Imaging of calcium transients in skeletal muscle fibers.

Epifluorescence images of Ca2+ transients elicited by electrical stimulation of single skeletal muscle fibers were studied with fast imaging techniques that take advantage of the large fluorescence signals emitted at relatively long wavelengths by the dyes fluo-3 and rhod-2 in response to binding of Ca2+ ions, and of the suitable features of a commercially available CCD video camera. The localized release of Ca2+ in response to microinjection of InsP3 was also monitored to demonstrate the adequate space and time resolutions of the imaging system. The time resolution of the imager system, although limited to the standard video frequency response, still proved to be adequate to investigate the fast Ca2+ release process in skeletal muscle fibers at low temperatures.     

Calcium transients in asymmetrically activated skeletal muscle fibers.

Skeletal muscle fibers of the frog Rana temporaria were held just taut and stimulated transversely by unidirectional electrical fields. We observed the reversible effects of stimulus duration (0.1-100 ms) and strength on action potentials, intracellular Ca2+ transients (monitored by aequorin), and contractile force during fixed-end contractions. Long duration stimuli (e.g., 10 ms) induced a maintained depolarization on the cathodal side of a cell and a maintained hyperpolarization on its anodal side. The hyperpolarization of the side facing the anode prevented the action potential from reaching mechanical threshold during strong stimuli. Variation of the duration or strength of a stimulus changed the luminescent response from a fiber injected with aequorin. Thus, the intracellular Ca2+ released during excitation-contraction coupling could be changed by the stimulus parameters. Prolongation of a stimulus at field strengths above 1.1 x rheobase decreased the amplitude of aequorin signals and the force of contractions. The decreases in aequorin and force signals from a given fiber paralleled one another and depended on the stimulus strength, but not on the stimulus polarity. These changes were completely reversible for stimulus strengths up to at least 4.2 x rheobase. The graded decreases in membrane depolarization, aequorin signals, and contractile force were correlated with the previously described folding of myofibrils in fibers allowed to shorten in response to the application of a long duration stimulus. The changes in aequorin signals and force suggest an absence of myofilament activation by Ca2+ in the section of the fiber closest to the anode. The results imply that injected aequorin distributes circumferentially in frog muscle with a coefficient of at least 10(-7) cm2/s, which is not remarkably different from the previously measured coefficient of 5 x 10(-8) cm2/s for its diffusion lengthwise.     

Force generation and phosphate release steps in skinned rabbit soleus slow-twitch muscle fibers.

The force-generation and phosphate-release steps of the cross-bridge cycle in rabbit soleus slow-twitch muscle fibers (STF) were investigated using sinusoidal analysis, and the results were compared with those of rabbit psoas fast-twitch fibers (FTF). Single fiber preparations were activated at pCa 4.40 and ionic strength 180 mM at 20 degrees C. The effects of inorganic phosphate (Pi) concentrations on three exponential processes, B, C, and D, were studied. Results are consistent with the following cross-bridge scheme: [formula: see text] where A is actin, M is myosin, D is MgADP, and P is inorganic phosphate. The values determined are k4 = 5.7 +/- 0.5 s-1 (rate constant of isomerization step, N = 9, mean +/- SE), k-4 = 4.5 +/- 0.5 s-1 (rate constant of reverse isomerization), K4 = 1.37 +/- 0.13 (equilibrium constant of the isomerization), and K5 = 0.18 +/- 0.01 mM-1 (Pi association constant). The isomerization step (k4) in soleus STF is 20 times slower, and its reversal (k-4) is 20 times slower than psoas fibers. Consequently, the equilibrium constant of the isomerization step (K4) is the same in these two types of fibers. The Pi association constant (K5) is slightly higher in STF than in FTF, indicating that Pi binds to cross-bridges slightly more tightly in STF than FTF. By correlating the cross-bridge distribution with isometric tension, it was confirmed that force is generated during the isomerization (step 4) of the AMDP state and before Pi release in soleus STF.     

Force generation and work production by covalently cross-linked actin-myosin cross-bridges in rabbit muscle fibers.

