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.

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.

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.

Prolonged force depression after mechanically demanding contractions is largely independent of Ca2+ and reactive oxygen species.

Increased production of reactive oxygen/nitrogen species (ROS) and impaired cellular Ca2+ handling are implicated in the prolonged low-frequency force depression (PLFFD) observed in skeletal muscle after both metabolically and mechanically demanding exercise. Metabolically demanding high-intensity exercise can induce PLFFD accompanied by ROS-dependent fragmentation of the sarcoplasmic reticulum Ca2+ release channels, the ryanodine receptor 1s (RyR1s). We tested whether similar changes occur after mechanically demanding eccentric contractions. Human subjects performed 100 repeated drop jumps, which require eccentric knee extensor contractions upon landing. This exercise caused a major PLFFD, such that maximum voluntary and electrically evoked forces did not recover within 24 h. Drop jumps induced only minor signs of increased ROS, and RyR1 fragmentation was observed in only 3 of 7 elderly subjects. Also, isolated mouse muscle preparations exposed to drop-jump-mimicking eccentric contractions showed neither signs of increased ROS nor RyR1 fragmentation. Still, the free cytosolic [Ca2+] during tetanic contractions was decreased by ∼15% 1 h after contractions, which can explain the exaggerated force decrease at low-stimulation frequencies but not the major frequency-independent force depression. In conclusion, PLFFD caused by mechanically demanding eccentric contractions does not involve any major increase in ROS or RyR1 fragmentation.-Kamandulis, S., de Souza Leite, F., Hernandez, A., Katz, A., Brazaitis, M., Bruton, J. D., Venckunas, T., Masiulis, N., Mickeviciene, D., Eimantas, N., Subocius, A., Rassier, D. E., Skurvydas, A., Ivarsson, N., Westerblad, H. Prolonged force depression after mechanically demanding contractions is largely independent of Ca2+ and reactive oxygen species.

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.

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.

Impaired Ca(2+) release contributes to muscle weakness in a rat model of critical illness myopathy.

BACKGROUND: Critical illness myopathy is an acquired skeletal muscle disorder with severe myosin loss and muscle weakness frequently seen in intensive care unit (ICU) patients. It is unknown if impaired excitation-contraction coupling contributes to the muscle weakness. METHODS: We used a unique ICU model where rats were deeply sedated, post-synaptically pharmacologically paralyzed, mechanically ventilated and closely monitored for up to ten days. Single intact fibers from the flexor digitorum brevis muscle were isolated and used to measure force and free myoplasmic [Ca(2+)] ([Ca(2+)]i) during tetanic contractions. RESULTS: Fibers from ICU rats had 80 % lower tetanic [Ca(2+)]i and produced only 15 % of the force seen in fibers from sham-operated (SHAM) rats. In the presence of 5 mM caffeine, tetanic [Ca(2+)]i was similar in fibers from ICU and SHAM rats but force was 50 % lower in fibers from ICU rats than SHAM rats. Confocal imaging showed disrupted tetanic [Ca(2+)]i transients in fibers from ICU rats compared to SHAM rats. Western blots showed similar levels of Na(+) channel and dihydropyridine receptor (DHPR) protein expression, whereas ryanodine receptor (RyR) and sarco-endoplasmic reticulum Ca(2+) ATPase 1 (SERCA1) expression was markedly lower in muscle of ICU rats than in SHAM rats. Immunohistochemical analysis showed that distribution of Na(+) channel and DHPR protein on the sarcolemma was disrupted in fibers from ICU rats compared with SHAM rats. CONCLUSIONS: These results suggest that impaired SR Ca(2+) release contributes to the muscle weakness seen in patients in ICU.

Antioxidant treatments do not improve force recovery after fatiguing stimulation of mouse skeletal muscle fibres.

