RESUMO
In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.
Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Miofibrilas/ultraestrutura , Actinas/química , Actinas/metabolismo , Animais , Tomografia com Microscopia Eletrônica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Proteínas Musculares/genética , Mutação , Miocárdio/química , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Miofibrilas/química , Miofibrilas/metabolismo , Miopatias da Nemalina/genética , Miopatias da Nemalina/metabolismo , Miosinas/química , Miosinas/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Músculos Psoas/química , Músculos Psoas/metabolismo , Músculos Psoas/ultraestrutura , Sarcômeros/química , Sarcômeros/metabolismo , Sarcômeros/ultraestruturaRESUMO
Skeletal muscle weakness is associated with oxidative stress and oxidative posttranslational modifications on contractile proteins. There is indirect evidence that reactive oxygen/nitrogen species (ROS/RNS) affect skeletal muscle myofibrillar function, although the details of the acute effects of ROS/RNS 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 ~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) and 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 on how ROS/RNS affect myofibrillar force production which may be of importance for conditions where increased oxidative stress is part of the pathophysiology.
Assuntos
Contração Isométrica/efeitos dos fármacos , Molsidomina/análogos & derivados , Miofibrilas/efeitos dos fármacos , Miosinas/antagonistas & inibidores , Doadores de Óxido Nítrico/farmacologia , Oxidantes/farmacologia , Ácido Peroxinitroso/farmacologia , Actinas/antagonistas & inibidores , Actinas/química , Actinas/fisiologia , Animais , Contração Isométrica/fisiologia , Molsidomina/química , Molsidomina/farmacologia , Miofibrilas/fisiologia , Miofibrilas/ultraestrutura , Miosinas/química , Miosinas/fisiologia , Doadores de Óxido Nítrico/química , Estresse Oxidativo , Músculos Psoas/efeitos dos fármacos , Músculos Psoas/fisiologia , Músculos Psoas/ultraestrutura , Coelhos , Técnicas de Cultura de TecidosRESUMO
In experiments on active muscle, we examined the tension decline and its temperature sensitivity at the onset of ramp shortening and at a range of velocities. A segment (â¼1.5â mm long) of a skinned muscle fibre isolated from rabbit psoas muscle was held isometrically (sarcomere length â¼2.5â µm) at 8-9°C, maximally Ca2+-activated and a ramp shortening applied. The tension decline with a ramp shortening showed an early decrease of slope (the P1 transition) followed by a slower decrease in slope (the P2 transition) to the steady (isotonic) force. The tension level at the initial P1 transition and the time to that transition decreased as the velocity was increased; the length change to this transition increased with shortening velocity to a steady value of â¼8â nmâ half-sarcomere-1 A small, rapid, temperature jump (T-jump) (3-4°C, <0.2â ms) applied coincident with the onset of ramp shortening showed force enhancement by T-jump and changed the tension decline markedly. Analyses showed that the rate of T-jump-induced force rise increased linearly with increase of shortening velocity. These results provide crucial evidence that the strain-sensitive cross-bridge force generation, or a step closely coupled to it, is endothermic.
Assuntos
Músculos Psoas/fisiologia , Temperatura , Animais , Fenômenos Biomecânicos , Técnicas In Vitro , Contração Isotônica , Masculino , Tono Muscular , Músculos Psoas/ultraestrutura , CoelhosRESUMO
The radial stability of the actomyosin filament lattice in skeletal myofibrils was examined by using atomic force microscopy. The diameter and the radial stiffness of the A-band region were examined based on force-distance curves obtained for single myofibrils adsorbed onto cover slips and compressed with the tip of a cantilever and with the Dextran treatment. The results obtained indicated that the A-band is composed of a couple of stiffness components having a rigid core-like component. It was further clarified that these radial components changed the thickness as well as the stiffness depending on the physiological condition of myofibrils. Notably, by decreasing the ionic strength, the diameter of the A-band region became greatly shrunken, but the rigid core-like component thickened, indicating that the electrostatic force distinctly affects the radial structure of actomyosin filament components. The results obtained were analyzed based on the elementary structures of the filament lattice composed of cross-bridges, thin filaments and thick filament backbones. It was clarified that the actomyosin filament lattice is radially deformable greatly and that (1), under mild compression, the filament lattice is stabilized primarily by the interactions of myosin heads with thin filaments and thick filament backbones, and (2), under severe compression, the electrostatic repulsive interactions between thin filaments and thick filament backbones became predominant.