To separate a fraction of the myosin cross-bridges that are attached to the thin filaments and that participate in the mechanical responses, muscle fibers were cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and then immersed in high-salt relaxing solution (HSRS) of 0.6 M ionic strength for detaching the unlinked myosin heads. The mechanical properties and force-generating ability of the cross-linked cross-bridges were tested with step length changes (L-steps) and temperature jumps (T-jumps) from 6-10 degrees C to 30-40 degrees C. After partial cross-linking, when instantaneous stiffness in HSRS was 25-40% of that in rigor, the mechanical behavior of the fibers was similar to that during active contraction. The kinetics of the T-jump-induced tension transients as well as the rate of the fast phase of tension recovery after length steps were close to those in unlinked fibers during activation. Under feedback force control, the T-jump initiated fiber shortening by up to 4 nm/half-sarcomere. Work produced by a cross-linked myosin head after the T-jump was up to 30 x 10(-21) J. When the extent of cross-linking was increased and fiber stiffness in HSRS approached that in rigor, the fibers lost their viscoelastic properties and ability to generate force with a rise in temperature.     

Strain-dependent modulation of phosphate transients in rabbit skeletal muscle fibers.

When inorganic phosphate (Pi) is photogenerated from caged Pi during isometric contractions of glycerinated rabbit psoas muscle fibers, the released Pi binds to cross-bridges and reverses the working stroke of cross-bridges. The consequent force decline, the Pi-transient, is exponential and probes the kinetics of the power-stroke and Pi release. During muscle shortening, the fraction of attached cross-bridges and the average strain on them decreases (Ford, L. E., A.F. Huxley, and R.M. Simmons, 1977. Tension responses to sudden length change in stimulated frog muscle fibers near slack length. J. Physiol. (Lond.). 269:441-515; Ford, L. E., A. F. Huxley, and R.M. Simmons, 1985. Tension transients during steady state shortening of frog muscle fibers. J. Physiol. (Lond.). 361:131-150. To learn to what extent the Pi transient is strain dependent, muscle fibers were activated and shortened or lengthened at a fixed velocity during the photogeneration of Pi. The Pi transients observed during changes in muscle length showed three primary characteristics: 1) during shortening the Pi transient rate, Kpi, increased and its amplitude decreased with shortening velocity; Kpi increased linearly with velocity to > 110 s-1 at 0.3 muscle lengths per second (ML/s). 2) At a specific shortening velocity, increases in [Pi] produce increases in Kpi that are nonlinear with [Pi] and approach an asymptote. 3) During forced lengthening Kpi and the amplitude of the Pi transient are little different from the isometric contractions. These data can be approximated by a strain-dependent three-state cross-bridge model. The results show that the power stroke's rate is strain-dependent, and are consistent with biochemical studies indicating that the rate-limiting step at low strains is a transition from a weakly to a strongly bound cross-bridge state.     

Fluctuations in tension during contraction of single muscle fibers.

We have searched for fluctuations in the steady-state tension developed by stimulated single muscle fibers. Such tension "noise" is expected to be present as a result of the statistical fluctuations in the number and/or state of myosin cross-bridges interacting with thin filament sites at any time. A sensitive electro-optical tension transducer capable of resolving the expected fluctuations in magnitude and frequency was constructed to search for the fluctuations. The noise was analyzed by computing the power spectra and amplitude of stochastic fluctuations in the photomultiplier counting rate, which was made proportional to muscle force. The optical system and electronic instrumentation together with the minicomputer software are described. Tensions were measured in single skinned glycerinated rabbit psoas muscle fibers in rigor and during contraction and relaxation. The results indicate the presence of fluctuations in contracting muscles and a complete absence of tension noise in eith rigor or relaxation. Also, a numerical method was developed to simulate the power spectra and amplitude of fluctuations, given the rate constants for association and dissociation of the cross-bridges and actin. The simulated power spectra and the frequency distributions observed experimentally are similar.     

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.     

Resting tension characteristics in differentiating intact rat fast- and slow-twitch muscle fibers.

