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.

Differential utilisation of subcellular skeletal muscle glycogen pools: a comparative analysis between 1 and 15 min of maximal exercise.

In skeletal muscle, glycogen particles are distributed both within and between myofibrils, as well as just beneath the sarcolemma. Their precise localisation may influence their degradation rate. Here, we investigated how exercise at different intensities and durations (1- and 15-min maximal exercise) with known variations in glycogenolytic rate and contribution from anaerobic metabolism affects utilisation of the distinct pools. Furthermore, we investigated how decreased glycogen availability achieved through lowering carbohydrate and energy intake after glycogen-depleting exercise affect the storage of glycogen particles (size, numerical density, localisation). Twenty participants were divided into two groups performing either a 1-min (n = 10) or a 15-min (n = 10) maximal cycling exercise test. In a randomised, counterbalanced, cross-over design, the exercise tests were performed following short-term consumption of two distinct diets with either high or moderate carbohydrate content (10 vs. 4 g kg-1 body mass (BM) day-1) mediating a difference in total energy consumption (240 vs. 138 g kg-1 BM day-1). Muscle biopsies from m. vastus lateralis were obtained before and after the exercise tests. Intermyofibrillar glycogen was preferentially utilised during the 1-min test, whereas intramyofibrillar glycogen was preferentially utilised during the 15-min test. Lowering carbohydrate and energy intake after glycogen-depleting exercise reduced glycogen availability by decreasing particle size across all pools and diminishing numerical density in the intramyofibrillar and subsarcolemmal pools. In conclusion, distinct subcellular glycogen pools were differentially utilised during 1-min and 15-min maximal cycling exercise. Additionally, lowered carbohydrate and energy consumption after glycogen-depleting exercise altered glycogen storage by reducing particle size and numerical density, depending on subcellular localisation. KEY POINTS: In human skeletal muscle, glycogen particles are localised in distinct subcellular compartments, referred to as intermyofibrillar, intramyofibrillar and subsarcolemmal pools. The intermyofibrillar and subsarcolemmal pools are close to mitochondria, while the intramyofibrillar pool is at a distance from mitochondria. We show that 1 min of maximal exercise is associated with a preferential utilisation of intermyofibrillar glycogen, and, on the other hand, that 15 min of maximal exercise is associated with a preferential utilisation of intramyofibrillar glycogen. Furthermore, we demonstrate that reduced glycogen availability achieved through lowering carbohydrate and energy intake after glycogen-depleting exercise is characterised by a decreased glycogen particle size across all compartments, with the numerical density only diminished in the intramyofibrillar and subsarcolemmal compartments. These results suggest that exercise intensity influences the subcellular pools of glycogen differently and that the dietary content of carbohydrates and energy is linked to the size and subcellular distribution of glycogen particles.

Skeletal muscle mitochondria demonstrate similar respiration per cristae surface area independent of training status and sex in healthy humans.

The impact of training status and sex on intrinsic skeletal muscle mitochondrial respiratory capacity remains unclear. We examined this by analysing human skeletal muscle mitochondrial respiration relative to mitochondrial volume and cristae density across training statuses and sexes. Mitochondrial cristae density was estimated in skeletal muscle biopsies originating from previous independent studies. Participants included females (n = 12) and males (n = 41) across training statuses ranging from untrained (UT, n = 8), recreationally active (RA, n = 9), active-to-elite runners (RUN, n = 27) and cross-country skiers (XC, n = 9). The XC and RUN groups demonstrated higher mitochondrial volume density than the RA and UT groups while all active groups (RA, RUN and XC) displayed higher mass-specific capacity of oxidative phosphorylation (OXPHOS) and mitochondrial cristae density than UT. Differences in OXPHOS diminished between active groups and UT when normalising to mitochondrial volume density and were lost when normalising to muscle cristae surface area density. Moreover, active females (n = 6-9) and males (n = 15-18) did not differ in mitochondrial volume and cristae density, OXPHOS, or when normalising OXPHOS to mitochondrial volume density and muscle cristae surface area density. These findings demonstrate: (1) differences in OXPHOS between active and untrained individuals may be explained by both higher mitochondrial volume and cristae density in active individuals, with no difference in intrinsic mitochondrial respiratory capacity (OXPHOS per muscle cristae surface area density); and (2) no sex differences in mitochondrial volume and cristae density or mass-specific and normalised OXPHOS. This highlights the importance of normalising OXPHOS to muscle cristae surface area density when studying skeletal muscle mitochondrial biology. KEY POINTS: Oxidative phosphorylation is the mitochondrial process by which ATP is produced, governed by the electrochemical gradient across the inner mitochondrial membrane with infoldings named cristae. In human skeletal muscle, the mass-specific capacity of oxidative phosphorylation (OXPHOS) can change independently of shifts in mitochondrial volume density, which may be attributed to variations in cristae density. We demonstrate that differences in skeletal muscle OXPHOS between healthy females and males, ranging from untrained to elite endurance athletes, are matched by differences in cristae density. This suggests that higher OXPHOS in skeletal muscles of active individuals is attributable to an increase in the density of cristae. These findings broaden our understanding of the variability in human skeletal muscle OXPHOS and highlight the significance of cristae, specific to mitochondrial respiration.

