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1.
Adv Exp Med Biol ; 1131: 7-26, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646505

RESUMO

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.


Assuntos
Cálcio , Citosol , Organelas , Animais , Cálcio/metabolismo , Técnicas Citológicas , Citosol/química , Citosol/metabolismo , Humanos , Organelas/química , Organelas/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-31553646

RESUMO

Skeletal muscle weakness is associated with oxidative stress and oxidative post-translational modifications on contractile proteins. There is indirect evidence that reactive oxygen/nitrogen species (ROS/RNS) affect skeletal muscle myofibrillar function, although the details of ROS/RNS' acute effects on myosin-actin interactions are not known. In this study, we examined the effects of peroxynitrite (ONOO-) on the contractile properties of individual skeletal muscle myofibrils by monitoring myofibril-induced displacements of an atomic force cantilever upon activation and relaxation. The isometric force decreased by approximately 50% in myofibrils treated with the ONOO-donor (SIN-1) or directly with ONOO-, which was independent of the cross-bridge abundancy condition (i.e. rigor or relaxing condition) during SIN-1 or ONOO-treatment. The force decrease was attributed to an increase in the cross-bridge detachment rate (gapp) in combination with a conservation of the force redevelopment rate (kTr), hence an increase in the population of cross-bridges transitioning from force-generating to non-force-generating cross-bridges during steady-state. Taken together, the results of this study provide important information of how ROS/RNS affect myofibrillar force production which may be of importance for conditions where increased oxidative stress is part of the pathophysiology.

3.
Am J Physiol Cell Physiol ; 317(5): C900-C909, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411922

RESUMO

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.

4.
J Physiol ; 597(17): 4615-4625, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31246276

RESUMO

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.

5.
Eur J Appl Physiol ; 119(9): 1951-1958, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31250088

RESUMO

PURPOSE: Mechanisms underlying the efficacy of sprint interval training (SIT) remain to be understood. We previously reported that an acute bout of SIT disrupts the integrity of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor 1 (RyR1), in recreationally active human subjects. We here hypothesize that in addition to improving the exercise performance of recreationally active humans, a period of repeated SIT sessions would make the RyR1 protein less vulnerable and accelerate recovery of contractile function after a SIT session. METHODS: Eight recreationally active males participated in a 3-week SIT program consisting of nine sessions of four-six 30-s all-out cycling bouts with 4 min of rest between bouts. RESULTS: Total work performed during a SIT session and maximal power (Wmax) reached during an incremental cycling test were both increased by ~ 7.5% at the end of the training period (P < 0.05). Western blots performed on vastus lateralis muscle biopsies taken before, 1 h, 24 h and 72 h after SIT sessions in the untrained and trained state showed some protection against SIT-induced reduction of full-length RyR1 protein expression in the trained state. SIT-induced knee extensor force deficits were similar in the untrained and trained states, with a major reduction in voluntary and electrically evoked forces immediately and 1 h after SIT (P < 0.05), and recovery after 24 h. CONCLUSIONS: Three weeks of SIT improves exercise performance and provides some protection against RyR1 modification, whereas it does not accelerate recovery of contractile function.

6.
J Gen Physiol ; 151(4): 567-577, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30635368

RESUMO

Effective practices to improve skeletal muscle fatigue resistance are crucial for athletes as well as patients with dysfunctional muscles. To this end, it is important to identify the cellular signaling pathway that triggers mitochondrial biogenesis and thereby increases oxidative capacity and fatigue resistance in skeletal muscle fibers. Here, we test the hypothesis that the stress induced in skeletal muscle fibers by endurance exercise causes a reduction in the association of FK506-binding protein 12 (FKBP12) with ryanodine receptor 1 (RYR1). This will result in a mild Ca2+ leak from the sarcoplasmic reticulum (SR), which could trigger mitochondrial biogenesis and improved fatigue resistance. After giving mice access to an in-cage running wheel for three weeks, we observed decreased FKBP12 association to RYR1, increased baseline [Ca2+]i, and signaling associated with greater mitochondrial biogenesis in muscle, including PGC1α1. After six weeks of voluntary running, FKBP12 association is normalized, baseline [Ca2+]i returned to values below that of nonrunning controls, and signaling for increased mitochondrial biogenesis was no longer present. The adaptations toward improved endurance exercise performance that were observed with training could be mimicked by pharmacological agents that destabilize RYR1 and thereby induce a modest Ca2+ leak. We conclude that a mild RYR1 SR Ca2+ leak is a key trigger for the signaling pathway that increases muscle fatigue resistance.

