Phosphorylating pathways and fatigue development in contracting Xenopus single skeletal muscle fibers

To investigate the differential contribution of oxidative and substrate-level phosphorylation to force production during repetitive, maximal tetanic contractions, single skeletal muscle fiber performance was examined under conditions of high-oxygen availability and anoxia. Tetanic force development (P) was measured in isolated, single type-1 muscle fibers (fast twitch; n = 6) dissected from Xenopus lumbrical muscle while being stimulated at increasing frequencies (0.25, 0.33, and 0.5 Hz), with each frequency lasting 2 min. Two separate work bouts were conducted, with the perfusate PO(2) being either 0 or 159 mmHg. No significant (P < 0. 05) difference was found in the initial peak tensions (P(0)) between the high (334 +/- 57 kPa) and the low (325 +/- 41 kPa) PO(2) treatment. No significant difference in P was observed between the treatments during the first 50 s. However, a significant difference in force production was observed between the high (P/P(0) = 0.96 +/- 0.02) and the low PO(2) condition (P/P(0) = 0.92 +/- 0.02) by 60 s of work. After 60 s, steady-state force production was maintained during the high compared with the low PO(2) condition until stimulation frequency was increased, at which point developed tension during the high PO(2) condition began to decline. Time to fatigue (P/P(0) = 0.3) was reached significantly sooner during the low (250 +/- 16 s) than the high PO(2) condition (367 +/- 28 s). These results demonstrate that during the first 50 s of 0.25-Hz contractions, substrate-level phosphorylation has the capacity to maintain force and ATP hydrolysis when oxidative phosphorylation is absent. This period was followed by an oxygen-dependent phase in which force generation was maintained during the high PO(2) condition (but not during the low PO(2) condition) until the onset of a final fatiguing phase, at which a calculated maximal rate of oxidative phosphorylation was reached.     

Residual force enhancement after stretch of contracting frog single muscle fibers

Single fibers from the tibialis anterior muscle of Rana temporaria at 0.8-3.8 degrees C were subjected to long tetani lasting up to 8 s. Stretch of the fiber early in the tetanus caused an enhancement of force above the isometric control level which decayed only slowly and stayed higher throughout the contraction. This residual enhancement was uninfluenced by velocity of stretch and occurred only on the descending limb of the length-tension curve. The absolute magnitude of the effect increased with sarcomere length to a maximum at approximately 2.9 micrometers and then declined. The phenomenon was further characterized by its dependence on the amplitude of stretch. The final force level reached after stretch was usually higher than the isometric force level corresponding to the starting length of the stretch. The possibility that the phenomenon was caused by nonuniformity of sarcomere length along the fiber was examined by (a) laser diffraction studies that showed sarcomere stretch at all locations and (b) studies of 9-10 segments of approximately 0.6-0.7 mm along the entire fiber, which all elongated during stretch. Length-clamped segments showed residual force enhancement after stretch when compared with the tetanus produced by the same segment held at the short length as well as at the long length. It is concluded that residual force enhancement after stretch is a property shown by all individual segments along the fiber.     

Excitation-calcium release uncoupling in aged single human skeletal muscle fibers

The biological mechanisms underlying decline in muscle power and fatigue with age are not completely understood. The contribution of alterations in the excitation-calcium release coupling in single muscle fibers was explored in this work. Single muscle fibers were voltage-clamped using the double Vaseline gap technique. The samples were obtained by needle biopsy of the vastus lateralis (quadriceps) from 9 young (25–35 years; 25.9 ± 9.1; 5 female and 4 male) and 11 old subjects (65–75 years; 70.5 ± 2.3; 6 f, 5 m). Data were obtained from 36 and 39 fibers from young and old subjects, respectively. Subjects included in this study had similar physical activity. Denervated and slow-twitch muscle fibers were excluded from this study. A significant reduction of maximum charge movement (Q_max) and DHP-sensitive Ca current were recorded in muscle fibers from the 65–75 group. Q_max values were 7.6 ± 0.9 and 3.2 ± 0.3 nC/F for young and old muscle fibers, respectively (P < 0.01). No evidences of charge inactivation or interconversion (charge 1 to charge 2) were found. The peak Ca current was (–)4.7 ± 0.08 and (–)2.15 ± 0.11 A/F for young and old fibers, respectively (P < 0.01). The peak calcium transient studied with mag-fura-2 (400 m) was 6.3 ± 0.4 m and 4.2 ± 0.3 m for young and old muscle fibers, respectively. Caffeine (0.5 mm) induced potentiation of the peak calcium transient in both groups. The decrease in the voltage-/ Ca-dependent Ca release ratio in old fibers (0.18 ± 0.02) compared to young fibers (0.47 ± 0.03) (P < 0.01), was recorded in the absence of sarcoplasmic reticulum calcium depletion. These data support a significant reduction of the amount of Ca available for triggering mechanical responses in aged skeletal muscle and, the reduction of Ca release is due to DHPR-ryanodine receptor uncoupling in fast-twitch fibers. These alterations can account, at least partially for the skeletal muscle function impairment associated with aging.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association, and National Institutes of Health (2-P60AG18484-06)     