Assuntos
Actomiosina/ultraestrutura , Miofibrilas/ultraestrutura , Animais , Dextranos/farmacologia , Microscopia de Força Atômica , Contração Muscular/fisiologia , Miofibrilas/efeitos dos fármacos , Miofibrilas/fisiologia , Músculos Psoas/ultraestrutura , CoelhosRESUMO
Mechanical and two-dimensional (2D) x-ray diffraction studies suggest that during isometric steady-state contraction, strongly bound cross-bridges mostly occupy early states in the power stroke, whereas rigor or rigor-like cross-bridges could not be detected. However, it remained unclear whether cross-bridges accumulate, at least transiently, in rigor or rigor-like states in response to rapid-length releases. We addressed this question using time-resolved recording of 2D x-ray diffraction patterns of permeabilized fibers from rabbit psoas muscles during isometric contraction and when small, ramp-shaped length-releases were applied to these fibers. This maneuver allows a transient accumulation of cross-bridges in states near the end of their power stroke. By lowering the temperature to 5 degrees C, force transients were slowed sufficiently to record diffraction patterns in several 2-4-ms time frames before and during such releases, using the RAPID detector (Refined ADC Per Input Detector) at beam line ID02 of the European Synchrotron Radiation Facility (Grenoble, France). The same sequence of frames was recorded in relaxation and rigor. Comparisons of 2D patterns recorded during isometric contraction, with patterns recorded at different [MgATPgammaS] and at 1 degrees C, showed that changes in intensity profiles along the first and sixth actin layer lines (ALL1 and ALL6, respectively) allowed for discernment of the formation of rigor or rigor-like cross-bridges. During ramp-shaped releases of activated fibers, intensity profiles along ALL1 and ALL6 did not reveal evidence for the accumulation of rigor-like cross-bridges. Instead, changes in the ALL6-profile suggest that during ramp-shaped releases, cross-bridges transiently accumulate in a structural state that, to our knowledge, was not previously seen, but that could well be a strongly bound state with the light-chain binding domain in a conformation between a near prepower-stroke (isometric) orientation and the orientation in rigor.
Assuntos
Actinas/metabolismo , Contração Isométrica/fisiologia , Miosinas/metabolismo , Músculos Psoas/fisiologia , Actinas/ultraestrutura , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Relaxamento Muscular/fisiologia , Músculos Psoas/ultraestrutura , Coelhos , Difração de Raios XRESUMO
The mechanical strength of sarcomere structures of skeletal muscle was studied by rupturing single myofibrils of rabbit psoas muscle by submicromanipulation techniques. Microbeads coated with alpha-actinin were attached to the surface of myofibrils immobilized to coverslip. By use of either optical tweezers or atomic force microscope, the attached beads were captured and detached from the myofibrils. During the detachment of the beads, the actin filaments bound specifically to the beads were peeled off from the bulk structures of myofibrils, thus rupturing the peripheral components of the myofibrils bound to the actin filaments. By analyzing the ruptures thus produced in various myofibril preparations, it was found that the sarcomere structure of myofibrils is maintained by numerous molecular components having the mechanical strength sufficient to sustain the contractile force produced by the actomyosin system. The present techniques could be applied to study the mechanical strength of cellular organelles containing actin filaments as their component.