7.
Am J Physiol Cell Physiol ; 316(2): C246-C251, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30566390

RESUMO

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.

8.
Cell Calcium ; 76: 87-100, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30390422

RESUMO

STIM1 and ORAI1 regulate store-operated Ca2+ entry (SOCE) in most cell types, and mutations in these proteins have deleterious and diverse effects. We established a mouse line expressing the STIM1 R304 W gain-of-function mutation causing Stormorken syndrome to explore effects on organ and cell physiology. While STIM1 R304 W was lethal in the homozygous state, surviving mice presented with reduced growth, skeletal muscle degeneration, and reduced exercise endurance. Variable STIM1 expression levels between tissues directly impacted cellular SOCE capacity. In contrast to patients with Stormorken syndrome, STIM1 was downregulated in fibroblasts from Stim1R304W/R304W mice, which maintained SOCE despite constitutive protein activity. In studies using foetal liver chimeras, STIM1 protein was undetectable in homozygous megakaryocytes and platelets, resulting in impaired platelet activation and absent SOCE. These data indicate that downregulation of STIM1 R304 W effectively opposes the gain-of-function phenotype associated with this mutation, and highlight the importance of STIM1 in skeletal muscle development and integrity.

9.
J Physiol ; 596(18): 4427-4442, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30062729

RESUMO

KEY POINTS: We examined the mechanisms underlying the positive effect of preconditioning contractions (PCs) on the recovery of muscle force after damaging eccentric contractions (ECCs). The mechanisms underlying the immediate force decrease after damaging ECCs differ from those causing depressed force with a few days' delay, where reactive oxygen species (ROS) produced by invading immune cells play an important causative role. PCs counteracted the delayed onset force depression and this could be explained by prevention of immune cell invasion, which resulted in decreased myeloperoxidase-mediated ROS production, hence avoiding cell membrane disruption, calpain activation and degenerative changes in myosin and actin molecules. ABSTRACT: Preconditioning contractions (PCs) have been shown to result in markedly improved contractile function during the recovery periods after muscle damage from eccentric contractions (ECCs). Here, we examined the mechanisms underlying the beneficial effect of PCs with a special focus on the myofibrillar function. Rat medial gastrocnemius muscles were exposed to 100 repeated damaging ECCs in situ and excised immediately (recovery 0, REC0) or after 4 days (REC4). PCs with 10 repeated non-damaging ECCs were applied 2 days before the damaging ECCs. PCs improved in situ maximal isometric torque at REC4. Skinned muscle fibres were used to directly assess changes in myofibrillar function. PCs prevented the damaging ECC-induced depression in maximum Ca2+ -activated force at REC4. PCs also prevented the following damaging ECC-induced effects at REC4: (i) the reduction in myosin heavy chain and actin content; (ii) calpain activation; (iii) changes in redox homeostasis manifested as increased expression levels of malondialdehyde-protein adducts, NADPH oxidase 2, superoxide dismutase 2 and catalase, and activation of myeloperoxidase (MPO); (iv) infiltration of immune cells and loss of cell membrane integrity. Additionally, at REC0, PCs enhanced the expression levels of heat shock protein (HSP) 70, HSP25, and αB-crystallin in the myofibrils and prevented the increased mRNA levels of granulocyte-macrophage colony-stimulating factor and interleukin-6. In conclusion, PCs prevent the delayed force depression after damaging ECCs by an HSP-dependent inhibition of degenerative changes in myosin and actin molecules caused by myeloperoxidase-induced membrane lysis and subsequent calpain activation, which were triggered by an inflammatory reaction with immune cells invading damaged muscles.

10.
Front Physiol ; 9: 1111, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30147660

RESUMO

Severe muscle weakness concomitant with preferential depletion of myosin has been observed in several pathological conditions. Here, we used the steroid-denervation (S-D) rat model, which shows dramatic decrease in myosin content and force production, to test whether electrical stimulation (ES) treatment can prevent these deleterious changes. S-D was induced by cutting the sciatic nerve and subsequent daily injection of dexamethasone for 7 days. For ES treatment, plantarflexor muscles were electrically stimulated to produce four sets of five isometric contractions each day. Plantarflexor in situ isometric torque, muscle weight, skinned muscle fiber force, and protein and mRNA expression were measured after the intervention period. ES treatment partly prevented the S-D-induced decreases in plantarflexor in situ isometric torque and muscle weight. ES treatment fully prevented S-D-induced decreases in skinned fiber force and ratio of myosin heavy chain (MyHC) to actin, as well as increases in the reactive oxygen/nitrogen species-generating enzymes NADPH oxidase (NOX) 2 and 4, phosphorylation of p38 MAPK, mRNA expression of the muscle-specific ubiquitin ligases muscle ring finger-1 (MuRF-1) and atrogin-1, and autolyzed active calpain-1. Thus, ES treatment is an effective way to prevent muscle impairments associated with loss of myosin.