Assessments of individual fiber glycogen and mitochondrial volume percentages reveal a graded reduction in muscle oxidative power during prolonged exhaustive exercise.

During submaximal exercise, there is a heterogeneous recruitment of skeletal muscle fibers, with an ensuing heterogeneous depletion of muscle glycogen both within and between fiber types. Here, we show that the mean (95% CI) mitochondrial volume as a percentage of fiber volume of non-glycogen-depleted fibers was 2 (-10:6), 5 (-21:11), and 12 (-21:-2)% lower than all the sampled fibers after continuing exercise for 1, 2 h, and until task failure, respectively. Therefore, a glycogen-dependent fatigue of individual fibers during submaximal exercise may reduce the muscular oxidative power. These findings suggest a relationship between glycogen and mitochondrial content in individual muscle fibers, which is important for understanding fatigue during prolonged exercise.

Substrate utilization and durability during prolonged intermittent exercise in elite road cyclists.

PURPOSE: This study investigated the effects of prolonged intermittent cycling exercise on peak power output (PPO) and 6-min time-trial (6 min-TT) performance in elite and professional road cyclists. Moreover, the study aimed to determine whether changes in performance in the fatigued state could be predicted from substrate utilization during exercise and laboratory measures obtained in a fresh state. METHODS: Twelve cyclists (age: 23 years [21;25]; body mass: 71.5 kg [66.7;76.8]; height: 181 cm [178;185]; V ˙ O2peak: 73.6 ml kg-1 min-1 [71.2;76.0]) completed a graded submaximal cycling test to determine lactate threshold (LT1), gross efficiency (GE), and maximal fat oxidation (MFO) as well as power output during a maximal 6 min-TT (MPO6 min) in a fresh condition. On a separate day, the cyclists completed a 4-h intermittent cycling protocol with a high CHO intake (100 g h-1). Substrate utilization and PPO was measured hourly during the protocol, which was followed by another 6 min-TT. RESULTS: MPO6 min and PPO was reduced by 10% [4;15] and 6% [0;6], respectively, after the cycling protocol. These reductions were accompanied by reductions in the anaerobic energy contribution and V ˙ O2peak, whereas the average V ˙ O2 during the 6 min-TT was unchanged. Correlation analyses showed no strong associations between reductions in MPO6 min and PPO and laboratory measures (i.e., LT1, GE, MFO, V ˙ O2peak) obtained in the fresh condition. Additionally, fat oxidation rates during the cycling protocol were not related to changes in neither PPO nor MPO6 min. CONCLUSION: PPO and MPO6 min were reduced following prolonged intermittent cycling, but the magnitude of these reductions could not be predicted from laboratory measures obtained in the fresh condition.

A century of exercise physiology: effects of muscle contraction and exercise on skeletal muscle Na+,K+-ATPase, Na+ and K+ ions, and on plasma K+ concentration-historical developments.

This historical review traces key discoveries regarding K+ and Na+ ions in skeletal muscle at rest and with exercise, including contents and concentrations, Na+,K+-ATPase (NKA) and exercise effects on plasma [K+] in humans. Following initial measures in 1896 of muscle contents in various species, including humans, electrical stimulation of animal muscle showed K+ loss and gains in Na+, Cl- and H20, then subsequently bidirectional muscle K+ and Na+ fluxes. After NKA discovery in 1957, methods were developed to quantify muscle NKA activity via rates of ATP hydrolysis, Na+/K+ radioisotope fluxes, [3H]-ouabain binding and phosphatase activity. Since then, it became clear that NKA plays a central role in Na+/K+ homeostasis and that NKA content and activity are regulated by muscle contractions and numerous hormones. During intense exercise in humans, muscle intracellular [K+] falls by 21 mM (range - 13 to - 39 mM), interstitial [K+] increases to 12-13 mM, and plasma [K+] rises to 6-8 mM, whilst post-exercise plasma [K+] falls rapidly, reflecting increased muscle NKA activity. Contractions were shown to increase NKA activity in proportion to activation frequency in animal intact muscle preparations. In human muscle, [3H]-ouabain-binding content fully quantifies NKA content, whilst the method mainly detects α2 isoforms in rats. Acute or chronic exercise affects human muscle K+, NKA content, activity, isoforms and phospholemman (FXYD1). Numerous hormones, pharmacological and dietary interventions, altered acid-base or redox states, exercise training and physical inactivity modulate plasma [K+] during exercise. Finally, historical research approaches largely excluded female participants and typically used very small sample sizes.