Assuntos
Micromanipulação/métodos , Miofibrilas/fisiologia , Sarcômeros/fisiologia , Citoesqueleto de Actina/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinina , Animais , Fenômenos Biomecânicos , Eletroforese em Gel de Poliacrilamida , Microscopia de Força Atômica , Microesferas , Contração Muscular/fisiologia , Miofibrilas/ultraestrutura , Músculos Psoas/fisiologia , Músculos Psoas/ultraestrutura , Coelhos , Sarcômeros/ultraestruturaRESUMO
Recent experiments have shown that shortening and stretching of sarcomeres in single activated and unactivated myofibrils occur in stepwise fashion (Yang et al. (1998) Biophys J 74: 1473-1483; Blyakhman et al. (2001) Biophys J 81: 1093-1100; Yakovenko et al. (2002) Am J Physiol Cell Physiol 283: 735-742). Here, we carried out measurements on single myofibrils from rabbit psoas muscle to investigate steps in unactivated specimens in more detail. Activated and unactivated myofibrils were released and stretched in ramp-like fashion. The time course of length change in the single sarcomere was consistently stepwise. We found that in the unactivated myofibrils, step size depended on initial sarcomere length, diminishing progressively with increase of initial sarcomere length, whereas in the case of activated sarcomeres, step size was consistently 2.7 nm.
Assuntos
Relaxamento Muscular/fisiologia , Músculos Psoas/fisiologia , Sarcômeros/química , Sarcômeros/fisiologia , Animais , Técnicas In Vitro , Contração Muscular/fisiologia , Miofibrilas/fisiologia , Miofibrilas/ultraestrutura , Músculos Psoas/ultraestrutura , Coelhos , Sarcômeros/ultraestrutura , Fatores de TempoRESUMO
We describe a procedure whereby structural changes that occur in muscle fibres after a rapid temperature jump can be captured by cryofixation. In the thick filament from rabbit and other mammalian skeletal muscles there is a rapid transition from a non-helical to a helical structure as the temperature is raised from 273 K towards physiological levels. This transition is accompanied by characteristic intensity changes in the X-ray diffraction pattern of the muscle. In our experiments to capture these changes, single fibres of glycerinated psoas muscle were subjected to a Joule temperature jump of 15-30 K from approximately 278 K in air. We have developed a freezing method using a modified Gatan cryosnapper in which a pair of liquid nitrogen-cooled copper jaws were projected under pressure and closed on the fibre between 50 and 100 ms after the temperature jump. The frozen fibres were freeze-substituted and embedded for electron microscopy. Transverse and longitudinal sections of relaxed 'cold' (approximately 278 K) and temperature-jumped fibres as well as rigor fibres were obtained. Fourier transforms of the images from the three preparations showed differences in the relative intensities of the reflections from the hexagonal filament lattice and in those of the helix-based layer lines, similar to the differences seen by X-ray diffraction. We conclude that we have preserved the 'hot' structure and that cryofixation is sufficiently fast to prevent the transition back to the 'cold' state.
Assuntos
Criopreservação , Fibras Musculares Esqueléticas , Animais , Criopreservação/instrumentação , Microscopia Eletrônica , Fibras Musculares Esqueléticas/ultraestrutura , Músculos Psoas/ultraestrutura , Coelhos , Fixação de TecidosRESUMO
The calcium-dependent contraction of vertebrate skeletal muscle is thought to be primarily controlled through the interaction of the thick and thin filaments. Through measurement of the Donnan potential, we have shown that an electrical switching mechanism (sensitive to both anions and cations) is present in both A- and I-bands [1]. Here we show that this mechanism is not confined to the contractile apparatus and report for the first time the presence of M-line potentials. The Z-line responds to Ca2+ ions in a similar manner to the A-band under the same solution conditions (phosphate-chloride and imidazole buffers), even though it has no reported Ca2+ binding sites. Z-line potentials were not observed in tris-acetate buffer. The M-line has a markedly different response to any of the other subsarcomeric regions, however, and can only be detected in the phosphate-chloride buffer. Preliminary observations of the M-line potential in creatine kinase-deficient mouse muscle (phosphate-chloride buffer) reveal significant differences in the calcium-induced transitions between two of the genotypes and demonstrate definitively that it is the M-line potential that is being recorded. From these results, it seems likely that the charge response of the Z-line and M-line is being mediated by titin in an anion-dependent manner. Our evidence comes from several observations. First, the similarity between the response of the Z-line potentials to the A-band potentials, where titin is the only link between these structures and second, the differential observation of M-line and Z-line potentials in a range of buffers containing different anion(s). Both Z-line and M-line potentials were seen in phosphate-chloride buffer, but only the Z-line potentials could be detected in chloride-only (imidazole) buffer and neither was observed in the acetate buffer. The latter observations can be attributed to two sources. The first is the effect of acetate buffer on the conformation of myosin [2]; the second is the absence of binding of the M-line protein, myomesin, to titin in the absence of phosphate ions [3].
