hypoxia can negatively affect skeletal muscle size and tissue oxidative capacity. Although skeletal muscle is a mitochondrial rich, oxygen sensitive tissue, the role of NDUFA4L2 in skeletal muscle has not previously been investigated. Here we ectopically expressed NDUFA4L2 in mouse skeletal muscles using adenovirus-mediated expression and in vivo electroporation. Moreover, femoral artery ligation (FAL) was used as a model of peripheral vascular disease to induce hind limb ischemia and muscle damage. Ectopic NDUFA4L2 expression resulted in reduced mitochondrial respiration and reactive oxygen species followed by lowered AMP, ADP, ATP, and NAD+ levels without affecting the overall protein content of the mitochondrial electron transport chain. Furthermore, ectopically expressed NDUFA4L2 caused a ~20% reduction in muscle mass that resulted in weaker muscles. The loss of muscle mass was associated with increased gene expression of atrogenes MurF1 and Mul1, and apoptotic genes caspase 3 and Bax. Finally, we showed that NDUFA4L2 was induced by FAL and that the Ndufa4l2 mRNA expression correlated with the reduced capacity of the muscle to generate force after the ischemic insult. These results show, for the first time, that mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force. Specifically, induced NDUFA4L2 reduces mitochondrial activity leading to lower levels of important intramuscular metabolites, including adenine nucleotides and NAD+ , which are hallmarks of mitochondrial dysfunction and hence shows that dysfunctional mitochondrial activity may drive muscle wasting.

Calcium sensitivity during staircase with sequential incompletely fused contractions.

Activity dependent potentiation is thought to result from phosphorylation of the regulatory light chains of myosin, increasing Ca2+ sensitivity. Yet, Ca2+ sensitivity decreases early in a period of intermittent contractions. The purpose of this study was to investigate the early change in Ca2+ sensitivity during intermittent submaximal tetanic contractions. Flexor digitorum brevis muscle fibres were dissected from mice after cervical disarticulation. Fibres were superfused with Tyrode solution at 32 °C. Length was set to yield maximal tetanic force. Indo-1 was microinjected into fibres and allowed to dissipate for 30 min. Fluorescence was measured at 405 and 495 nm wavelength and the ratio was used to estimate [Ca2+]. A control force-Ca2+ relationship was determined with stimulation over a range of frequencies, yielding constants for slope, max force, and half-maximal [Ca2+] (pCa2 +50). Data were collected for sequential contractions at 40 Hz at 2 s intervals. Active force decreased over the first 1-4 contractions then increased. A force-pCa2+ curve was fit to each contraction, using the control values for the Hill slope and max force by adjusting pCa2+50 until the curve passed through the target contraction. Data are presented for three contractions for each fibre: first, maximum shift to the right, and last contraction. There was a significant shift to the right for pCa2+50 (decreased Ca2+ sensitivity), usually early in the series of intermittent contractions, then pCa2 +50 shifted to the left, but remained significantly different from the control value. Although potentiation is associated with increased Ca2+ sensitivity, this increase begins only after Ca2+ sensitivity has decreased and, in most cases, Ca2+ sensitivity does not increase above the control level.

Carbohydrate restriction following strenuous glycogen-depleting exercise does not potentiate the acute molecular response associated with mitochondrial biogenesis in human skeletal muscle.

PURPOSE: Carbohydrate (CHO) restriction could be a potent metabolic regulator of endurance exercise-induced muscle adaptations. Here, we determined whether post-exercise CHO restriction following strenuous exercise combining continuous cycling exercise (CCE) and sprint interval exercise could affect the gene expression related to mitochondrial biogenesis and oxidative metabolism in human skeletal muscle. METHODS: In a randomized cross-over design, 8 recreationally active males performed two cycling exercise sessions separated by 4 weeks. Each session consisted of 60-min CCE and six 30-s all-out sprints, which was followed by ingestion of either a CHO or placebo beverage in the post-exercise recovery period. Muscle glycogen concentration and the mRNA levels of several genes related to mitochondrial biogenesis and oxidative metabolism were determined before, immediately after, and at 3 h after exercise. RESULTS: Compared to pre-exercise, strenuous cycling led to a severe muscle glycogen depletion (> 90%) and induced a large increase in PGC1A and PDK4 mRNA levels (~ 20-fold and ~ 10-fold, respectively) during the acute recovery period in both trials. The abundance of the other transcripts was not changed or was only moderately increased during this period. CHO restriction during the 3-h post-exercise period blunted muscle glycogen resynthesis but did not increase the mRNA levels of genes associated with muscle adaptation to endurance exercise, as compared with abundant post-exercise CHO consumption. CONCLUSION: CHO restriction after a glycogen-depleting and metabolically-demanding cycling session is not effective for increasing the acute mRNA levels of genes involved in mitochondrial biogenesis and oxidative metabolism in human skeletal muscle.