A-band shortening in single fibers of frog skeletal muscle.

The question of whether A-bands shorten during contraction was investigated using two methods: high-resolution polarization microscopy and electron microscopy. During shortening from extended sarcomere lengths in the passive state, sarcomere-length changes were essentially accounted for by I-band shortening. During active shortening under otherwise identical conditions, the sarcomere length change was taken up approximately equally by A- and I-bands. Several potential artifacts that could give rise to apparent A-band shortening were considered and judged unlikely. Results obtained with polarization microscopy were similar to those obtained with electron microscopy. Thus, modest but significant thick filament shortening appears to occur during active sarcomere shortening under physiological conditions.     

Quasi-elastic light-scattering studies of single skeletal muscle fibers.

Measurements were made of the intensity autocorrelation function, g(2)[tau], of light scattered from intact frog muscle fibers. During the tension plateau of an isometric tenanus, scattered field statistics were approximately Gaussian and intensity fluctuations were quasi-stationary. The half time, tau 1/2, for the decay of g(2)[tau] was typically 70 ms at a scattering angle of 30 degrees. The decay rate, 1/tau 1/2, of g(2)[tau] varied roughly linearly with the projection of the scattering vector on the fiber axis. 1/tau 1/2 was greater during the tension creep phase of tetani of highly stretched fibers, but was roughly independent of sarcomere length during the tension plateau. g(2)[tau] measured during rest or on diffraction pattern maxima during isometric contraction were flat with low amplitudes. These results are consistent with a model of a 200-mu m segment of an isometrically contracting fiber in which scattering material possesses relative axial velocities of 1-2 mu m/s accompanied by relative axial displacements greater than 0.1 mu m. The slow (1-2 mu m/s) motion of one portion of the fiber relative to another observed under the microscope (500X) during isometric contraction is consistent with the light-scattering results. Structural fluctuations on the scale of the myofibrillar sarcomere which may arise from asynchronous cycling of cross-bridges must involve relative axial velocities less than 3 mu m/s or relative axial displacements less than 0.05 mu m.     

Influence of ionic strength on the actomyosin reaction steps in contracting skeletal muscle fibers

Muscle contraction occurs as the result of actin-myosin interaction, which is mediated by the intermolecular forces exerted at the actin-myosin interface. To obtain information about the nature of these intermolecular forces, we tested the sensitivity of various contractile parameters of skinned skeletal muscle fibers to ionic strength (IS) at 3-5 degrees C; IS variation is a useful technique for distinguishing between ionic and nonionic (primarily hydrophobic) types of intermolecular forces. The most striking effect of elevated IS was the strong suppression of isometric tension. However, none of the measured parameters suggested a corresponding decrease in the number of force-generating myosin heads on actin. The rate of actin-myosin association seemed to be only modestly IS-sensitive. The following force-generating isomerization was apparently IS-insensitive. The dissociation of the force-generating actomyosin complex was decelerated by elevated IS, contrary to the expectation from the suppressed isometric tension. These results led us to conclude that an IS-sensitive step, responsible for the large suppression of tension, occurs after force-generating isomerization but before dissociation. The present study suggests that the actomyosin interaction is generally nonionic in nature, but there are at least two ionic processes, one at the beginning and the other close to the end of the actomyosin interaction.     

Potassium efflux from single skinned skeletal muscle fibers.

