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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.
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.

3.
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.

4.
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
5.
Methods Mol Biol ; 1601: 79-87, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28470519

RESUMO

Mitochondrial respiration is the most important generator of cellular energy under most circumstances. It is a process of energy conversion of substrates into ATP. The Seahorse equipment allows measuring oxygen consumption rate (OCR) in living cells and estimates key parameters of mitochondrial respiration in real-time mode. Through use of mitochondrial inhibitors, four key mitochondrial respiration parameters can be measured: basal, ATP production-linked, maximal, and proton leak-linked OCR. This approach requires application of mitochondrial inhibitors-oligomycin to block ATP synthase, FCCP-to make the inner mitochondrial membrane permeable for protons and allow maximum electron flux through the electron transport chain, and rotenone and antimycin A-to inhibit complexes I and III, respectively. This chapter describes the protocol of OCR assessment in the culture of primary myotubes obtained upon satellite cell fusion.


Assuntos
Trifosfato de Adenosina/metabolismo , Bioensaio/instrumentação , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio , Animais , Antimicina A/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Respiração Celular , Sobrevivência Celular , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Camundongos , Mitocôndrias/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Oligomicinas/farmacologia , Cultura Primária de Células , Ionóforos de Próton/farmacologia , Rotenona/farmacologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Desacopladores/farmacologia
6.
Crit Care ; 20(1): 254, 2016 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-27510990

RESUMO

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.


Assuntos
Ácido Edético/provisão & distribução , Força Muscular/fisiologia , Debilidade Muscular/fisiopatologia , Doenças Musculares/induzido quimicamente , Animais , Estado Terminal , Modelos Animais de Doenças , Feminino , Masculino , Contração Muscular/fisiologia , Doenças Musculares/fisiopatologia , Ratos , Ratos Sprague-Dawley
7.
Proc Natl Acad Sci U S A ; 112(50): 15492-7, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26575622

RESUMO

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.


Assuntos
Cálcio/metabolismo , Exercício/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Adulto , Animais , Atletas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/fisiologia , Resistência Física , Espécies Reativas de Oxigênio/metabolismo , Recreação
8.
Biomed Res Int ; 2015: 594751, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26380282

RESUMO

Muscular dystrophies caused by defects in various genes are often associated with impairment of calcium homeostasis. Studies of calcium currents are hampered because of the lack of a robust cellular model. Primary murine myotubes, formed upon satellite cell fusion, were examined for their utilization as a model of adult skeletal muscle. We enzymatically isolated satellite cells and induced them to differentiation to myotubes. Myotubes displayed morphological and physiological properties resembling adult muscle fibers. Desmin and myosin heavy chain immunoreactivity in the differentiated myotubes were similar to the mature muscle cross-striated pattern. The myotubes responded to electrical and chemical stimulations with sarcoplasmic reticulum calcium release. Presence of L-type calcium channels in the myotubes sarcolemma was confirmed via whole-cell patch-clamp technique. To assess the use of myotubes for studying functional mutation effects lentiviral transduction was applied. Satellite cells easily underwent transduction and were able to retain a positive expression of lentivirally encoded GFP up to and after the formation of myotubes, without changes in their physiological and morphological properties. Thus, we conclude that murine myotubes may serve as a fruitful cell model for investigating calcium homeostasis in muscular dystrophy and the effects of gene modifications can be assessed due to lentiviral transduction.


Assuntos
Diferenciação Celular/genética , Fibras Musculares Esqueléticas/citologia , Distrofias Musculares/metabolismo , Cultura Primária de Células , Células Satélites de Músculo Esquelético/citologia , Animais , Sinalização do Cálcio/genética , Humanos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Distrofias Musculares/patologia , Cadeias Pesadas de Miosina/metabolismo , Retículo Sarcoplasmático/metabolismo , Células Satélites de Músculo Esquelético/metabolismo
9.
Hum Mol Genet ; 24(23): 6580-7, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26374844

RESUMO

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.


Assuntos
Ciclofilinas/antagonistas & inibidores , Ciclosporina/uso terapêutico , Mitocôndrias/metabolismo , Miopatias Mitocondriais/tratamento farmacológico , Músculo Esquelético/metabolismo , Animais , Cálcio/metabolismo , Ciclofilinas/efeitos dos fármacos , Ciclofilinas/genética , DNA Mitocondrial , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/metabolismo , Músculo Esquelético/efeitos dos fármacos , Mutação
10.
Skelet Muscle ; 5: 26, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26301072