Impaired sarcoplasmic reticulum Ca2+ release is the major cause of fatigue-induced force loss in intact single fibres from human intercostal muscle.

KEY POINTS: Changes in intramuscular Ca2+ handling contribute to development of fatigue and disease-related loss of muscle mass and function. To date, no data on human intact living muscle fibres have been described. We manually dissected intact single fibres from human intercostal muscle and simultaneously measured force and myoplasmic free [Ca2+ ] at physiological temperature. Based on their fatigue resistance, two distinct groups of fibres were distinguished: fatigue sensitive and fatigue resistant. Force depression in fatigue and during recovery was due to impaired sarcoplasmic reticulum Ca2+ release in both groups of fibres. Acidification did not affect force production in unfatigued fibres and did not affect fatigue development in fatigue-resistant fibres. The current study provides novel insight into the mechanisms of fatigue in human intercostal muscle. ABSTRACT: Changes in intracellular Ca2+ handling of individual skeletal muscle fibres cause a force depression following physical activity and are also implicated in disease-related loss of function. The relation of intracellular Ca2+ handling with muscle force production and fatigue tolerance is best studied in intact living single fibres that allow continuous measurements of force and myoplasmic free [Ca2+ ] during repeated contractions. To this end, manual dissections of human intercostal muscle biopsies were performed to isolate intact single fibres. Based on the ability to maintain tetanic force at >40% of the initial value during 500 fatiguing contractions, fibres were classified as either fatigue sensitive or fatigue resistant. Following fatigue all fibres demonstrated a marked reduction in sarcoplasmic reticulum Ca2+ release, while myofibrillar Ca2+ sensitivity was either unaltered or increased. In unfatigued fibres, acidosis caused a reduction in myofibrillar Ca2+ sensitivity that was offset by increased tetanic myoplasmic free [Ca2+ ] so that force remained unaffected. Acidification did not affect the fatigue tolerance of fatigue-resistant fibres, whereas uncertainties remain whether or not fatigue-sensitive fibres were affected. Following fatigue, a prolonged force depression at preferentially low-frequency stimulation was evident in fatigue-sensitive fibres and this was caused exclusively by an impaired sarcoplasmic reticulum Ca2+ release. We conclude that impaired sarcoplasmic reticulum Ca2+ release is the predominant mechanism of force depression both in the development of, and recovery from, fatigue in human intercostal muscle.

Carbohydrates do not accelerate force recovery after glycogen-depleting followed by high-intensity exercise in humans.

Prolonged low-frequency force depression (PLFFD) induced by fatiguing exercise is characterized by a persistent depression in submaximal contractile force during the recovery period. Muscle glycogen depletion is known to limit physical performance during prolonged low- and moderate-intensity exercise, and accelerating glycogen resynthesis with post-exercise carbohydrate intake can facilitate recovery and improve repeated bout exercise performance. Short-term, high-intensity exercise, however, can cause PLFFD without any marked decrease in glycogen. Here, we studied whether recovery from PLFFD was accelerated by carbohydrate ingestion after 60 minutes of moderate-intensity glycogen-depleting cycling exercise followed by six 30-seconds all-out cycling sprints. We used a randomized crossover study design where nine recreationally active males drank a beverage containing either carbohydrate or placebo after exercise. Blood glucose and muscle glycogen concentrations were determined at baseline, immediately post-exercise, and during the 3-hours recovery period. Transcutaneous electrical stimulation of the quadriceps muscle was performed to determine the extent of PLFFD by eliciting low-frequency (20 Hz) and high-frequency (100 Hz) stimulations. Muscle glycogen was severely depleted after exercise, with a significantly higher rate of muscle glycogen resynthesis during the 3-hours recovery period in the carbohydrate than in the placebo trials (13.7 and 5.4 mmol glucosyl units/kg wet weight/h, respectively). Torque at 20 Hz was significantly more depressed than 100 Hz torque during the recovery period in both conditions, and the extent of PLFFD (20/100 Hz ratio) was not different between the two trials. In conclusion, carbohydrate supplementation enhances glycogen resynthesis after glycogen-depleting exercise but does not improve force recovery when the exercise also involves all-out cycling sprints.

Measuring Ca2+ in Living Cells.