Assuntos
Cálcio/farmacologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Acetatos/farmacologia , Animais , Cloretos/farmacologia , Conectina , Imidazóis/farmacologia , Queratinas/metabolismo , Magnésio/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fibras Musculares Esqueléticas/ultraestrutura , Proteínas Musculares/metabolismo , Músculo Esquelético/ultraestrutura , Proteínas Quinases/metabolismo , Músculos Psoas/fisiologia , Músculos Psoas/ultraestrutura , CoelhosRESUMO
A mechanical study on skinned rat psoas muscle fibers was performed at about 16 degrees C with X-ray diffraction and caged-ATP photolysis. The amount of photoreleased ATP was set < 0.2 mM for analysis of a 'single turnover' of the cross-bridge ATPase. With regard to the phase of activation, the results under the single turn-over condition were generally consistent with previous results obtained with larger amount of photoreleased ATP. Formation of the ADP-rigor state was mechanically monitored by the 90 degrees out-of-phase component of stiffness at 500 Hz, which was elevated on activation and then decreased to zero with a half-time of 0.2-0.3 s. Intensity changes of the X-ray reflections (e.g. equatorial reflections, actin layer lines and a myosin meridional reflection) indicated that a large number of cross-bridges returned to the rigor structure with a half-time of 0.5-0.7 s. During this phase, tension did not increase but slowly decreased with a half-time of about 1.0 s. The in-phase stiffness increased only 20-30% at the most. These results indicate that, even if the number of cross-bridges formed at any moment during full contraction is small, they can interact with actin and form rigor bonds with a rate of 1 s(-1). The force developed in the rigor formation is probably lost due to the presence of rigor bridges and compliance in the preparation.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Fibras Musculares Esqueléticas/enzimologia , Miosinas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/efeitos da radiação , Animais , Fenômenos Químicos , Físico-Química , Ativação Enzimática , Contração Isométrica/fisiologia , Substâncias Macromoleculares , Fotólise , Mapeamento de Interação de Proteínas , Músculos Psoas/enzimologia , Músculos Psoas/ultraestrutura , Ratos , Difração de Raios XRESUMO
The effect of thin filament compliance on our ability to detect the cross-bridge kinetics was examined. Our experiment is based on the facts that in rabbit psoas the thin filament (1.12 micrometer) is longer than half the thick filament length (0.82 micrometer) and that the thick filament has a central bare zone (0.16 micrometer). Consequently, when sarcomere length is increased from 2.1 to 2.4 micrometer, the same number of cross-bridges is involved in force generation but extra series compliance is introduced in the I-band. Three apparent rate constants (2pia, 2pib, and 2pic) were characterized by sinusoidal analysis at pCa 4.66. Our results demonstrate that 2pia and 2pib increased 13-16% when sarcomere length was increased from 2.0 to 2.5 micrometer, and 2pic decreased slightly (9%). This slight decrease can be explained by compression of the lattice spacing. These observations are at variance with the expectation based on increased series compliance, which predicts that the rate constants will decrease. We also determined compliance of the I-band during rigor. I-band compliance during rigor induction was 35% of sarcomere compliance at sarcomere length 2.4 micrometer, and 24% at sarcomere length 2.1 micrometer. We conclude that the presence of thin filament compliance does not seriously interfere with our ability to detect cross-bridge kinetics using sinusoidal analysis.