11.
Artigo em Inglês | MEDLINE | ID: mdl-28432118

RESUMO

The contractile function of skeletal muscle declines during intense or prolonged physical exercise, that is, fatigue develops. Skeletal muscle fibers fatigue acutely during highly intense exercise when they have to rely on anaerobic metabolism. Early stages of fatigue involve impaired myofibrillar function, whereas decreased Ca2+ release from the sarcoplasmic reticulum (SR) becomes more important in later stages. SR Ca2+ release can also become reduced with more prolonged, lower intensity exercise, and it is then related to glycogen depletion. Increased reactive oxygen/nitrogen species can cause long-lasting impairments in SR Ca2+ release resulting in a prolonged force depression after exercise. In this article, we discuss molecular and cellular mechanisms of the above fatigue-induced changes, with special focus on multiple mechanisms to decrease SR Ca2+ release to avoid energy depletion and preserve muscle fiber integrity. We also discuss fatigue-related effects of exercise-induced Ca2+ fluxes over the sarcolemma and between the cytoplasm and mitochondria.

12.
J Physiol ; 595(24): 7413-7426, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28980321

RESUMO

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.


Assuntos
Exercício , Hipertermia Induzida/métodos , Hipotermia Induzida/efeitos adversos , Contração Muscular , Músculo Esquelético/fisiologia , Recuperação de Função Fisiológica , Adulto , Animais , Cálcio/metabolismo , Células Cultivadas , Feminino , Glicogênio/metabolismo , Humanos , Hipertermia Induzida/efeitos adversos , Hipotermia Induzida/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fadiga Muscular , Músculo Esquelético/metabolismo
13.
Front Physiol ; 8: 712, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28979214

RESUMO

Electrically-evoked low-frequency (submaximal) force is increased immediately following high-frequency stimulation in human skeletal muscle. Although central mechanisms have been suggested to be the major cause of this low-frequency force potentiation, intramuscular factors might contribute. Thus, we hypothesized that two intramuscular Ca2+-dependent mechanisms can contribute to the low-frequency force potentiation: increased sarcoplasmic reticulum Ca2+ release and increased myofibrillar Ca2+ sensitivity. Experiments in humans were performed on the plantar flexor muscles at a shortened, intermediate, and long muscle length and electrically evoked contractile force and membrane excitability (i.e., M-wave amplitude) were recorded during a stimulation protocol. Low-frequency force potentiation was assessed by stimulating with a low-frequency tetanus (25 Hz, 2 s duration), followed by a high-frequency tetanus (100 Hz, 2 s duration), and finally followed by another low-frequency (25 Hz, 2 s duration) tetanus. Similar stimulation protocols were performed on intact mouse single fibers from flexor digitorum brevis muscle, whereby force and myoplasmic free [Ca2+] ([Ca2+]i) were assessed. Our data show a low-frequency force potentiation that was not muscle length-dependent in human muscle and it was not accompanied by any increase in M-wave amplitude. A length-independent low-frequency force potentiation could be replicated in mouse single fibers, supporting an intramuscular mechanism. We show that at physiological temperature (31°C) this low-frequency force potentiation in mouse fibers corresponded with an increase in sarcoplasmic reticulum (SR) Ca2+ release. When mimicking the slower contractile properties of human muscle by cooling mouse single fibers to 18°C, the low-frequency force potentiation was accompanied by minimally increased SR Ca2+ release and hence it could be explained by increased myofibrillar Ca2+ sensitivity. Finally, introducing a brief 200 ms pause between the high- and low-frequency tetanus in human and mouse muscle revealed that the low-frequency force potentiation is abolished, arguing that increased myofibrillar Ca2+ sensitivity is the main intramuscular mechanism underlying the low-frequency force potentiation in humans.