The efflux of 42K from single, skinned (sarcolemma removed) skeletal muscle fibers has been determined. Isotope washout curves are kinetically complex and can be fit as the sum of three exponentials, including a fast component (k = 0.25 s-1) with a pool size equivalent to 91% of the fiber volume, an intermediate component (k = 0.08 s-1) equivalent to 6% of the fiber volume, and a slow component (k = 0.008 s-1) equivalent to 0.5% of fiber volume. Only the intermediate kinetic component is significantly affected by pretreatment of fibers with detergent. Efflux curves from detergent-treated fibers could be fit as the sum of two exponentials with coefficients and rate constants comparable to those of the fast and slow component of washout of untreated controls. Similarly the washout of [14C]sucrose can be described as the sum of two exponentials. We conclude that the intermediate component of 42K washout results from the movement of ions from a membrane bound space within the skinned fiber. Because of its relative volume, the sarcoplasmic reticulum seems to be a reasonable choice as a structural correlate for this component. Our estimate of the potassium permeability for the sarcoplasmic reticulum (SR) based on the efflux data is 10(-7) cm/s. This value is less than previous estimates from isolated preparations.     

Vasodilatory mechanisms in contracting skeletal muscle.

Skeletal muscle blood flow is closely coupled to metabolic demand, and its regulation is believed to be mainly the result of the interplay of neural vasoconstrictor activity and locally derived vasoactive substances. Muscle blood flow is increased within the first second after a single contraction and stabilizes within approximately 30 s during dynamic exercise under normal conditions. Vasodilator substances may be released from contracting skeletal muscle, vascular endothelium, or red blood cells. The importance of specific vasodilators is likely to vary over the time course of flow, from the initial rapid rise to the sustained elevation during steady-state exercise. Exercise hyperemia is therefore thought to be the result of an integrated response of more than one vasodilator mechanism. To date, the identity of vasoactive substances involved in the regulation of exercise hyperemia remains uncertain. Numerous vasodilators such as adenosine, ATP, potassium, hypoxia, hydrogen ion, nitric oxide, prostanoids, and endothelium-derived hyperpolarizing factor have been proposed to be of importance; however, there is little support for any single vasodilator being essential for exercise hyperemia. Because elevated blood flow cannot be explained by the failure of any single vasodilator, a consensus is beginning to emerge for redundancy among vasodilators, where one vasoactive compound may take over when the formation of another is compromised. Conducted vasodilation or flow-mediated vasodilation may explain dilation in vessels (i.e., feed arteries) not directly exposed to vasodilator substances in the interstitium. Future investigations should focus on identifying novel vasodilators and the interaction between vasodilators by simultaneous inhibition of multiple vasodilator pathways.     

Theory of light diffraction by single skeletal muscle fibers.

A theoretical discussion is presented describing the diffraction of laser light by a single fiber of striated muscle. The complete three-dimensional geometry of the fiber has been taken into consideration. The basic repeated unit is taken as the sarcomere of a single myofibril, including its cylindrical geometry. The single fiber is considered as the sum of myofibrils up to the fiber dimensions. When proper phasing is taken into account, three cases of interest are analyzed. (a) When the adjacent myofibrils are totally aligned with respect to their index of refraction regions (e.g., A and I bands), then the diffraction pattern reflects that of a larger striated cylinder with the dimensions of the fiber. (b) When a particular skew plane develops for the myofibril elements, additional Bragg reflection occurs at certain specific sarcomere lengths, and intensity asymmetry amongst the diffracted orders occurs. (c) When the myofibril phasing changes in a random fashion, while all sarcomeres remain at the same length, then intensity decrease is directly related to the phase deviation from a reference phase point. This condition may well describe a fiber undergoing active isometric contraction.     