Assuntos
Fibras Musculares Esqueléticas/ultraestrutura , Músculos Psoas/ultraestrutura , Sarcômeros/ultraestrutura , Animais , Elasticidade , Cinética , CoelhosRESUMO
The modulatory effect of myosin regulatory light chain phosphorylation in mammalian skeletal muscle, first documented as posttetanic potentiation of twitch tension, was subsequently shown to enhance the expression and development of tension at submaximal levels of activating calcium. Structural analyses demonstrated that thick filaments with phosphorylated myosin regulatory light chains appeared disordered: they lost the near-helical, periodic arrangement of myosin head characteristic of the relaxed state. We suggested that disordered heads may be more mobile than ordered heads and are likely to spend more time close to their binding sites on thin filaments. In this study we determined that the physiological effects of phosphorylation could be mimicked by decreasing the lattice spacing between the thick and the thin filaments, either by osmotic compression with dextran or by increasing the sarcomere length of permeabilized rabbit psoas fibers. Phosphorylation of regulatory light chains by incubation of permeabilized fibers with myosin light chain kinase and calmodulin, followed by low levels of activating calcium, potentiated tension development at resting or lower sarcomere lengths in the absence of dextran but had no additional effect on tension potentiation or development in fibers with decreased lattice spacing due to either osmotic compression or increased sarcomere length.
Assuntos
Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/enzimologia , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Animais , Cálcio/metabolismo , Feminino , Microscopia Eletrônica , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/ultraestrutura , Fosforilação , Músculos Psoas/enzimologia , Músculos Psoas/ultraestrutura , Coelhos , Sarcômeros/metabolismo , Sarcômeros/ultraestruturaRESUMO
The giant molecule titin/connectin was demonstrated to connect the ends of thick filaments with the Z-disks and thus to provide an elastic connection that seems to be responsible for passive tension in striated muscle. To investigate the physiological limits of I-band titin extension in skeletal muscle, we have measured sarcomere lengths of a number of mouse postural and clonal muscles in situ under the constraints imposed by the skeletal, ligamentous and tendinous components of the motile apparatus. These values now give upper limits for the extension of the I-band and therefore for the maximal degree of titin extension under physiological constraints. We find that I-band extension in all muscles investigated does not exceed a factor of approximately 2.5 in situ, which is well below values obtainable in isolated fibre preparations. Approach to the yield-point is therefore prevented by extramuscular mechanisms. Sarcomere lengths near the tendinous junction and within the muscle are virtually identical in extended muscle, suggesting that a major function of titin in intact muscle is to ensure uniform sarcomere lengths over the entire muscle length and thus to prevent localized myofibril overstretch during isometric contraction.
Assuntos
Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Sarcômeros/fisiologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Músculo Esquelético/ultraestrutura , Músculos Psoas/fisiologia , Músculos Psoas/ultraestrutura , Sarcômeros/ultraestruturaRESUMO
Fluorescently labelled myofibrils were imaged in physiological salt solution by near-field scanning optical microscopy and shear-force microscopy. These myofibrils were imaged in vitro, naturally adhering to glass while retaining their ability to contract. The Z-line protein structure of the myofibrils was antibody labelled and easily identified in the near-field fluorescence images. The distinctive protein banding structure of the myofibril was also seen clearly in the shear-force images without any labelling requirement. With the microscope in the transmission mode, resolution of the fluorescence images was degraded significantly by excessive specimen thickness (> 1 micron), whereas the shear-force images were less affected by specimen thickness and more affected by poor adherence to the substrate. Although the exact mechanism generating contrast in the shear-force images is still unknown, shear-force imaging appears to be a promising new imaging modality.
Assuntos
Microscopia/métodos , Miofibrilas/ultraestrutura , Animais , Fluorescência , Imunofluorescência , Processamento de Imagem Assistida por Computador , Microscopia/instrumentação , Microscopia Eletrônica , Músculos Psoas/ultraestrutura , CoelhosRESUMO
There is increasing evidence that Ca2+ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl- on the release of Ca2+ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca2+ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca2+ release from isolated SR was measured by rapid filtration of vesicles passively loaded with 45Ca2+. Ca2+ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca2+-loading solution that had propionate as the major anion. Controls using saponin (50 microg/ml), indicated that the release of Ca2+ was due to direct action of Cl- on the SR rather than via depolarization of T-tubules. Procaine (10 mM) totally blocked Cl-- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na+] + [K+]) x [Cl-] product of 6487.69 mm2 and 12361.52 mm2, respectively, and blocked 60% of Ca2+ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl- and caffeine together. The data from both preparations suggests that Cl- induces a relatively small amount of Ca2+ release from the SR by activating receptors other than RYR-1. In addition, Cl- may increase the Ca2+ sensitivity of RYR-1, which would then allow the small initial release of Ca2+ to facilitate further release of Ca2+ from the SR by Ca2+-induced Ca2+ release.