Altered intramuscular network of lipid droplets and mitochondria in type 2 diabetes.

Excessive storage of lipid droplets (LDs) in skeletal muscles is a hallmark of type 2 diabetes. However, LD morphology displays a high degree of subcellular heterogeneity and varies between single muscle fibers, which impedes the current understanding of lipid-induced insulin resistance. Using quantitative transmission electron microscopy (TEM), we conducted a comprehensive single-fiber morphological analysis to investigate the intramuscular network of LDs and mitochondria, and the effects of 8 wk of high-intensity interval training (HIIT) targeting major muscle groups, in patients with type 2 diabetes and nondiabetic obese and lean controls. We found that excessive storage of intramuscular lipids in patients with type 2 diabetes was exclusively explained by extremely large LDs situated in distinct muscle fibers with a location-specific deficiency in subsarcolemmal mitochondria. After HIIT, this intramuscular deficiency was improved by a remodeling of LD size and subcellular distribution and mitochondrial content. Analysis of LD morphology further revealed that individual organelles were better described as ellipsoids than spheres. Moreover, physical contact between LD and mitochondrial membranes indicated a dysfunctional interplay between organelles in the diabetic state. Taken together, type 2 diabetes should be recognized as a metabolic disease with high cellular heterogeneity in intramuscular lipid storage, underlining the relevance of single-cell technologies in clinical research. Furthermore, HIIT changed intramuscular LD storage toward nondiabetic characteristics.

Increased mitochondrial surface area and cristae density in the skeletal muscle of strength athletes.

Mitochondria are the cellular organelles responsible for resynthesising the majority of ATP. In skeletal muscle, there is an increased ATP turnover during resistance exercise to sustain the energetic demands of muscle contraction. Despite this, little is known regarding the mitochondrial characteristics of chronically strength-trained individuals and any potential pathways regulating the strength-specific mitochondrial remodelling. Here, we investigated the mitochondrial structural characteristics in skeletal muscle of strength athletes and age-matched untrained controls. The mitochondrial pool in strength athletes was characterised by increased mitochondrial cristae density, decreased mitochondrial size, and increased surface-to-volume ratio, despite similar mitochondrial volume density. We also provide a fibre-type and compartment-specific assessment of mitochondria morphology in human skeletal muscle, which reveals across groups a compartment-specific influence on mitochondrial morphology that is largely independent of fibre type. Furthermore, we show that resistance exercise leads to signs of mild mitochondrial stress, without an increase in the number of damaged mitochondria. Using publicly available transcriptomic data we show that acute resistance exercise increases the expression of markers of mitochondrial biogenesis, fission and mitochondrial unfolded protein responses (UPRmt ). Further, we observed an enrichment of the UPRmt in the basal transcriptome of strength-trained individuals. Together, these findings show that strength athletes possess a unique mitochondrial remodelling, which minimises the space required for mitochondria. We propose that the concurrent activation of markers of mitochondrial biogenesis and mitochondrial remodelling pathways (fission and UPRmt ) with resistance exercise may be partially responsible for the observed mitochondrial phenotype of strength athletes. KEY POINTS: Untrained individuals and strength athletes possess comparable skeletal muscle mitochondrial volume density. In contrast, strength athletes' mitochondria are characterised by increased cristae density, decreased size and increased surface-to-volume ratio. Type I fibres have an increased number of mitochondrial profiles with minor differences in the mitochondrial morphological characteristics compared with type II fibres. The mitochondrial morphology is distinct across the subcellular compartments in both groups, with subsarcolemmal mitochondria being bigger in size when compared with intermyofibrillar. Acute resistance exercise leads to signs of mild morphological mitochondrial stress accompanied by increased gene expression of markers of mitochondrial biogenesis, fission and mitochondrial unfolded protein response (UPRmt ).

Acute exercise increases the contact between lipid droplets and mitochondria independently of obesity and type 2 diabetes.