The postnatal changes in resting muscle tension were investigated at 20 degrees C by using small muscle fiber bundles isolated from either the extensor digitorum longus or the soleus of both neonatal (7-21 days old) and adult rats. The results show that the tension-extension characteristics of the bundles depended on the age of the rats. For example, both the extensor digitorum longus and soleus bundles of rats older than 14 days showed characteristic differences that were absent in bundles from younger rats. Furthermore, the tension-extension relation of the adult slow muscle fiber bundles were similar to those of the two neonatal muscles and were shifted to longer sarcomere lengths relative to those of the adult fast-fiber bundles. Thus, at the extended sarcomere length of 2.9 microm, the adult fast muscle fiber bundles developed higher resting tensions (5.6 +/- 0.5 kN/m2) than either the two neonatal ( approximately 3 kN/m2) or the adult slow (3.1 +/- 0.4 kN/m2) muscle fiber bundles. At all ages examined, the resting tension responses to a ramp stretch were qualitatively similar and consisted of three components: a viscous, a viscoelastic, and an elastic tension. However, in rats older than 14 days, all three tension components showed clear fast- and slow-fiber type differences that were absent in younger rats. Bundles from 7-day-old rats also developed significantly lower resting tensions than the corresponding adult ones. Additionally, the resting tension characteristics of the adult muscles were not affected by chemical skinning. From these results, we conclude that in rats resting muscle tension, like active tension, differentiates within the first 3 wk after birth.     

Force generation,but not myosin ATPase activity,declines with age in rat muscle fibers

We tested the hypothesis thatage-associated decline in muscle function is related to a change inmyosin ATPase activity. Single, glycerinated semimembranosus fibersfrom young (8-12 mo) and aged (32-37 mo) Fischer 344 × Brown Norway male rats were analyzed simultaneously for force andmyosin ATPase activity over a range of Ca²⁺ concentrations.Maximal force generation was ~20% lower in fibers from aged animals(P = 0.02), but myosin ATPase activity was not different between fibers from young and aged rats: 686 ± 46 (n = 30) and 697 ± 46 µM/s (n = 33) (P = 0.89). The apparent rate constant for thedissociation of strong-binding myosin from actin was calculated to be~30% greater in fibers from aged animals (P = 0.03),indicating that the lower force produced by fibers from aged animals isdue to a greater flux of myosin heads from the strong-binding state tothe weak-binding state during contraction. This is in agreement withour previous electron paramagnetic resonance experiments that showed areduced fraction of myosin heads in the strong-binding state during amaximal isometric contraction in fibers from older rats.

     

Effects of a non-divalent cation binding mutant of myosin regulatory light chain on tension generation in skinned skeletal muscle fibers.

Each myosin molecule contains two heavy chains and a total of four low-molecular weight light chain subunits, two "essential" and two "regulatory" light chains (RLCs). Although the roles of myosin light chains in vertebrate striated muscle are poorly understood at present, recent studies on the RLC have suggested that it has a modulatory role with respect to Ca2+ sensitivity of tension and the rate of tension development, effects that may be mediated by Ca2+ binding to the RLC. To examine possible roles of the RLC Ca2+/Mg2+ binding site in tension development by skeletal muscle, we replaced endogenous RLC in rabbit skinned psoas fibers with an avian mutant RLC (D47A) having much reduced affinity for divalent cations. After replacement of up to 80% of the endogenous RLC with D47A RLC, maximum tension (at pCa 4.5) was significantly reduced compared with preexchange tension, and the amount of decrease was directly related to the extent of D47A exchange. Fiber stiffness changed in proportion to tension, indicating that the decrease in tension was due to a decrease in the number of tension-generating cross-bridges. Decreases in both tension and stiffness were substantially, although incompletely, reversed after reexchange of native RLC for D47A. RLC exchange was also performed using a wild-type RLC. Although a small decrease in tension was observed after wild-type RLC exchange, the decrease was not proportional to the extent of RLC exchange and was not reversed by reexchange of the native RLC. D47A exchange also decreased the Ca2+ sensitivity of tension and reduced the apparent cooperativity of tension development. The results suggest that divalent cation binding to myosin RLC plays an important role in tension generation in skeletal muscle fibers.     