Endocytosis in skeletal muscle fibers

Defining the organization of endocytic pathway in multinucleated skeletal myofibers is crucial to understand the routing of membrane proteins, such as receptors and glucose transporters, through this system. Here we analyzed the organization of the endocytic trafficking pathways in isolated rat myofibers. We found that sarcolemmal-coated pits and transferrin receptors were concentrated in the I band areas. Fluid phase markers were taken up into vesicles in the same areas along the whole length of the fibers and were then delivered into structures around and between the nuclei. These markers also accumulated beneath the neuromuscular and myotendinous junctions. The recycling compartment, labeled with transferrin, appeared as perinuclear and interfibrillar dots that partially colocalized with the GLUT4 compartment. Low-density lipoprotein, a marker of the lysosome-directed pathway, was transported into sparsely distributed perinuclear and interfibrillar dots that contacted microtubules. A majority of these dots did not colocalize with internalized transferrin, indicating that the recycling and the lysosome-directed pathways were distinct. In conclusion, the I band areas were active in endocytosis along the whole length of the multinucleated myofibers. The sorting endosomes distributed in a cross-striated fashion while the recycling and late endosomal compartments showed perinuclear and interfibrillar localizations and followed the course of microtubules.     

Orientational dynamics of indane dione spin-labeled myosin heads in relaxed and contracting skeletal muscle fibers.

We have used electron paramagnetic resonance (EPR) spectroscopy to study the orientation and rotational motions of spin-labeled myosin heads during steady-state relaxation and contraction of skinned rabbit psoas muscle fibers. Using an indane-dione spin label, we obtained EPR spectra corresponding specifically to probes attached to Cys 707 (SH1) on the catalytic domain of myosin heads. The probe is rigidly immobilized, so that it reports the global rotation of the myosin head, and the probe's principal axis is aligned almost parallel with the fiber axis in rigor, making it directly sensitive to axial rotation of the head. Numerical simulations of EPR spectra showed that the labeled heads are highly oriented in rigor, but in relaxation they have at least 90 degrees (Gaussian full width) of axial disorder, centered at an angle approximately equal to that in rigor. Spectra obtained in isometric contraction are fit quite well by assuming that 79 +/- 2% of the myosin heads are disordered as in relaxation, whereas the remaining 21 +/- 2% have the same orientation as in rigor. Computer-simulated spectra confirm that there is no significant population (> 5%) of heads having a distinct orientation substantially different (> 10 degrees) from that in rigor, and even the large disordered population of heads has a mean orientation that is similar to that in rigor. Because this spin label reports axial head rotations directly, these results suggest strongly that the catalytic domain of myosin does not undergo a transition between two distinct axial orientations during force generation. Saturation transfer EPR shows that the rotational disorder is dynamic on the microsecond time scale in both relaxation and contraction. These results are consistent with models of contraction involving 1) a transition from a dynamically disordered preforce state to an ordered (rigorlike) force-generating state and/or 2) domain movements within the myosin head that do not change the axial orientation of the SH1-containing catalytic domain relative to actin.     

Sarcomere length dispersion in single skeletal muscle fibers and fiber bundles.

Light diffraction patterns produced by single skeletal muscle fibers and small fiber bundles of Rana pipiens semitendinosus have been examined at rest and during tetanic contraction. The muscle diffraction patterns were recorded with a vidicon camera interfaced to a minicomputer. Digitized video output was analyzed on-line to determine mean sarcomere length, line intensity, and the distribution of sarcomere lengths. The occurrence of first-order line intensity and peak amplitude maxima at approximately 3.0 mum is interpreted in terms of simple scattering theory. Measurements made along the length of a singel fiber reveal small variations in calculated mean sarcomere length (SD about 1.2%) and its percent dispersion (2.1% +/- 0.8%). Dispersion in small multifiber preparations increases approximately linearly with fiber number (about 0.2% per fiber) to a maximum of 8-10% in large bundles. Dispersion measurements based upon diffraction line analysis are comparable to SDs calculated from length distribution histograms obtained by light micrography of the fiber. First-order line intensity decreases by about 40% during tetanus; larger multifibered bundles exhibit substantial increases in sarcomere dispersion during contraction, but single fibers show no appreciable dispersion change. These results suggest the occurrence of asynchronous static or dynamic axial disordering of thick filaments, with a persistence in long range order of sarcomere spacing during contraction in single fibers.     