Intramuscular lipid droplets (LDs) and mitochondria are essential organelles in cellular communication and metabolism, supporting local energy demands during muscle contractions. While insulin resistance impacts cellular functions and systems within the skeletal muscle, it remains unclear whether the interaction of LDs and mitochondria is affected by exercise and the role of obesity and type 2 diabetes. By employing transmission electron microscopy (TEM), we aimed to investigate the effects of 1 h of ergometry cycling on LD morphology, subcellular distribution and mitochondrial contact in skeletal muscle fibres of patients with type 2 diabetes and glucose-tolerant lean and obese controls, matched for equal exercise intensities. Exercise did not change LD volumetric density, numerical density, profile size or subcellular distribution. However, evaluated as the magnitude of inter-organelle contact, exercise increased the contact between LDs and mitochondria with no differences between the three groups. This effect was most profound in the subsarcolemmal space of type 1 muscle fibres, and here the absolute contact length increased on average from ∼275 to ∼420 nm. Furthermore, the absolute contact length before exercise (ranging from ∼140 to ∼430 nm) was positively associated with the fat oxidation rate during exercise. In conclusion, we showed that acute exercise did not mediate changes in the LD volume fractions, numbers or size but increased the contact between LDs and mitochondria, irrespective of obesity or type 2 diabetes. These data suggest that the increased LD-mitochondria contact with exercise is not disturbed in obesity or type 2 diabetes. KEY POINTS: Type 2 diabetes is associated with altered interactivity between lipid droplets (LDs) and mitochondria in the skeletal muscle. Physical contact between the surface of LDs and the surrounding mitochondrial network is considered favourable for fat oxidation. We show that 1 h of acute exercise increases the length of contact between LDs and mitochondria, irrespective of obesity or type 2 diabetes. This contact length between LDs and mitochondria is not associated with a net decrease in the LD volumetric density after the acute exercise. However, it correlates with the fat oxidation rate during exercise. Our data establish that exercise mediates contact between LDs and the mitochondrial network and that this effect is not impaired in individuals with type 2 diabetes or obesity.

No net utilization of intramuscular lipid droplets during repeated high-intensity intermittent exercise.

Intramuscular lipids are stored as subsarcolemmal or intramyofibrillar droplets with potential diverse roles in energy metabolism. We examined intramuscular lipid utilization through transmission electron microscopy during repeated high-intensity intermittent exercise, an aspect that is hitherto unexplored. Seventeen moderately to well-trained males underwent three periods (EX1-EX3) of 10 × 45-s high-intensity cycling [∼100%-120% Wattmax (Wmax)] combined with maximal repeated sprints (∼250%-300% Wmax). M. vastus lateralis biopsies were obtained at baseline, after EX1, and EX3. During the complete exercise session, no net decline in either subsarcolemmal or intermyofibrillar lipid volume density occurred. However, a temporal relationship emerged for subsarcolemmal lipids with an ∼11% increase in droplet size after EX1 (P = 0.024), which reverted to baseline levels after EX3 accompanied by an ∼30% reduction in the numerical density of subsarcolemmal lipid droplets compared with both baseline (P = 0.019) and after EX1 (P = 0.018). Baseline distinctions were demonstrated with an approximately twofold higher intermyofibrillar lipid volume in type 1 versus type 2 fibers (P = 0.008), mediated solely by a higher number rather than the size of lipid droplets (P < 0.001). No fiber-type-specific differences were observed in subsarcolemmal lipid volume although type 2 fibers exhibited ∼17% larger droplets (P = 0.034) but a lower numerical density (main effect; P = 0.010) including 3% less droplets at baseline. Collectively, these findings suggest that intramuscular lipids do not serve as an important substrate during high-intensity intermittent exercise; however, the repeated exercise pattern mediated a temporal remodeling of the subsarcolemmal lipid pool. Furthermore, fiber-type- and compartment-specific differences were found at baseline underscoring the heterogeneity in lipid droplet deposition.NEW & NOTEWORTHY Undertaking a severe repeated high-intensity intermittent exercise protocol led to no net decline in neither subsarcolemmal nor intermyofibrillar lipid content in the thigh muscle of young moderately to well-trained participants. However, a temporal remodeling of the subsarcolemmal pool of lipid droplets did occur indicative of potential transient lipid accumulation. Moreover, baseline fiber-type distinctions in subcellular lipid droplet deposition were present underscoring the diversity in lipid droplet storage among fiber types and subcellular regions.

Lowered muscle glycogen reduces body mass with no effect on short-term exercise performance in men.

Performance in short-duration sports is highly dependent on muscle glycogen, but the total degradation is only moderate and considering the water-binding property of glycogen, unnecessary storing of glycogen may cause an unfavorable increase in body mass. To investigate this, we determined the effect of manipulating dietary carbohydrates (CHO) on muscle glycogen content, body mass, and short-term exercise performance. In a randomized and counterbalanced cross-over design, twenty-two men completed two maximal cycle tests of either 1-min (n = 10) or 15-min (n = 12) duration with different pre-exercise muscle glycogen levels. Glycogen manipulation was initiated three days prior to the tests by exercise-induced glycogen depletion followed by ingestion of a moderate (M-CHO) or high (H-CHO) CHO-diet. Subjects were weighed before each test, and muscle glycogen content was determined in biopsies from m. vastus lateralis before and after each test. Pre-exercise muscle glycogen content was lower following M-CHO than H-CHO (367 mmol · kg-1 DW vs. 525 mmol · kg-1 DW, p < 0.00001), accompanied by a 0.7 kg lower body mass (p < 0.00001). No differences were observed in performance between diets in neither the 1-min (p = 0.33) nor the 15-min (p = 0.99) test. In conclusion, pre-exercise muscle glycogen content and body mass were lower after ingesting moderate compared with high amounts of CHO, while short-term exercise performance was unaffected. This demonstrates that adjusting pre-exercise glycogen levels to the requirements of competition may provide an attractive weight management strategy in weight-bearing sports, particularly in athletes with high resting glycogen levels.