RESUMO

BACKGROUND: In skeletal muscle, intracellular Ca(2+) is an important regulator of contraction as well as gene expression and metabolic processes. Because of the difficulties to obtain intact human muscle fibers, human myotubes have been extensively employed for studies of Ca(2+)-dependent processes in human adult muscle. Despite this, it is unknown whether the Ca(2+)-handling properties of myotubes adequately represent those of adult muscle fibers. METHODS: To enable a comparison of the Ca(2+)-handling properties of human muscle fibers and myotubes, we developed a model of dissected intact single muscle fibers obtained from human intercostal muscle biopsies. The intracellular Ca(2+)-handling of human muscle fibers was compared with that of myotubes generated by the differentiation of primary human myoblasts obtained from vastus lateralis muscle biopsies. RESULTS: The intact single muscle fibers all demonstrated strictly regulated cytosolic free [Ca(2+)] ([Ca(2+)]i) transients and force production upon electrical stimulation. In contrast, despite a more mature Ca(2+)-handling in myotubes than in myoblasts, myotubes lacked fundamental aspects of adult Ca(2+)-handling and did not contract. These functional differences were explained by discrepancies in the quantity and localization of Ca(2+)-handling proteins, as well as ultrastructural differences between muscle fibers and myotubes. CONCLUSIONS: Intact single muscle fibers that display strictly regulated [Ca(2+)]i transients and force production upon electrical stimulation can be obtained from human intercostal muscle biopsies. In contrast, human myotubes lack important aspects of adult Ca(2+)-handling and are thus an inappropriate model for human adult muscle when studying Ca(2+)-dependent processes, such as gene expression and metabolic processes.

11.
Skelet Muscle ; 5: 20, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26161253

RESUMO

BACKGROUND: In addition to the primary symptoms arising from inflamed joints, muscle weakness is prominent and frequent in patients with rheumatoid arthritis (RA). Here, we investigated the mechanisms of arthritis-induced muscle dysfunction in rats with adjuvant-induced arthritis (AIA). METHODS: AIA was induced in the knees of rats by injection of complete Freund's adjuvant and was allowed to develop for 21 days. Muscle contractile function was assessed in isolated extensor digitorum longus (EDL) muscles. To assess mechanisms underlying contractile dysfunction, we measured redox modifications, redox enzymes and inflammatory mediators, and activity of actomyosin ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase. RESULTS: EDL muscles from AIA rats showed decreased tetanic force per cross-sectional area and slowed twitch contraction and relaxation. These contractile dysfunctions in AIA muscles were accompanied by marked decreases in actomyosin ATPase and SR Ca(2+)-ATPase activities. Actin aggregates were observed in AIA muscles, and these contained high levels of 3-nitrotyrosine and malondialdehyde-protein adducts. AIA muscles showed increased protein expression of NADPH oxidase 2/gp91(phox), neuronal nitric oxide synthase, tumor necrosis factor α (TNF-α), and high-mobility group box 1 (HMGB1). Treatment of AIA rats with EUK-134 (3 mg/kg/day), a superoxide dismutase/catalase mimetic, prevented both the decrease in tetanic force and the formation of actin aggregates in EDL muscles without having any beneficial effect on the arthritis development. CONCLUSIONS: Antioxidant treatment prevented the development of oxidant-induced actin aggregates and contractile dysfunction in the skeletal muscle of AIA rats. This implies that antioxidant treatment can be used to effectively counteract muscle weakness in inflammatory conditions.

12.
J Physiol ; 593(2): 457-72, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25630265

RESUMO

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.


Assuntos
Antioxidantes/farmacologia , Fadiga Muscular , Fibras Musculares Esqueléticas/efeitos dos fármacos , Recuperação de Função Fisiológica , Animais , Cálcio/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiologia , NADPH Oxidases/antagonistas & inibidores , Óxido Nítrico Sintase/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo
13.
PLoS One ; 9(9): e108601, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25259575

RESUMO

In cells, such as neurones and immune cells, mitochondria can form dynamic and extensive networks that change over the minute timescale. In contrast, mitochondria in adult mammalian skeletal muscle fibres show little motility over several hours. Here, we use a novel three channelled microflow device, the multifunctional pipette, to test whether mitochondria in mouse skeletal muscle connect to each other. The central channel in the pipette delivers compounds to a restricted region of the sarcolemma, typically 30 µm in diameter. Two channels on either side of the central channel use suction to create a hydrodynamically confined flow zone and remove compounds completely from the bulk solution to internal waste compartments. Compounds were delivered locally to the end or side of single adult mouse skeletal muscle fibres to test whether changes in mitochondrial membrane potential were transmitted to more distant located mitochondria. Mitochondrial membrane potential was monitored with tetramethylrhodamine ethyl ester (TMRE). Cytosolic free [Ca2+] was monitored with fluo-3. A pulse of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 100 µM) applied to a small area of the muscle fibre (30 µm in diameter) produced a rapid decrease in the mitochondrial TMRE signal (indicative of depolarization) to 38% of its initial value. After washout of FCCP, the TMRE signal partially recovered. At distances greater than 50 µm away from the site of FCCP application, the mitochondrial TMRE signal was unchanged. Similar results were observed when two sites along the fibre were pulsed sequentially with FCCP. After a pulse of FCCP, cytosolic [Ca2+] was unchanged and fibres contracted in response to electrical stimulation. In conclusion, our results indicate that extensive networks of interconnected mitochondria do not exist in skeletal muscle. Furthermore, the limited and reversible effects of targeted FCCP application with the multifunctional pipette highlight its advantages over bulk application of compounds to isolated cells.