The contractile performance of skeletal muscle declines during intense activities, i.e. fatigue develops. Fatigued muscle can enter a state of prolonged low-frequency force depression (PLFFD). PLFFD can be due to decreased tetanic free cytosolic [Ca(2+) ] ([Ca(2+) ]i ) and/or decreased myofibrillar Ca(2+) sensitivity. Increases in reactive oxygen and nitrogen species (ROS/RNS) may contribute to fatigue-induced force reductions. We studied whether pharmacological ROS/RNS inhibition delays fatigue and/or counteracts the development of PLFFD. Mechanically isolated mouse fast-twitch fibres were fatigued by sixty 150 ms, 70 Hz tetani given every 1 s. Experiments were performed in standard Tyrode solution (control) or in the presence of: NADPH oxidase (NOX) 2 inhibitor (gp91ds-tat); NOX4 inhibitor (GKT137831); mitochondria-targeted antioxidant (SS-31); nitric oxide synthase (NOS) inhibitor (l-NAME); the general antioxidant N-acetylcysteine (NAC); a cocktail of SS-31, l-NAME and NAC. Spatially and temporally averaged [Ca(2+) ]i and peak force were reduced by ∼20% and ∼70% at the end of fatiguing stimulation, respectively, with no marked differences between groups. PLFFD was similar in all groups, with 30 Hz force being decreased by ∼60% at 30 min of recovery. PLFFD was mostly due to decreased tetanic [Ca(2+) ]i in control fibres and in the presence of NOX2 or NOX4 inhibitors. Conversely, in fibres exposed to SS-31 or the anti ROS/RNS cocktail, tetanic [Ca(2+) ]i was not decreased during recovery so PLFFD was only caused by decreased myofibrillar Ca(2+) sensitivity. The cocktail also increased resting [Ca(2+) ]i and ultimately caused cell death. In conclusion, ROS/RNS-neutralizing compounds did not counteract the force decline during or after induction of fatigue.

Effects of N-acetylcysteine on isolated mouse skeletal muscle: contractile properties, temperature dependence, and metabolism.

The effects of the general antioxidant N-acetylcysteine (NAC) on muscle function and metabolism were examined. Isolated paired mouse extensor digitorum longus muscles were studied in the absence or presence of 20 mM NAC. Muscles were electrically stimulated to perform 100 isometric tetanic contractions (300 ms duration) at frequencies resulting in ∼85% of maximal force (70-150 Hz at 25-40 °C). NAC did not significantly affect peak force in the unfatigued state at any temperature but significantly slowed tetanic force development in a temperature-dependent fashion (e.g., time to 50% of peak tension averaged 35 ± 2 ms [control] and 37 ± 1 ms [NAC] at 25 °C vs. 21 ± 1 ms [control] and 52 ± 6 ms [NAC, P < 0.01] at 40 °C). During repeated contractions, NAC maximally enhanced peak force by the fifth tetanus at all temperatures (by ∼30%). Thereafter, the effect of NAC disappeared rapidly at high temperatures (35-40 °C) and more slowly at the lower temperatures (25-30 °C). At all temperatures, the enhancing effect of NAC on peak force was associated with a slowing of relaxation. NAC did not significantly affect myosin light chain phosphorylation at rest or after five contractions (∼50% increase vs. rest). After five tetani, lactate and inorganic phosphate increased about 20-fold and 2-fold, respectively, both in control and NAC-treated muscles. Interestingly, after five tetani, the increase in glucose 6-P was ∼2-fold greater, whereas the increase in malate was inhibited by ∼75% with NAC vs. control, illustrating the metabolic effects of NAC. NAC slightly decreased the maximum shortening velocity in early fatigue (five to seven repeated tetani). These data demonstrate that the antioxidant NAC transiently enhances muscle force generation by a mechanism that is independent of changes in myosin light chain phosphorylation and inorganic phosphate. The slowing of relaxation suggests that NAC enhances isometric force by facilitating fusion (i.e., delaying force decline between pulses). The initial slowing of tension development and subsequent slowing of relaxation suggest that NAC would result in impaired performance during a high-intensity dynamic exercise.

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.

Doublet discharge stimulation increases sarcoplasmic reticulum Ca2+ release and improves performance during fatiguing contractions in mouse muscle fibres.

Double discharges (doublets) of motor neurones at the onset of contractions increase both force and rate of force development during voluntary submaximal contractions. The purpose of this study was to examine the role of doublet discharges on force and myoplasmic free [Ca(2+)] ([Ca(2+)]i) during repeated fatiguing contractions, using a stimulation protocol mimicking the in vivo activation pattern during running. Individual intact fibres from the flexor digitorum brevis muscle of mice were stimulated at 33°C to undergo 150 constant-frequency (five pulses at 70 Hz) or doublet (an initial, extra pulse at 200 Hz) contractions at 300 ms intervals. In the unfatigued state, doublet stimulation resulted in a transient (∼10 ms) approximate doubling of [Ca(2+)]i, which was accompanied by a greater force-time integral (∼70%) and peak force (∼40%) compared to constant frequency contractions. Moreover, doublets markedly increased force-time integral and peak force during the first 25 contractions of the fatiguing stimulation. In later stages of fatigue, addition of doublets increased force production but the increase in force production corresponded to only a minor portion of the fatigue-induced reduction in force. In conclusion, double discharges at the onset of contractions effectively increase force production, especially in early stages of fatigue. This beneficial effect occurs without additional force loss in later stages of fatigue, indicating that the additional energy cost induced by doublet discharges to skeletal muscle is limited.