Exercise and fatigue: integrating the role of K+, Na+ and Cl- in the regulation of sarcolemmal excitability of skeletal muscle.

Perturbations in K+ have long been considered a key factor in skeletal muscle fatigue. However, the exercise-induced changes in K+ intra-to-extracellular gradient is by itself insufficiently large to be a major cause for the force decrease during fatigue unless combined to other ion gradient changes such as for Na+. Whilst several studies described K+-induced force depression at high extracellular [K+] ([K+]e), others reported that small increases in [K+]e induced potentiation during submaximal activation frequencies, a finding that has mostly been ignored. There is evidence for decreased Cl- ClC-1 channel activity at muscle activity onset, which may limit K+-induced force depression, and large increases in ClC-1 channel activity during metabolic stress that may enhance K+ induced force depression. The ATP-sensitive K+ channel (KATP channel) is also activated during metabolic stress to lower sarcolemmal excitability. Taking into account all these findings, we propose a revised concept in which K+ has two physiological roles: (1) K+-induced potentiation and (2) K+-induced force depression. During low-moderate intensity muscle contractions, the K+-induced force depression associated with increased [K+]e is prevented by concomitant decreased ClC-1 channel activity, allowing K+-induced potentiation of sub-maximal tetanic contractions to dominate, thereby optimizing muscle performance. When ATP demand exceeds supply, creating metabolic stress, both KATP and ClC-1 channels are activated. KATP channels contribute to force reductions by lowering sarcolemmal generation of action potentials, whilst ClC-1 channel enhances the force-depressing effects of K+, thereby triggering fatigue. The ultimate function of these changes is to preserve the remaining ATP to prevent damaging ATP depletion.

Prolonged loss of force and power following fatiguing contractions in rat soleus muscles. Is low-frequency fatigue an issue during dynamic contractions?

Low-frequency fatigue (LFF) is defined by a relatively larger deficit in isometric force elicited by low-frequency electrical stimulation compared with high-frequency stimulation. However, the effects of LFF on power during dynamic contractions elicited at low and high frequencies have not been thoroughly characterized. In the current study, rat soleus muscles underwent fatiguing either concentric, eccentric, or isometric contractions. Before and 1 h after the fatiguing contractions, a series of brief isometric and dynamic contractions elicited at 20 and 80 Hz stimulation to establish force-velocity relationships. Maximal force (Fmax), velocity (Vmax), and power (Pmax) were assessed for each frequency. Sarcoplasmic reticulum (SR) Ca2+ release and reuptake rates were assessed pre- and postfatigue. Prolonged fatigue was observed as a loss of Fmax and Pmax in muscles fatigued by concentric or eccentric, but not by isometric contractions. When quantified as a decrease in the ratio between 20 Hz and 80 Hz contractile output, LFF was more pronounced for isometric force than for power (-21% vs. -16% for concentrically fatigued muscles, P = 0.003; 29 vs. 13% for eccentrically fatigued muscles, P < 0.001). No changes in SR Ca2+ release or reuptake rates were observed. We conclude that LFF is less pronounced when expressed in terms of power deficits than when expressed in terms of force deficits, and that LFF, therefore, likely affects performance to a lesser degree during fast concentric contractions than during static or slow contractions.

Fibre type- and localisation-specific muscle glycogen utilisation during repeated high-intensity intermittent exercise.