14.
Nat Protoc ; 12(9): 1763-1776, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28771237

RESUMO

Mechanical dissection of single intact mammalian skeletal muscle fibers permits real-time measurement of intracellular properties and contractile function of living fibers. A major advantage of mechanical over enzymatic fiber dissociation is that single fibers can be isolated with their tendons remaining attached, which allows contractile forces (in the normal expected range of 300-450 kN/m2) to be measured during electrical stimulation. Furthermore, the sarcolemma of single fibers remains fully intact after mechanical dissection, and hence the living fibers can be studied with intact intracellular milieu and normal function and metabolic properties, as well as ionic control. Given that Ca2+ is the principal regulator of the contractile force, measurements of myoplasmic free [Ca2+] ([Ca2+]i) can be used to further delineate the intrinsic mechanisms underlying changes in skeletal muscle function. [Ca2+]i measurements are most commonly performed in intact single fibers using ratiometric fluorescent indicators such as indo-1 or fura-2. These Ca2+ indicators are introduced into the fiber by pressure injection or by using the membrane-permeable indo-1 AM, and [Ca2+]i is measured by calculating a ratio of the fluorescence at specific wavelengths emitted for the Ca2+-free and Ca2+-bound forms of the dye. We describe here the procedures for mechanical dissection, and for force and [Ca2+]i measurement in intact single fibers from mouse flexor digitorum brevis (FDB) muscle, which is the most commonly used muscle in studies using intact single fibers. This technique can also be used to isolate intact single fibers from various muscles and from various species. As an alternative to Ca2+ indicators, single fibers can also be loaded with fluorescent indicators to measure, for instance, reactive oxygen species, pH, and [Mg2+], or they can be injected with proteins to change functional properties. The entire protocol, from dissection to the start of an experiment on a single fiber, takes ∼3 h.


Assuntos
Cálcio/metabolismo , Citoplasma/metabolismo , Fenômenos Mecânicos , Fibras Musculares Esqueléticas/citologia , Análise de Célula Única/métodos , Animais , Fenômenos Biomecânicos , Camundongos , Camundongos Endogâmicos C57BL
15.
FASEB J ; 31(11): 4809-4820, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28716970

RESUMO

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.


Assuntos
Cálcio/metabolismo , Contração Muscular/fisiologia , Força Muscular/fisiologia , Músculo Esquelético/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Adulto , Animais , Humanos , Masculino , Camundongos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
16.
PLoS One ; 12(6): e0179925, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28636643

RESUMO

Skeletal muscle weakness is a prominent feature in patients with rheumatoid arthritis (RA). In this study, we investigated whether neuromuscular electrical stimulation (NMES) training protects against skeletal muscle dysfunction in rats with adjuvant-induced arthritis (AIA). AIA was produced by intraarticular injection of complete Freund's adjuvant into the knees of Wistar rats. For NMES training, dorsiflexor muscles were stimulated via a surface electrode (0.5 ms pulse, 50 Hz, 2 s on/4 s off). NMES training was performed every other day for three weeks and consisted of three sets produced at three min intervals. In each set, the electrical current was set to achieve 60% of the initial maximum isometric torque and the current was progressively increased to maintain this torque; stimulation was stopped when the 60% torque could no longer be maintained. After the intervention period, extensor digitorum longus (EDL) muscles were excised and used for physiological and biochemical analyses. There was a reduction in specific force production (i.e. force per cross-sectional area) in AIA EDL muscles, which was accompanied by aggregation of the myofibrillar proteins actin and desmin. Moreover, the protein expressions of the pro-oxidative enzymes NADPH oxidase, neuronal nitric oxide synthase, p62, and the ratio of the autophagosome marker LC3bII/LC3bI were increased in AIA EDL muscles. NMES training prevented all these AIA-induced alterations. The present data suggest that NMES training prevents AIA-induced skeletal muscle weakness presumably by counteracting the formation of actin and desmin aggregates. Thus, NMES training can be an effective treatment for muscle dysfunction in patients with RA.


Assuntos
Artrite Experimental/terapia , Músculo Esquelético/metabolismo , Actinas/metabolismo , Animais , Artrite Experimental/metabolismo , Artrite Experimental/fisiopatologia , Desmina/metabolismo , Terapia por Estimulação Elétrica , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/efeitos dos fármacos , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Ácido Peroxinitroso/farmacologia , Ratos , Ratos Wistar , Proteína Sequestossoma-1/metabolismo , Superóxido Dismutase/metabolismo , Ubiquitinação
17.
PLoS One ; 12(2): e0169146, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28152009