Effects of shortening on stretch-induced force enhancement in single skeletal muscle fibers

The main purpose of this study was to evaluate the effects of shortening on the stretch-induced force enhancement in single muscle fibers, and indirectly test the hypothesis that force enhancement may be associated with the engagement of a passive element upon activation. Fibers were placed on the descending limb of the force-length relationship, and stretch and shortening contractions were performed. Fibers underwent two sets of shortening-stretch cycles. First, fibers were shortened by a fixed amplitude and speed (10% fiber length, and at 40% fiber length/s), and then were stretched (10% fiber length, and at 40% fiber length/s) immediately following shortening, or 500 or 1000 ms following the shortening. Second, fibers were shortened by varying amounts (5%, 10% and 15% fiber length) and at a constant speed (40% fiber length/s) immediately preceding a given fiber stretch (10% fiber length, and at 40% fiber length/s). When stretching was immediately preceded by shortening, force enhancement was decreased proportionally with the shortening magnitude. When intervals were introduced between shortening and stretch, the effects of shortening on the stretch-induced force enhancement became less prominent. We concluded that, in contrast to published suggestions, shortening affects the stretch-induced force enhancement in an amplitude-dependent manner in single fibers, as it does in whole muscles, but this effect is diminished by increasing the time period between the shortening and stretch phases.     

Finite element modelling of contracting skeletal muscle

To describe the mechanical behaviour of biological tissues and transport processes in biological tissues, conservation laws such as conservation of mass, momentum and energy play a central role. Mathematically these are cast into the form of partial differential equations. Because of nonlinear material behaviour, inhomogeneous properties and usually a complex geometry, it is impossible to find closed-form analytical solutions for these sets of equations. The objective of the finite element method is to find approximate solutions for these problems. The concepts of the finite element method are explained on a finite element continuum model of skeletal muscle. In this case, the momentum equations have to be solved with an extra constraint, because the material behaves as nearly incompressible. The material behaviour consists of a highly nonlinear passive part and an active part. The latter is described with a two-state Huxley model. This means that an extra nonlinear partial differential equation has to be solved. The problems and solutions involved with this procedure are explained. The model is used to describe the mechanical behaviour of a tibialis anterior of a rat. The results have been compared with experimentally determined strains at the surface of the muscle. Qualitatively there is good agreement between measured and calculated strains, but the measured strains were higher.     

Light diffraction studies of sarcomere dynamics in single skeletal muscle fibers.

A position-sensitive optical diffractometer has been used to examine the diffraction spectra produced by single skeletal muscle fibers during twitch and tetanic contraction. First-order diffraction lines were computer-analyzed for mean sarcomere length, line intensity, and percent dispersion in sarcomere length. Line intensity was observed to decrease rapidly by about 60 percent during a twitch, with an exponential recovery to resting intensity persisting well beyond cessation of sarcomere shortening; recovery was particularly prolonged at zero myofilament overlap. A number of single fibers at initial lengths from 2.5 to 3.5 MICRON EXHIBITED a splitting of the first-order line into two or more components during relaxation, with components merging back into a single peak by 200 ms after stimulation. This splitting reflects the asynchronous nature of myofibrillar relaxation within a single fiber. During tetanus, the dispersion decreased by more than 10 percent from onset to plateau, implying a gradual stabilization of sarcomeres.     

Effect of temperature on residual force enhancement in single skeletal muscle fibers

It is well accepted that the steady-state isometric force following active stretching of a muscle is greater than the steady-state isometric force obtained in a purely isometric contraction at the same length. This property of skeletal muscle has been called residual force enhancement (FE). Despite decades of research the mechanisms responsible for FE have remained largely unknown. Based on previous studies showing increases in FE in fibers in which cross-bridges were biased towards weakly bound states, we hypothesized that FE might be associated with a stretch-induced facilitation of transitioning from weakly to strongly bound cross-bridges. In order to test this hypothesis, single fibers (n=11) from the lumbrical muscles of frog (Rana pipiens) were used to determine FE at temperatures of 7 and 20 degrees C. At the cold temperature, cross-bridges are biased towards weakly bound states, therefore we expected FE to be greater at 7 degrees C compared to 20 degrees C. The average FE was significantly greater at 7 degrees C (11.5+/-1.1%) than at 20 degrees C (7.8+/-1.0%), as expected. The enhancement of force/stiffness was also significantly greater at the low (13.3+/-1.4%) compared to the high temperature (5.6+/-1.7%), indicating an increased conversion from weakly to strongly bound cross-bridges at the low temperature. We conclude from the results of this study that muscle preparations that are biased towards weakly bound cross-bridge states show increased FE for given stretch conditions, thereby supporting the idea that FE might be caused, in part, by a stretch-induced facilitation of the conversion of weakly to strongly bound cross-bridges.     