Glycogen particles are situated in key areas of the muscle cell in the vicinity of the main energy-consumption sites and may be utilised heterogeneously dependent on the nature of the metabolic demands. The present study aimed to investigate the time course of fibre type-specific utilisation of muscle glycogen in three distinct subcellular fractions (intermyofibrillar, IMF; intramyofibrillar, Intra; and subsarcolemmal, SS) during repeated high-intensity intermittent exercise. Eighteen moderately to well-trained male participants performed three periods of 10 × 45 s cycling at ∼105% watt max (EX1-EX3) coupled with 5 × 6 s maximal sprints at baseline and after each period. Muscle biopsies were sampled at baseline and after EX1 and EX3. A higher glycogen breakdown rate in type 2 compared to type 1 fibres was found during EX1 for the Intra (-72 vs. -45%) and IMF (-59 vs. -35%) glycogen fractions (P < 0.001) but with no differences for SS glycogen (-52 vs. -40%). In contrast, no fibre type differences were observed during EX2-EX3, where the utilisation of Intra and IMF glycogen in type 2 fibres was reduced, resulting in depletion of all three subcellular fractions to very low levels post-exercise within both fibre types. Importantly, large heterogeneity in single-fibre glycogen utilisation was present with an early depletion of especially Intra glycogen in individual type 2 fibres. In conclusion, there is a clear fibre type- and localisation-specific glycogen utilisation during high-intensity intermittent exercise, which varies with time course of exercise and is characterised by exacerbated pool-specific glycogen depletion at the single-fibre level. KEY POINTS: Muscle glycogen is the major fuel during high-intensity exercise and is stored in distinct subcellular areas of the muscle cell in close vicinity to the main energy consumption sites. In the present study quantitative electron microscopy imaging was used to investigate the utilisation pattern of three distinct subcellular muscle glycogen fractions during repeated high-intensity intermittent exercise. It is shown that the utilisation differs dependent on fibre type, subcellular localisation and time course of exercise and with large single-fibre heterogeneity. These findings expand on our understanding of subcellular muscle glycogen metabolism during exercise and may help us explain how reductions in muscle glycogen can attenuate muscle function even at only moderately lowered whole-muscle glycogen concentrations.

Muscle metabolism and impaired sprint performance in an elite women's football game.

The present study examined skeletal muscle metabolism and changes in repeated sprint performance during match play for n = 20 competitive elite women outfield players. We obtained musculus vastus lateralis biopsies and blood samples before, after, and following intense periods in each half of a friendly match, along with 5 × 30-meter sprint tests and movement pattern analyses (10-Hz S5 Global Positioning System [GPS]). Muscle glycogen decreased by 39% and 42% after an intense period of the second half and after the match, respectively, compared to baseline (p < 0.05). Post-match, 80% type I fibers and 69% type II fibers were almost empty or completely empty of glycogen. Muscle lactate was higher (p < 0.05) after the intense period of the first half and post-match compared to baseline (14.3 ± 4.6 (±SEM) and 12.9 ± 5.7 vs. 6.4 ± 3.7 mmol/kg d.w.). Muscle phosphocreatine was reduced (p < 0.05) by 16% and 12%, respectively, after an intense period in the first and second half compared to baseline. Blood lactate and glucose increased during the match and peaked at 8.4 ± 2.0 and 7.9 ± 1.2 mmol/L, respectively. Mean 5 × 30 m sprint time declined by 3.2 ± 1.7 and 7.0 ± 2.1% after the first and second half, respectively, and 4.7 ± 1.6% (p < 0.05) after an intense period in the first half compared to baseline. In conclusion, match play in elite female football players resulted in marked glycogen depletion in both fiber types, which may explain fatigue at the end of a match. Repeated sprint ability was impaired after intense periods in the first half and after both halves, which may be associated with the observed muscle metabolite perturbations.

The expression of HSP70 in skeletal muscle is not associated with glycogen availability during recovery following prolonged exercise in elite endurance athletes.

The 70-kDa heat shock protein (HSP70) is a ubiquitous molecular chaperone which is highly inducible by cellular stress such as exercise. To investigate the role of muscle glycogen content on the HSP70 expression, muscle glycogen was manipulated by consumption of either water (H2O) or a carbohydrate-enriched diet (CHO) during recovery from 4 h of glycogen-depleting cycling exercise in fourteen elite endurance athletes. Muscle biopsies were obtained pre- and post-exercise, and after 4 and 24 h of recovery, and analyzed for HSP70 mRNA expression, as well as HSP70 protein expression and muscle glycogen within the same skeletal muscle fibers using immunohistochemistry. Exercise reduced glycogen by 59 ± 10% (P < 0.0001). After 4 h of recovery, glycogen approached resting levels in the CHO group (86% of pre, P = 0.28) but remained suppressed in the H2O group (41% of pre, P < 0.001) (group × time interaction: P = 0.002). Importantly, both the HSP70 mRNA (+ 1.6-fold (+ 0.28/- 0.24), P = 0.02) and protein expression (+ 147 ± 99%, P < 0.0001) was substantially increased after exercise and remained elevated in both groups after 4 h of recovery, despite clear differences in muscle glycogen content. Thus, muscle glycogen content was not related to the variation in single fiber HSP70 expression at the 4-h time-point (r2 = 0.004). In conclusion, muscle HSP70 expression remained elevated during recovery from prolonged exercise in highly trained skeletal muscle, irrespective of muscle glycogen availability.