Assuntos
Cálcio/metabolismo , Colina/farmacologia , Músculos Psoas/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Músculos Psoas/ultraestrutura , CoelhosRESUMO
A new image-analysis-based method is described for assessing sarcomere heterogeneity in skinned rabbit psoas muscle fiber segments. This method consists of off-line, two-dimensional Fourier spectral analysis of video-taped muscle images. Local sarcomere length is assessed by partitioning the muscle images into half and quarter images spanning the original image and analyzing the associated spectra. The spectra are analyzed in two different ways, yielding two measures of sarcomere length. The first measure is obtained by calculating and inverting the centroid frequency of the first-order peak associated with each two-dimensional Fourier spectrum. The second measure is obtained in a similar manner, the only difference being that the two-dimensional spectra are first collapsed into one-dimensional line spectra by summing the pixels perpendicular to the fiber axis. Comparison of the two measures provides a measure of striation skewness that cannot be obtained by other image analysis based methods that determine sarcomere length by analyzing selected line luminance profiles.
Assuntos
Fibras Musculares Esqueléticas/ultraestrutura , Sarcômeros/ultraestrutura , Animais , Fenômenos Biomecânicos , Fenômenos Biofísicos , Biofísica , Análise de Fourier , Processamento de Imagem Assistida por Computador , Técnicas In Vitro , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/fisiologia , Músculos Psoas/química , Músculos Psoas/fisiologia , Músculos Psoas/ultraestrutura , Coelhos , Sarcômeros/química , Sarcômeros/fisiologia , Processamento de Sinais Assistido por ComputadorRESUMO
1. We analysed the effect of calmodulin on Ca(2+)-induced Ca2+ release (CICR) in the sarcoplasmic reticulum (SR) using chemically skinned fibres of rabbit psoas muscle. Ca2+ release was measured using fura-2 microfluorometry. 2. In saponin-skinned fibres, calmodulin potentiated Ca2+ release at low Ca2+ concentrations (< 3 microM), while it showed an inhibitory effect at high Ca2+ concentrations (3-30 microM). 3. Co-application of ryanodine and calmodulin at 0.3 microM Ca2+, but not ryanodine alone, induced a decline in the Ca2+ uptake capacity of the SR, an effect expected from the open-lock of active CICR channels by ryanodine. Thus, potentiation of Ca2+ release by calmodulin at low Ca2+ concentrations can be regarded as a result of the activation of the ryanodine receptor. 4. Greater concentrations of calmodulin were required for potentiation of CICR at low Ca2+ concentrations (1 microM) than for inhibition at high Ca2+ concentrations (10 microM). 5. In beta-escin-permeabilized fibres in which intrinsic calmodulin was retained, the rates of CICR were similar to those measured in the presence of 1 microM calmodulin in saponin-permeabilized fibres. 6. These results suggest that calmodulin plays an important role in the regulation of CICR channels in intact skeletal muscle fibres.