Arsenazo III and antipyrylazo III calcium transients in single skeletal muscle fibers

The metallochrome calcium indicators arsenazo III and antipyrylazo III have been introduced individually into cut single frog skeletal muscle fibers from which calcium transients have been elicited either by action potential stimulation or by voltage-clamp pulses of up to 50 ms in duration. Calcium transients recorded with both dyes at selected wavelengths have similar characteristics when elicited by action potentials. Longer voltage-clamp pulse stimulation reveals differences in the late phases of the optical signals obtained with the two dyes. The effects of different tension blocking methods on Ca transients were compared experimentally. Internal application of EGTA at concentrations up to 3 mM was demonstrated to be efficient in blocking movement artifacts without affecting Ca transients. Higher EGTA concentrations affect the Ca signals' characteristics. Differential effects of internally applied EGTA on tension development as opposed to calcium transients suggest that diffusion with binding from Ca++ release sites to filament overlap sites may be significant. The spectral characteristics of the absorbance transients recorded with arsenazo III suggest that in situ recorded signals cannot be easily interpreted in terms of Ca concentration changes. A more exhaustic knowledge of the dye chemistry and/or in situ complications in the use of the dye will be necessary.     

Intracellular pH recovery from lactic acidosis of single skeletal muscle fibers

Intracellular pH (pHi), measured with H+-selective microelectrodes, in quiescent frog sartorius muscle fibres was 7.29 +/- 0.09 (n = 13). Frog muscle fibres were superfused with a modified Ringer solution containing 30 mM HEPES buffer, at extracellular pH (pHo) 7.35. Intracellular pH decreased to 6.45 +/- 0.14 (n = 13) following replacement of 30 mM NaCl with sodium lactate (30 mM MES, pHo 6.20). Intracellular pH recovery, upon removal of external lactic acid, depended on the buffer concentration of the modified Ringer solution. The measured values of the pHi recovery rates was 0.06 +/- 0.01 delta pHi/min (n = 5) in 3 mM HEPES and was 0.18 +/- 0.06 delta pHi/min (n = 13) in 30 mM HEPES, pHo 7.35. The Na+-H+ exchange inhibitor amiloride (2 mM) slightly reduced pHi recovery rate. The results indicate that the net proton efflux from lactic acidotic frog skeletal muscle is mainly by lactic acid efflux and is limited by the transmembrane pH gradient which, in turn, depends on the extracellular buffer capacity in the diffusion limited space around the muscle fibres.     

Polarization changes in light diffracted from contracting muscle fibers

Single fibers were isolated from the semitendinosus muscle of frog and illuminated with an He-Ne laser. The polarization of the laser beam was varied by a photoelastic modulator. The time course of the degree of polarization of light diffracted from the muscle fiber during an isometric contraction was measured directly with a time resolution of 1 ms. Tension, sarcomere length, and diffraction intensity were also measured. During the contraction cycle, the degree of polarization of the active fiber exhibited a biphasic variation relative to that of the resting fiber. Analysis identifies the movement of heavy meromyosin toward actin and the rise in myoplasmic calcium ion concentration as the main contributors to the polarization transient of active fibers. A quantitative theory describing the polarized diffraction from muscle fibers is formulated. There is good agreement between the theory and measurements.     

Polarization changes in light diffracted from contracting muscle fibers

Single fibers were isolated from the semitendinosus muscle of frog and illuminated with an He-Ne laser. The polarization of the laser beam was varied by a photoelastic modulator. The time course of the degree of polarization of light diffracted from the muscle fiber during an isometric contraction was measured directly with a time resolution of 1 ms. Tension, sarcomere length, and diffraction intensity were also measured. During the contraction cycle, the degree of polarization of the active fiber exhibited a biphasic variation relative to that of the resting fiber. Analysis identifies the movement of heavy meromyosin toward actin and the rise in myoplasmic calcium ion concentration as the main contributors to the polarization transient of active fibers. A quantitative theory describing the polarized diffraction from muscle fibers is formulated. There is good agreement between the theory and measurements.