Myocardial subcellular glycogen distribution and sarcoplasmic reticulum Ca2+ handling: effects of ischaemia, reperfusion and ischaemic preconditioning.

Ischaemic preconditioning (IPC) protects against myocardial ischaemia-reperfusion injury. The metabolic and ionic effects of IPC remain to be clarified in detail. We aimed to investigate the effect of IPC (2 times 5 min ischaemia) on the subcellular distribution of glycogen and Ca2+-uptake and leakiness by the sarcoplasmic reticulum (SR) in response to ischaemia-reperfusion in cardiomyocytes of isolated perfused rat hearts (Wistar rats, 335 ± 25 g). As estimated by quantitative transmission electron microscopy, the pre-ischaemic contribution [%, mean (95% CI)] of three sub-fractions of glycogen relative to total glycogen was 50 (39:61) as subsarcolemmal, 41 (31:50) as intermyofibrillar, and 9 (5:13) as intramyofibrillar glycogen. After 25 min of ischaemia, the relative contribution (%) of subsarcolemmal glycogen decreased to 39 (32:47) in control hearts (Con) and to 38 (31:45) in IPC. After 15 min reperfusion the contribution of subsarcolemmal glycogen was restored to pre-ischaemic levels in IPC hearts, but not in Con hearts. IPC increased the left ventricular developed pressure following ischaemia-reperfusion compared with Con. In saponin-skinned cardiomyocyte bundles, ischaemia reduced the SR Ca2+-uptake rate, with no effect of IPC. However, IPC reduced a SR Ca2+-leakage at pre-ischaemia, after ischaemia and during reperfusion. In conclusion, subsarcolemmal glycogen was preferentially utilised during sustained myocardial ischaemia. IPC improved left ventricular function reflecting reduced ischaemia-reperfusion injury, mediated a re-distribution of glycogen towards a preferential storage within the subsarcolemmal space during reperfusion, and lowered SR Ca2+-leakage. Under the present conditions, we found no temporal associations between alterations in glycogen localisation and SR Ca2+ kinetics.

Glycogen supercompensation is due to increased number, not size, of glycogen particles in human skeletal muscle.

NEW FINDINGS: What is the central question of this study? Glycogen supercompensation after glycogen-depleting exercise can be achieved by consuming a carbohydrate-enriched diet, but the associated effects on the size, number and localization of intramuscular glycogen particles are unknown. What is the main finding and its importance? Using transmission electron microscopy to inspect individual glycogen particles visually, we show that glycogen supercompensation is achieved by increasing the number of particles while keeping them at submaximal sizes. This might be a strategy to ensure that glycogen particles can be used fast, because particles that are too large might impair utilization rate. ABSTRACT: Glycogen supercompensation after glycogen-depleting exercise can be achieved by consuming a carbohydrate-enriched diet, but the associated effects on the size, number and localization of intramuscular glycogen particles are unknown. We investigated how a glycogen-loading protocol affects fibre type-specific glycogen volume density, particle diameter and numerical density in three subcellular pools: between (intermyofibrillar) or within (intramyofibrillar) the myofibrils or beneath the sarcolemma (subsarcolemmal). Resting muscle biopsies from 11 physically active men were analysed using transmission electron microscopy after mixed (MIX), LOW or HIGH carbohydrate consumption separated by glycogen-lowering cycling at 75% of maximal oxygen consumption until exhaustion. After HIGH, the total volumetric glycogen content was 40% [95% confidence interval 16, 68] higher than after MIX in type I fibres (P < 0.001), with little to no difference in type II fibres (9% [95% confidence interval -9, 27]). Median particle diameter was 22.5 (interquartile range 20.8-24.7) nm across glycogen pools and fibre types, and the numerical density was 61% [25, 107] and 40% [9, 80] higher in the subsarcolemmal (P < 0.001) and intermyofibrillar (P < 0.01) pools of type I fibres, respectively, with little to no difference in the intramyofibrillar pool (3% [-20, 32]). In LOW, total glycogen was in the range of 21-23% lower, relative to MIX, in both fibre types, reflected in a 21-46% lower numerical density across pools. In comparison to MIX, particle diameter was unaffected by other diets ([-1.4, 1.3] nm). In conclusion, glycogen supercompensation after prolonged cycling is exclusive to type I fibres, predominantly in the subsarcolemmal pool, and involves an increase in the numerical density rather than the size of existing glycogen particles.

Inhibition of glycogenolysis prolongs action potential repriming period and impairs muscle function in rat skeletal muscle.