Measuring free Ca2+ concentration ([Ca2+]) in the cytosol or organelles is routine in many fields of research. The availability of membrane permeant forms of indicators coupled with the relative ease of transfecting cell lines with biological Ca2+ sensors have led to the situation where cellular and subcellular [Ca2+] is examined by many non-specialists. In this chapter, we evaluate the most used Ca2+ indicators and highlight what their major advantages and disadvantages are. We stress the potential pitfalls of non-ratiometric techniques for measuring Ca2+ and the clear advantages of ratiometric methods. Likely improvements and new directions for Ca2+ measurement are discussed.

Moderately elevated extracellular [K+] potentiates submaximal force and power in skeletal muscle via increased [Ca2+]i during contractions.

The extracellular K+ concentration ([K+]o) increases during physical exercise. We here studied whether moderately elevated [K+]o may increase force and power output during contractions at in vivo-like subtetanic frequencies and whether such potentiation was associated with increased cytosolic free Ca2+ concentration ([Ca2+]i) during contractions. Isolated whole soleus and extensor digitorum longus (EDL) rat muscles were incubated at different levels of [K+]o, and isometric and dynamic contractility were tested at various stimulation frequencies. Furthermore, [Ca2+]i at rest and during contraction was measured along with isometric force in single mouse flexor digitorum brevis (FDB) fibers exposed to elevated [K+]o. Elevating [K+]o from 4 mM up to 8 mM (soleus) and 11 mM (EDL) increased isometric force at subtetanic frequencies, 2-15 Hz in soleus and up to 50 Hz in EDL, while inhibition was seen at tetanic frequency in both muscle types. Elevating [K+]o also increased peak power of dynamic subtetanic contractions, with potentiation being more pronounced in EDL than in soleus muscles. The force-potentiating effect of elevated [K+]o was transient in FDB single fibers, reaching peak after ~4 and 2.5 min in 9 and 11 mM [K+]o, respectively. At the time of peak potentiation, force and [Ca2+]i during 15-Hz contractions were significantly increased, whereas force was slightly decreased and [Ca2+]i unchanged during 50-Hz contractions. Moderate elevation of [K+]o can transiently potentiate force and power during contractions at subtetanic frequencies, which can be explained by a higher [Ca2+]i during contractions.

Toxic doses of caffeine are needed to increase skeletal muscle contractility.

Discrepant results have been reported regarding an intramuscular mechanism underlying the ergogenic effect of caffeine on neuromuscular function in humans. Here, we reevaluated the effect of caffeine on muscular force production in humans and combined this with measurements of the caffeine dose-response relationship on force and cytosolic free [Ca2+] ([Ca2+]i) in isolated mouse muscle fibers. Twenty-one healthy and physically active men (29 ± 9 yr, 178 ± 6 cm, 73 ± 10 kg, mean ± SD) took part in the present study. Nine participants were involved in two experimental sessions during which supramaximal single and paired electrical stimulations (at 10 and 100 Hz) were applied to the femoral nerve to record evoked forces. Evoked forces were recorded before and 1 h after ingestion of 1) 6 mg caffeine/kg body mass or 2) placebo. Caffeine plasma concentration was measured in 12 participants. In addition, submaximal tetanic force and [Ca2+]i were measured in single mouse flexor digitorum brevis (FDB) muscle fibers exposed to 100 nM up to 5 mM caffeine. Six milligrams of caffeine per kilogram body mass (plasma concentration ~40 µM) did not increase electrically evoked forces in humans. In superfused FDB single fibers, millimolar caffeine concentrations (i.e., 15- to 35-fold above usual concentrations observed in humans) were required to increase tetanic force and [Ca2+]i. Our results suggest that toxic doses of caffeine are required to increase muscle contractility, questioning the purported intramuscular ergogenic effect of caffeine in humans.

Intact single muscle fibres from SOD1G93A amyotrophic lateral sclerosis mice display preserved specific force, fatigue resistance and training-like adaptations.