RESUMO

Patients with pulmonary hypertension (PH) suffer from inspiratory insufficiency, which has been associated with intrinsic contractile dysfunction in diaphragm muscle. Here, we examined the role of redox stress in PH-induced diaphragm weakness by using the novel antioxidant, EUK-134. Male Wistar rats were randomly divided into control (CNT), CNT + EUK-134 (CNT + EUK), monocrotaline-induced PH (PH), and PH + EUK groups. PH was induced by a single intraperitoneal injection of monocrotaline (60 mg/kg body weight). EUK-134 (3 mg/kg body weight/day), a cell permeable mimetic of superoxide dismutase (SOD) and catalase, was daily intraperitoneally administered starting one day after induction of PH. After four weeks, diaphragm muscles were excised for mechanical and biochemical analyses. There was a decrease in specific tetanic force in diaphragm bundles from the PH group, which was accompanied by increases in: protein expression of NADPH oxidase 2/gp91phox, SOD2, and catalase; 3-nitrotyrosine content and aggregation of actin; glutathione oxidation. Treatment with EUK-134 prevented the force decrease and the actin modifications in PH diaphragm bundles. These data show that redox stress plays a pivotal role in PH-induced diaphragm weakness. Thus, antioxidant treatment can be a promising strategy for PH patients with inspiratory failure.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Debilidade Muscular/prevenção & controle , Compostos Organometálicos/farmacologia , Salicilatos/farmacologia , Actinas/metabolismo , Animais , Antioxidantes/farmacologia , Materiais Biomiméticos/farmacologia , Catalase/metabolismo , Diafragma/efeitos dos fármacos , Diafragma/fisiopatologia , Modelos Animais de Doenças , Glutationa/metabolismo , Hipertensão Pulmonar/induzido quimicamente , Masculino , Monocrotalina/toxicidade , Contração Muscular/efeitos dos fármacos , Debilidade Muscular/fisiopatologia , Estresse Oxidativo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo
18.
Physiol Behav ; 168: 55-61, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27794435

RESUMO

Nitrate supplementation is shown to increase submaximal force in human and mouse skeletal muscles. In this study, we test the hypothesis that the increased submaximal force induced by nitrate supplementation reduces the effort of submaximal voluntary running, resulting in increased running speed and distance. C57Bl/6N male mice were fed nitrate in the drinking water and housed with or without access to an in-cage running wheel. Nitrate supplementation in sedentary mice had no effect on endurance in a treadmill test, nor did it enhance mitochondrial function. However, after three weeks with in-cage running wheel, mice fed nitrate ran on average 20% faster and 30% further than controls (p<0.01). Compared to running controls, this resulted in ~13% improved endurance on a subsequent treadmill test (p<0.05) and increased mitochondrial oxidative capacity, as judged from a mean increase in citrate synthase activity of 14% (p<0.05). After six weeks with nitrate, the mice were running 58% longer distances per night. When nitrate supplementation was removed from the diet, the running distance and speed decreased to the control level, despite the improved endurance achieved during nitrate supplementation. In conclusion, low-frequency force improvement due to nitrate supplementation facilitates submaximal exercise such as voluntary running.


Assuntos
Suplementos Nutricionais , Músculo Esquelético/fisiologia , Nitratos/administração & dosagem , Corrida/fisiologia , Trifosfato de Adenosina/metabolismo , Análise de Variância , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Teste de Esforço , Locomoção/fisiologia , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Músculo Esquelético/ultraestrutura , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Estatísticas não Paramétricas
19.
J Physiol ; 595(5): 1657-1670, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27869319

RESUMO

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.


Assuntos
Cálcio/fisiologia , Imidazóis/farmacologia , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Pirazinas/farmacologia , Adenosina Trifosfatases/fisiologia , Animais , Bovinos , Feminino , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiologia , Camundongos Endogâmicos C57BL , Fibras Musculares de Contração Rápida/fisiologia , Miofibrilas/fisiologia , Coelhos , Ratos Sprague-Dawley , Troponina C/fisiologia
20.
PLoS One ; 11(12): e0167090, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27907040

RESUMO

The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute ß-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: ß-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to ß-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after ß-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute ß-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.


Assuntos
Cardiomiopatias/metabolismo , Síndrome Metabólica/metabolismo , Miócitos Cardíacos/metabolismo , Obesidade/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Animais , Cálcio/metabolismo , Cardiomiopatias/etiologia , Cardiomiopatias/patologia , Dieta Hiperlipídica/efeitos adversos , Estimulação Elétrica , Peróxido de Hidrogênio/farmacologia , Isoproterenol/farmacologia , Masculino , Síndrome Metabólica/etiologia , Síndrome Metabólica/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Obesidade/etiologia , Obesidade/patologia , Técnicas de Cultura de Órgãos , Cultura Primária de Células
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