KEY POINTS: Muscle glycogen content is associated with muscle function, but the physiological link between the two is poorly understood. This study investigated the effects of inhibiting glycogenolysis, while maintaining high overall energy status, on different aspects of muscle function. We demonstrate here that Na+ ,K+ -ATPase activity depends on glycogenolytically derived ATP regardless of high global ATP, with a decrease in activity leading to reduced force production and accelerated fatigue development. The results support the concept of compartmentalized energy transfer with glycogen metabolism playing a crucial role in intramuscular ATP resynthesis and ion regulation. This study gives specific insights into muscular function and may help towards a better understanding of glycogen storage diseases and muscle fatigue. ABSTRACT: Skeletal muscle glycogen content is associated with muscle function and fatigability. However, little is known about the physiological link between glycogen content and muscle function. Here we aimed to investigate the importance of glycogenolytically derived ATP per se on muscle force and action potential (AP) repriming period, i.e. the time before a second AP can be produced (indicative of Na+ ,K+ -ATPase activity). Single fibres from rat extensor digitorum longus muscles were isolated and mechanically skinned in order to investigate force production and the AP repriming period while global ATP and PCr concentrations were kept high. The importance of glycogenolytically derived ATP was studied by inhibition of glycogen phosphorylase (1,4-dideoxy-1,4-imino-d-arabinitol (DAB; 2 mm) or CP-316,819 (CP; 10 µm)) or glycogen removal (amyloglucosidase, 20 U ml-1 ). Tetanic force decreased by (mean (SD)) 21 (15)% (P < 0.001) and 76 (28)% (DAB) or 94 (6)% (CP, P < 0.001) in well-polarized and partially depolarized fibres, respectively. In depolarized fibres, twitch force decreased by 16 (10)% and 55 (26)% with DAB and CP, respectively, with no effect in well-polarized fibres (84 (10)%, P = 0.14). There was no effect of glycogen phosphorylase inhibition on repriming period in well-polarized fibres (median (25th, 75th percentile): 5 (4, 5) vs. 4 (4, 5) ms, P = 0.26), while the repriming period was prolonged from 6 (5, 7) to 8 (7, 10) ms (P = 0.01) in partially depolarized fibres. In line with this, glycogen removal increased repriming period from 5 (5, 6) to 6 (5, 7) ms (P = 0.003) in depolarized fibres. Together, these data strongly indicate that blocking glycogenolysis attenuates Na+ ,K+ -ATPase activity, which in turn increases the repriming period and reduces force, demonstrating a functional link between glycogenolytically derived ATP and force production.

Heterogeneity in subcellular muscle glycogen utilisation during exercise impacts endurance capacity in men.

KEY POINTS: When muscle biopsies first began to be used routinely in research on exercise physiology five decades ago, it soon become clear that the muscle content of glycogen is an important determinant of exercise performance. Glycogen particles are stored in distinct pools within the muscles, but the role of each pool during exercise and how this is affected by diet is unknown. Here, the effects of diet and exercise on these pools, as well as their relation to endurance during prolonged cycling were examined. We demonstrate here that an improved endurance capacity with high carbohydrate loading is associated with a temporal shift in the utilisation of the distinct stores of glycogen pools and is closely linked to the content of the glycogen pool closest to actin and myosin (intramyofibrillar glycogen). These findings highlight the functional importance of distinguishing between different subcellular microcompartments of glycogen in individual muscle fibres. ABSTRACT: In muscle cells, glycogen is stored in three distinct subcellular pools: between or within myofibrils (inter- and intramyofibrillar glycogen, respectively) or beneath the sarcolemma (subsarcolemmal glycogen) and these pools may well have different functions. Here, we investigated the effect of diet and exercise on the content of these distinct pools and their relation to endurance capacity in type 1 and 2 muscle fibres. Following consumption of three different diets (normal, mixed diet = MIX, high in carbohydrate = HIGH, or low in carbohydrate = LOW) for 72 h, 11 men cycled at 75% of V̇O2 max until exhaustion. The volumetric content of the glycogen pools in muscle biopsies obtained before, during, and after exercise were quantified by transmission electron micrographs. The mean (SD) time to exhaustion was 150 (30), 112 (22), and 69 (18) minutes in the HIGH, MIX and LOW trials, respectively (P < 0.001). As shown by multiple regression analyses, the intramyofibrillar glycogen content in type 1 fibres, particularly after 60 min of exercise, correlated most strongly with time to exhaustion. In the HIGH trial, intramyofibrillar glycogen was spared during the initial 60 min of exercise, which was associated with levels and utilisation of subsarcolemmal glycogen above normal. In all trials, utilisation of subsarcolemmal and intramyofibrillar glycogen was more pronounced than that of intermyofibrillar glycogen in relative terms. In conclusion, the muscle pool of intramyofibrillar glycogen appears to be the most important for endurance capacity in humans. In addition, a local abundance of subsarcolemmal glycogen reduces the utilisation of intramyofibrillar glycogen during exercise.