Assuntos
Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias Musculares/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Animais , Cálcio/metabolismo , Membranas Intracelulares/metabolismo , Camundongos
14.
Cell Calcium ; 56(4): 269-75, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25171807

RESUMO

Desmin, being a major intermediate filament of mature muscle cell, interacts with mitochondria within the cell and participates in mitochondria proper localization. The goal of the present study was to assess the effect of aggregate-prone and non-aggregate-prone desmin mutations on mitochondrial calcium uptake. Primary murine satellite cells were transduced with lentiviruses carrying desmin in wild type or mutant form, and were induced to differentiate into myotubes. Four mutations resulting in different degree of desmin aggregates formation were analyzed. Tail domain mutation Asp399Tyr has the mildest impact on desmin filament polymerization, rod domain mutation Ala357Pro causes formation of large aggregates composed of filamentous material, and Leu345Pro and Leu370Pro are considered to be the most severest in their impact on desmin polymerization and structure. For mitochondrial calcium measurement cells were loaded with rhod 2-AM. We found that aggregate-prone mutations significantly decreased [Ca(2+)]mit, whereas non-aggregate-prone mutations did not decrease [Ca(2+)]mit. Moreover aggregate-prone desmin mutations resulted in increased resting cytosolic [Ca(2+)]. However this increase was not accompanied by any alterations in sarcoplasmic reticulum calcium release. We suggest that the observed decline in [Ca(2+)]mit was due to desmin aggregate accumulation resulting in the loss of desmin mitochondria interactions.


Assuntos
Cálcio/metabolismo , Citoesqueleto/metabolismo , Desmina/genética , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mutação/genética , Animais , Células Cultivadas , Desmina/metabolismo , Compostos Heterocíclicos com 3 Anéis/metabolismo , Masculino , Camundongos Endogâmicos C57BL
15.
Pflugers Arch ; 466(3): 577-85, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23912895

RESUMO

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.


Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Temperatura Alta , Contração Isométrica , Músculo Esquelético/efeitos dos fármacos , Animais , Feminino , Ácido Láctico/metabolismo , Malatos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Relaxamento Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Cadeias Leves de Miosina/metabolismo , Fosfatos/metabolismo , Fosforilação
16.
J Physiol ; 591(15): 3739-48, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23690559

RESUMO

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.


Assuntos
Cálcio/fisiologia , Fadiga Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Retículo Sarcoplasmático/fisiologia , Potenciais de Ação/fisiologia , Animais , Estimulação Elétrica , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL
17.
Ann Rheum Dis ; 72(8): 1390-9, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23148306

RESUMO

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.


Assuntos
Proteína HMGB1/farmacologia , Fadiga Muscular/efeitos dos fármacos , Miosite/metabolismo , Receptor 4 Toll-Like/metabolismo , Adulto , Idoso , Animais , Cálcio/metabolismo , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas , Cadeias Pesadas de Miosina/metabolismo , Miosite/patologia , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/deficiência , Receptores Imunológicos/metabolismo , Proteínas Recombinantes , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Receptor 4 Toll-Like/deficiência
18.
J Physiol ; 590(23): 6187-97, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22988144

RESUMO

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.


Assuntos
Senilidade Prematura/fisiopatologia , Mitocôndrias Musculares/fisiologia , Fadiga Muscular/fisiologia , Debilidade Muscular/fisiopatologia , Músculo Esquelético/fisiopatologia , Animais , Cálcio/fisiologia , DNA Mitocondrial/genética , Camundongos , Camundongos Mutantes , Espécies Reativas de Oxigênio/metabolismo , Retículo Sarcoplasmático/fisiologia
19.
J Physiol ; 590(15): 3575-83, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22687611

RESUMO

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.


Assuntos
Cálcio/fisiologia , Contração Muscular/efeitos dos fármacos , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Nitratos/administração & dosagem , Animais , Canais de Cálcio Tipo L/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Calsequestrina/fisiologia , Dieta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares de Contração Rápida/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia
20.
Adv Exp Med Biol ; 740: 27-43, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22453937

RESUMO

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.


Assuntos
Cálcio/análise , Animais , Cálcio/metabolismo , Calibragem , Sobrevivência Celular , Citosol/metabolismo , Fluorescência , Humanos , Microscopia Confocal
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