Assuntos
Cálcio/metabolismo , Cálcio/farmacologia , Calmodulina/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Cafeína/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Citofotometria , Técnicas In Vitro , Magnésio/fisiologia , Masculino , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/ultraestrutura , Proteínas Musculares/efeitos dos fármacos , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/ultraestrutura , Inibidores de Fosfodiesterase/farmacologia , Músculos Psoas/efeitos dos fármacos , Músculos Psoas/metabolismo , Músculos Psoas/ultraestrutura , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina , Saponinas/farmacologia , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
Length-dependence of myofilament Ca2+ sensitivity is now considered to be an important component of the steep relationship between active force and sarcomere length along the ascending limb of the cardiac force-length curve. Studies with skinned cardiac muscle preparations have demonstrated that Ca(2+)-troponin C affinity is significantly increased as sarcomere length is increased over the range 1.7-2.3 microns. Increase in sarcomere length is accompanied by a reduction in interfilament spacing. In skinned fiber preparations from both cardiac and skeletal muscle osmotic compression of the filament lattice enhances myofilament Ca2+ sensitivity. This study was undertaken to evaluate the hypothesis that a change in filament separation may contribute to the length-dependent activation seen in cardiac muscle. Moderate reduction in interfilament spacing caused by exposure to Dextran T-500 (5-10%) produced an increase in force generation in both maximally activated and partially activated preparations of skinned bovine ventricular muscle. With fiber bundles of mean sarcomere length 1.7 microns the addition of 5% Dextran T-500 produced an increase in Ca2+ sensitivity of about 0.25 pCa units and a significant increase in Ca2+ binding in the pCa range (6.0-5.0) in which the single regulatory site of cardiac troponin C is titrated. This concentration of Dextran T-500 produced a reduction in fiber width equivalent to that produced by stretching fibers from sarcomere length 1.7 microns to sarcomere length 2.3 microns Osmotic compression of skinned rabbit psoas muscle fibers also enhanced Ca2+ sensitivity but there was no significant change in Ca(2+)-troponin C affinity. These data suggest that 1) an important component of length-dependent Ca2+ sensitivity in both cardiac and skeletal muscle is the change in interfilament spacing, and 2) in cardiac muscle a reduction in spacing, like increase in length, leads to a specific increase in Ca(2+)-troponin C affinity. Thus both filament overlap and filament separation contribute to the length dependence of Ca2+ sensitivity and Ca2+ binding in cardiac muscle.
Assuntos
Cálcio/metabolismo , Cálcio/farmacologia , Coração/efeitos dos fármacos , Coração/fisiologia , Miocárdio/metabolismo , Músculos Psoas/efeitos dos fármacos , Músculos Psoas/fisiologia , Animais , Bovinos , Dextranos , Técnicas In Vitro , Contração Muscular/fisiologia , Contração Miocárdica/fisiologia , Miocárdio/ultraestrutura , Pressão Osmótica , Músculos Psoas/ultraestrutura , Coelhos , Sarcômeros/fisiologia , Sarcômeros/ultraestrutura , Troponina/metabolismo , Troponina CRESUMO
The early, rapid phase of tension recovery (phase 2) after a step change in sarcomere length is thought to reflect the force-generating transition of myosin bound to actin. We have measured the relation between the rate of tension redevelopment during phase 2 (r), estimated from the half-time of tension recovery during phase 2 (r = t0.5(-1)), and steady-state force at varying [Ca2+] in single fibers from rabbit psoas. Sarcomere length was monitored continuously by laser diffraction of fiber segments (length approximately 1.6 mm), and sarcomere homogeneity was maintained using periodic length release/restretch cycles at 13-15 degrees C. At lower [Ca2+] and forces, r was elevated relative to that at pCa 4.0 for both releases and stretches (between +/- 8 nm). For releases of -3.4 +/- 0.7 nm.hs-1 at pCa 6.6 (where force was 10-20% of maximum force at pCa 4.0), r was 3.3 +/- 1.0 ms-1 (mean +/- SD; N = 5), whereas the corresponding value of r at pCa 4.0 was 1.0 +/- 0.2 ms-1 for releases of -3.5 +/- 0.5 nm.hs-1 (mean +/- SD; N = 5). For stretches of 1.9 +/- 0.7 nm.hs-1, r was 1.0 +/- 0.3 ms-1 (mean +/- SD; N = 9) at pCa 6.6, whereas r was 0.4 +/- 0.1 ms-1 at pCa 4.0 for stretches of 1.9 +/- 0.5 (mean +/- SD; N = 14). Faster phase 2 transients at submaximal Ca(2+)-activation were not caused by changes in myofilament lattice spacing because 4% Dextran T-500, which minimizes lattice spacing changes, was present in all solutions. The inverse relationship between phase 2 kinetics and force obtained during steady-state activation of skinned fibers appears to be qualitatively similar to observations on intact frog skeletal fibers during the development of tetanic force. The data are consistent with models that incorporate a direct effect of [Ca2+] on phase 2 kinetics of individual cross-bridges or, alternatively, in which phase 2 kinetics depend on cooperative interactions between cross-bridges.