KEY POINTS: How defects in muscle contractile function contribute to weakness in amyotrophic lateral sclerosis (ALS) were systematically investigated. Weakness in whole muscles from late stage SOD1G93A mice was explained by muscle atrophy as seen by reduced mass and maximal force. On the other hand, surviving single muscle fibres in late stage SOD1G93A have preserved intracellular Ca2+ handling, normal force-generating capacity and increased fatigue resistance. These intriguing findings provide a substrate for therapeutic interventions to potentiate muscular capacity and delay the progression of the ALS phenotype. ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by degeneration and loss of motor neurons, leading to severe muscle weakness and paralysis. The SOD1G93A mouse model of ALS displays motor neuron degeneration and a phenotype consistent with human ALS. The purpose of this study was to determine whether muscle weakness in ALS can be attributed to impaired intrinsic force generation in skeletal muscles. In the current study, motor neuron loss and decreased force were evident in whole flexor digitorum brevis (FDB) muscles of mice in the late stage of disease (125-150 days of age). However, in intact single muscle fibres, specific force, tetanic myoplasmic free [Ca2+ ] ([Ca2+ ]i ), and resting [Ca2+ ]i remained unchanged with disease. Fibre-type distribution was maintained in late-stage SOD1G93A FDB muscles, but remaining muscle fibres displayed greater fatigue resistance compared to control and showed increased expression of myoglobin and mitochondrial respiratory chain proteins that are important determinants of fatigue resistance. Expression of genes central to both mitochondrial biogenesis and muscle atrophy where increased, suggesting that atrophic and compensatory adaptive signalling occurs simultaneously within the muscle tissue. These results support the hypothesis that muscle weakness in SOD1G93A is primarily attributed to neuromuscular degeneration and not intrinsic muscle fibre defects. In fact, surviving muscle fibres displayed maintained adaptive capacity with an exercise training-like phenotype, which suggests that compensatory mechanisms are activated that can function to delay disease progression.

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.

Role of Ca2+ in changing active force during intermittent submaximal stimulation in intact, single mouse muscle fibers.

Fatigue of single mouse fibers during repeated high-frequency stimulation results initially from decreased Ca2+ sensitivity while free myoplasmic calcium concentration ([Ca2+]m) increases, followed by decreasing [Ca2+]m. Recovery of active force with low-frequency stimulation is slow and persistent fatigue results from low [Ca2+]m. However, the consequences of intermittent submaximal contractions are not known. The aim of the present study was to investigate the changes in [Ca2+]m and active force during intermittent submaximal contractions and subsequent recovery. Single fibers of mouse flexor digitorum brevis muscles at 32 °C were stimulated with 40 or 50 Hz, for 350 ms every 2 s for 2 min and then every 1 s until < 40% of initial force. Values obtained during the intermittent stimulation were compared with a control force-[Ca2+]m relationship. A "P"-shaped pattern in the force-[Ca2+]m relationship was observed during intermittent stimulation. Early in the intermittent stimulation, [Ca2+]m increased while active force decreased. Subsequent force potentiation was accompanied by increased Ca2+ sensitivity. Later, as active force declined, [Ca2+]m decreased significantly (p < 0.001). This was followed, in the final phase, by a significant decrease in Ca2+ sensitivity determined by [Ca2+]m at half-maximal force (Ca50) (p = 0.001). Low-frequency fatigue persisted during recovery while Ca50 was not significantly different from prefatigue (p > 0.5). In conclusion, the main mechanism of fatigue is due to decreases in both [Ca2+]m and Ca2+ sensitivity following the initial force potentiation. The intermittent submaximal contractions resulted in persistent low-frequency fatigue seen during recovery, which was explained by depressed [Ca2+]m with no change in Ca2+ sensitivity.

Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise.

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

Post-exercise recovery of contractile function and endurance in humans and mice is accelerated by heating and slowed by cooling skeletal muscle.

KEY POINTS: We investigated whether intramuscular temperature affects the acute recovery of exercise performance following fatigue-induced by endurance exercise. Mean power output was better preserved during an all-out arm-cycling exercise following a 2 h recovery period in which the upper arms were warmed to an intramuscular temperature of Ì´ 38°C than when they were cooled to as low as 15°C, which suggested that recovery of exercise performance in humans is dependent on muscle temperature. Mechanisms underlying the temperature-dependent effect on recovery were studied in intact single mouse muscle fibres where we found that recovery of submaximal force and restoration of fatigue resistance was worsened by cooling (16-26°C) and improved by heating (36°C). Isolated whole mouse muscle experiments confirmed that cooling impaired muscle glycogen resynthesis. We conclude that skeletal muscle recovery from fatigue-induced by endurance exercise is impaired by cooling and improved by heating, due to changes in glycogen resynthesis rate. ABSTRACT: Manipulation of muscle temperature is believed to improve post-exercise recovery, with cooling being especially popular among athletes. However, it is unclear whether such temperature manipulations actually have positive effects. Accordingly, we studied the effect of muscle temperature on the acute recovery of force and fatigue resistance after endurance exercise. One hour of moderate-intensity arm cycling exercise in humans was followed by 2 h recovery in which the upper arms were either heated to 38°C, not treated (33°C), or cooled to ∼15°C. Fatigue resistance after the recovery period was assessed by performing 3 × 5 min sessions of all-out arm cycling at physiological temperature for all conditions (i.e. not heated or cooled). Power output during the all-out exercise was better maintained when muscles were heated during recovery, whereas cooling had the opposite effect. Mechanisms underlying the temperature-dependent effect on recovery were tested in mouse intact single muscle fibres, which were exposed to ∼12 min of glycogen-depleting fatiguing stimulation (350 ms tetani given at 10 s interval until force decreased to 30% of the starting force). Fibres were subsequently exposed to the same fatiguing stimulation protocol after 1-2 h of recovery at 16-36°C. Recovery of submaximal force (30 Hz), the tetanic myoplasmic free [Ca2+ ] (measured with the fluorescent indicator indo-1), and fatigue resistance were all impaired by cooling (16-26°C) and improved by heating (36°C). In addition, glycogen resynthesis was faster at 36°C than 26°C in whole flexor digitorum brevis muscles. We conclude that recovery from exhaustive endurance exercise is accelerated by raising and slowed by lowering muscle temperature.