TLR4 as receptor for HMGB1 induced muscle dysfunction in myositis.

OBJECTIVES: Polymyositis and dermatomyositis are characterised by muscle weakness and fatigue even in patients with normal muscle histology via unresolved pathogenic mechanisms. In this study, we investigated the mechanisms by which high mobility group box protein 1 (HMGB1) acts to accelerate muscle fatigue development. METHODS: Intact single fibres were dissociated from flexor digitorum brevis (FDB) of wild type, receptor for advanced glycation endproduct (RAGE) knockout and toll like receptor 4 (TLR4) knockout mice and cultured in the absence or presence of recombinant HMGB1. A decrease in sarcoplasmic reticulum Ca(2+) release during a series of 300 tetanic contractions, which reflects the development of muscle fatigue, was determined by measuring myoplasmic free tetanic Ca(2+). TLR4 and major histocompatibility complex (MHC)-class I expression in mouse FDB fibres were investigated by immunofluorescence and confocal microscopy. Immunohistochemistry was used to investigate TLR4, MHC-class I and myosin heavy chain expression in muscle fibres of patients. RESULTS: Our results demonstrate that TLR4 is expressed in human and mouse skeletal muscle fibres, and coexpressed with MHC-class I in muscle fibres of patients with myositis. Furthermore, we show that HMGB1 acts via TLR4 but not RAGE to accelerate muscle fatigue and to induce MHC-class I expression in vitro. In order to bind and signal via TLR4, HMGB1 must have a reduced cysteine 106 and a disulphide linkage between cysteine 23 and 45. CONCLUSIONS: The HMGB1-TLR4 pathway may play an important role in causing muscle fatigue in patients with polymyositis or dermatomyositis and thus is a potential novel target for future therapy.

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.

Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle.

Dietary inorganic nitrate has profound effects on health and physiological responses to exercise. Here, we examined if nitrate, in doses readily achievable via a normal diet, could improve Ca(2+) handling and contractile function using fast- and slow-twitch skeletal muscles from C57bl/6 male mice given 1 mm sodium nitrate in water for 7 days. Age matched controls were provided water without added nitrate. In fast-twitch muscle fibres dissected from nitrate treated mice, myoplasmic free [Ca(2+)] was significantly greater than in Control fibres at stimulation frequencies from 20 to 150 Hz, which resulted in a major increase in contractile force at ≤ 50 Hz. At 100 Hz stimulation, the rate of force development was ∼35% faster in the nitrate group. These changes in nitrate treated mice were accompanied by increased expression of the Ca(2+) handling proteins calsequestrin 1 and the dihydropyridine receptor. No changes in force or calsequestrin 1 and dihydropyridine receptor expression were measured in slow-twitch muscles. In conclusion, these results show a striking effect of nitrate supplementation on intracellular Ca(2+) handling in fast-twitch muscle resulting in increased force production. A new mechanism is revealed by which nitrate can exert effects on muscle function with applications to performance and a potential therapeutic role in conditions with muscle weakness.

Impaired mitochondrial respiration and decreased fatigue resistance followed by severe muscle weakness in skeletal muscle of mitochondrial DNA mutator mice.

Mitochondrial dysfunction can drastically impair muscle function, with weakness and exercise intolerance as key symptoms. Here we examine the time course of development of muscle dysfunction in a mouse model of premature ageing induced by defective proofreading function of mitochondrial DNA (mtDNA) polymerase (mtDNA mutator mouse). Isolated fast-twitch muscles and single muscle fibres from young (3-5 months) and end-stage (11 months) mtDNA mutator mice were compared to age-matched control mice. Force and free myoplasmic [Ca(2+)] ([Ca(2+)](i)) were measured under resting conditions and during fatigue induced by repeated tetani. Muscles of young mtDNA mutator mice displayed no weakness in the rested state, but had lower force and [Ca(2+)](i) than control mice during induction of fatigue. Muscles of young mtDNA mutator mice showed decreased activities of citrate synthase and ß-hydroxyacyl-coenzyme A dehydrogenase, reduced expression of cytochrome c oxidase, and decreased expression of triggers of mitochondrial biogenesis (PGC-1α, PPARα, AMPK). Muscles from end-stage mtDNA mutator mice showed weakness under resting conditions with markedly decreased tetanic [Ca(2+)](i), force per cross-sectional area and protein expression of the sarcoplasmic reticulum Ca(2+) pump (SERCA1). In conclusion, fast-twitch muscles of prematurely ageing mtDNA mutator mice display a sequence of deleterious mitochondrial-to-nucleus signalling with an initial decrease in oxidative capacity, which was not counteracted by activation of signalling to increase mitochondrial biogenesis. This was followed by severe muscle weakness in the end stage. These results have implication for normal ageing and suggest that decreased mitochondrial oxidative capacity due to a sedentary lifestyle may predispose towards muscle weakness developing later in life.