Tension transients initiated by photogeneration of MgADP in skinned skeletal muscle fibers

Addition of MgADP to skinned skeletal muscle fibers causes a rise in Ca(2+)-activated isometric tension. Mechanisms underlying this tension increase have been investigated by rapid photogeneration of ADP within skinned single fibers of rabbit psoas muscle. Photolysis of caged ADP (P2-1(2-nitrophenyl)ethyladenosine 5'-diphosphate) resulted in an exponential increase in isometric tension with an apparent rate constant, kADP, of 9.6 +/- 0.3 s-1 (mean +/- SE, n = 28) and an amplitude, PADP, of 4.9 +/- 0.3% Po under standard conditions (0.5 mM photoreleased MgADP, 4 mM MgATP, pH 7.0, pCa 4.5, 0.18 M ionic strength, 15 degrees C). PADP depended upon the concentration of photoreleased MgADP as well as the concentration of MgATP. A plot of 1/PADP vs. 1/[MgADP] at three MgATP concentrations was consistent with competition between MgADP and MgATP for the same site on the crossbridge. The rate of the transient, kADP, also depended upon the concentration of MgADP and MgATP. At both 4 and 1 mM MgATP, kADP was not significantly different after photorelease of 0.1-0.5 mM MgADP, but was reduced by 28-40% when 3.5 mM MgADP was added before photorelease of 0.5 mM MgADP. kADP was accelerated by about twofold when MgATP was varied from 0.5 to 8 mM MgATP. These effects of MgATP and MgADP were not readily accounted for by population of high force-producing states resulting from reversal of the ADP dissociation process. Rather, the results suggest that competition between MgADP and MgATP for crossbridges at the end of the cycle slows detachment leading to accumulation of force-generating crossbridges. Elevation of steady- state Pi concentration from 0.5 to 30 mM caused acceleration of kADP from 10.2 +/- 0.5 to 27.8 +/- 1.8 s-1, indicating that the tension rise involved crossbridge flux through the Pi dissociation step of the cycle.     

Calcium transients in single fibers of low-frequency stimulated fast-twitch muscle of rat

Ca²⁺transients were investigated in single fibers isolated from ratextensor digitorum longus muscles exposed to chronic low-frequency stimulation for different time periods up to 10 days. Approximately 2.5-fold increases in resting Ca²⁺ concentration([Ca²⁺]) were observed 2 h after stimulationonset and persisted throughout the stimulation period. The elevated[Ca²⁺] levels were in the range characteristicof slow-twitch fibers from soleus muscle. In addition, we noticed atransitory elevation of the integral[Ca²⁺] per pulse with a maximum (~5-fold)after 1 day. Steep decreases in rate constant of[Ca²⁺] decay could be explained by an immediateimpairment of Ca²⁺ uptake and, with longer stimulationperiods, by an additional loss of cytosolic Ca²⁺ bindingcapacity resulting from a decay in parvalbumin content. A partialrecovery of the rate constant of [Ca²⁺] decayin 10-day stimulated muscle could be explained by an increasing mitochondrial contribution to Ca²⁺ sequestration.

     

Lattice spacing changes accompanying isometric tension development in intact single muscle fibers.

The myosin lattice spacing of single intact muscle fibers of the frog, Rana temporaria, was studied in Ringer's solution (standard osmolarity 230 mOsm) and hyper- and hypotonic salines (1.4 and 0.8 times standard osmolarity respectively) in the relaxed state, during "fixed end" tetani, and during shortening, using synchrotron radiation. At standard tonicity, a tetanus was associated with an initial brief lattice expansion (and a small amount of sarcomere shortening), followed by a slow compression (unaccompanied by sarcomere length changes). In hypertonic saline (myosin lattice compressed by 8.1%), these spacing changes were suppressed, in hypotonic saline (lattice spacing increased by 7.5%), they were enhanced. During unloaded shortening of activated fibers, a rapid lattice expansion occurred at all tonicities, but became larger as tonicity was reduced. This expansion was caused in part by the change in length of the preparation, but also by a recoil of a stressed radial compliance associated with axial force. The lattice spacing during unloaded shortening was equal to or occasionally greater than predicted for a relaxed fiber at that sarcomere length, indicating that the lattice compression associated with activation is rapidly reversed upon loss of axial force. Lattice recompression occurred upon termination of shortening under standard and hypotonic conditions, but was almost absent under hypertonic conditions. These observations indicate that axial cross-bridge tension is associated with a compressive radial force in intact muscle fibers at full overlap; however, this radial force exhibits a much greater sensitivity to lattice spacing than does the axial force.