The Ca2+ sensitizer CK-2066260 increases myofibrillar Ca2+ sensitivity and submaximal force selectively in fast skeletal muscle.

KEY POINTS: We report that the small molecule CK-2066260 selectively slows the off-rate of Ca2+ from fast skeletal muscle troponin, leading to increased myofibrillar Ca2+ sensitivity in fast skeletal muscle. Rodents dosed with CK-2066260 show increased hindlimb muscle force and power in response to submaximal rates of nerve stimulation in situ. CK-2066260 has no effect on free cytosolic [Ca2+ ] during contractions of isolated muscle fibres. We conclude that fast skeletal muscle troponin sensitizers constitute a potential therapy to address an unmet need of improving muscle function in conditions of weakness and premature muscle fatigue. ABSTRACT: Skeletal muscle dysfunction occurs in many diseases and can lead to muscle weakness and premature muscle fatigue. Here we show that the fast skeletal troponin activator, CK-2066260, counteracts muscle weakness by increasing troponin Ca2+ affinity, thereby increasing myofibrillar Ca2+ sensitivity. Exposure to CK-2066260 resulted in a concentration-dependent increase in the Ca2+ sensitivity of ATPase activity in isolated myofibrils and reconstituted hybrid sarcomeres containing fast skeletal muscle troponin C. Stopped-flow experiments revealed a ∼2.7-fold decrease in the Ca2+ off-rate of isolated troponin complexes in the presence of CK-2066260 (6 vs. 17 s-1 under control conditions). Isolated mouse flexor digitorum brevis fibres showed a rapidly developing, reversible and concentration-dependent force increase at submaximal stimulation frequencies. This force increase was not accompanied by any changes in the free cytosolic [Ca2+ ] or its kinetics. CK-2066260 induced a slowing of relaxation, which was markedly larger at 26°C than at 31°C and could be linked to the decreased Ca2+ off-rate of troponin C. Rats dosed with CK-2066260 showed increased hindlimb isometric and isokinetic force in response to submaximal rates of nerve stimulation in situ producing significantly higher absolute forces at low isokinetic velocities, whereas there was no difference in force at the highest velocities. Overall muscle power was increased and the findings are consistent with a lack of effect on crossbridge kinetics. In conclusion, CK-2066260 acts as a fast skeletal troponin activator that may be used to increase muscle force and power in conditions of muscle weakness.

Cyclophilin D, a target for counteracting skeletal muscle dysfunction in mitochondrial myopathy.

Muscle weakness and exercise intolerance are hallmark symptoms in mitochondrial disorders. Little is known about the mechanisms leading to impaired skeletal muscle function and ultimately muscle weakness in these patients. In a mouse model of lethal mitochondrial myopathy, the muscle-specific Tfam knock-out (KO) mouse, we previously demonstrated an excessive mitochondrial Ca(2+) uptake in isolated muscle fibers that could be inhibited by the cyclophilin D (CypD) inhibitor, cyclosporine A (CsA). Here we show that the Tfam KO mice have increased CypD levels, and we demonstrate that this increase is a common feature in patients with mitochondrial myopathy. We tested the effect of CsA treatment on Tfam KO mice during the transition from a mild to terminal myopathy. CsA treatment counteracted the development of muscle weakness and improved muscle fiber Ca(2+) handling. Importantly, CsA treatment prolonged the lifespan of these muscle-specific Tfam KO mice. These results demonstrate that CsA treatment is an efficient therapeutic strategy to slow the development of severe mitochondrial myopathy.