Non-crossbridge calcium-dependent stiffness in slow and fast skeletal fibres from mouse muscle.

We showed previously that force development in frog and FDB mouse skeletal muscle fibres is preceded by an increase of fibre stiffness occurring well before crossbridge attachment and force generation. This stiffness increase, referred to as static stiffness, is due to a Ca(2+)-dependent stiffening of a non-crossbridge sarcomere structure which we suggested could be attributed to the titin filaments. To investigate further the role of titin in static stiffness, we measured static stiffness properties at 24 and 35°C in soleus and EDL mouse muscle fibres which are known to express different titin isoforms. We found that static stiffness was present in both soleus and EDL fibres, however, its value was about five times greater in EDL than in soleus fibres. The rate of development of static stiffness on stimulation increased with temperature and was slightly faster in EDL than in soleus in agreement with previously published data on the time course of the intracellular Ca(2+) transients in these muscles. The present results show that the presence of a non-crossbridge Ca(2+)-dependent stiffening of the muscle fibre is a physiological general characteristic of skeletal muscle. Static stiffness depends on fibre type, being greater and developing faster in fast than in slow fibres. Our observations are consistent with the idea that titin stiffening on contraction improves the sarcomere structure stability. Such an action in fact seems to be more important in EDL fast fibre than in soleus slow fibres.

Methods to detect Ca(2+) in living cells.

Measurements of free cytosolic Ca(2+) concentration ([Ca(2+)](i)) or free Ca(2+) concentration in cellular organelles have become more routine. The primary reason for this is the availability of membrane permeant forms of Ca(2+) indicators that can easily enter cells. In this chapter, the properties required of an ideal Ca(2+) indicator are identified and the advantages and disadvantages of available Ca(2+) indicators are pointed out. The pitfalls associated with usage of Ca(2+) indicators together with the clear advantages of ratiometric over non-ratiometric indicators are discussed. The excitation of Ca(2+) indicators and detection of the emitted fluorescence light require dedicated equipment; epifluorescence or confocal microscopes are most frequently used for this purpose and the advantages and disadvantages of these are discussed. Calibration experiments are required to translate changes in the fluorescence of Ca(2+) indicators into real [Ca(2+)](i) changes, but this procedure is non-trivial and potential sources of error are identified. Future developments in the field of Ca(2+) detection are discussed.

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.

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.

Force decline during fatigue is due to both a decrease in the force per individual cross-bridge and the number of cross-bridges.

Fatigue occurring during exercise can be defined as the inability to maintain the initial force or power output. As fatigue becomes pronounced, force and maximum velocity of shortening are greatly reduced and force relaxation is prolonged. In principle, force loss during fatigue can result from a decrease in the number of cross-bridges generating force or a decrease of the individual cross-bridge force or to both mechanisms. The present experiments were made to investigate this point in single fibres or small fibre bundles isolated from flexor digitorum brevis (FDB) of C57BL/6 mice at 22-24◦C. During a series of 105 tetanic contractions, we measured force and fibre stiffness by applying small sinusoidal length oscillations at 2.5 or 4 kHz frequency to the activated preparation and measuring the resulting force changes. Stiffness data were corrected for the influence of compliance in series with the cross-bridge ensemble. The results show that the force decline during the first 20 tetani is due to the reduction of force developed by the individual cross-bridges and thereafter as fatigue becomes more severe, the number of cross-bridges decreases. In spite of the force reduction in the early phase of fatigue, there was an increased rate of tetanic force development and relaxation. In the latter stages of fatigue, the rate of force development and relaxation became slower. Thus, the start of fatigue is characterised by decreased cross-bridge force development and as fatigue becomes more marked, the number of cross-bridges decreases. These findings are discussed in the context of the current hypotheses about fatigue mechanisms.