Myofilament Space Research Articles

Spatiotemporal Features of Ca 2+ Buffering and Diffusion in Atrial Cardiac Myocytes with Inhibited Sarcoplasmic Reticulum

Ca 2+ signaling in cells is largely governed by Ca 2+ diffusion and Ca 2+ binding to mobile and stationary Ca 2+ buffers, including organelles. To examine Ca 2+ signaling in cardiac atrial myocytes, a mathematical model of Ca 2+ diffusion was developed which represents several subcellular compartments, including a subsarcolemmal space with restricted diffusion, a myofilament space, and the cytosol. The model was used to quantitatively simulate experimental Ca 2+ signals in terms of amplitude, time course, and spatial features. For experimental reference data, L-type Ca 2+ currents were recorded from atrial cells with the whole-cell voltage-clamp technique. Ca 2+ signals were simultaneously imaged with the fluorescent Ca 2+ indicator Fluo-3 and a laser-scanning confocal microscope. The simulations indicate that in atrial myocytes lacking T-tubules, Ca 2+ movement from the cell membrane to the center of the cells relies strongly on the presence of mobile Ca 2+ buffers, particularly when the sarcoplasmic reticulum is inhibited pharmacologically. Furthermore, during the influx of Ca 2+ large and steep concentration gradients are predicted between the cytosol and the submicroscopically narrow subsarcolemmal space. In addition, the computations revealed that, despite its low Ca 2+ affinity, ATP acts as a significant buffer and carrier for Ca 2+, even at the modest elevations of [Ca 2+] i reached during influx of Ca 2+.

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres.

The present study was carried out to investigate the contribution of the Ca2+-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca2+ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca2+ concentration of 0.36 microM to load the SR with Ca2+. Release of Ca2+ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = -log10 [Ca2+]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca2+ transient. The calculated Ca2+ transients were fitted by a double exponential function: Y0 + A1 . (-t/t1) + A2 . exp(t/t2), with A1 < 0 < A2, t1 < t2 and Y0, A1, A2 in micromolar. Ca2+ transients in the presence of the SR Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca2+-ATPase during caffeine-induced Ca2+ release causes an increase in the peak Ca2+ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 +/- 3 microM CPA and a Hill slope of 1.1 +/- 0.2, saturating at 100 microM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca2+-ATPase influences the Ca2+ gradient across the SR membrane in particular during the early stages of the Ca2+ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca2+ into the bathing solution.

Calcium transients and calcium release in rat fast‐twitch skeletal muscle fibres.

1. Calcium transients were recorded from cut segments of fast-twitch rat skeletal muscle fibres stretched to 3.7-4.0 microns per sarcomere and voltage clamped at a holding potential of -80 mV using the double Vaseline-gap technique. Calcium transients were monitored simultaneously with the two calcium indicators antipyrylazo III (AP III) and fura-2. AP III was used to record the calcium changes in response to 10-200 ms depolarizing pulses to different membrane potentials while fura-2 monitored the slow decay of the transient (during 16-20 s) and the resting calcium concentration. Experiments were performed at 14-17 degrees C. 2. For 50-100 ms depolarizing pulses calcium transients were first detected between -30 and -20 mV in a total of twenty-one fibres. The transients recorded with AP III showed a plateau for small pulses (-20 mV) and a steady increase during stronger pulses (-10 mV and more positive). Upon repolarization the transients decayed towards the baseline. The signal recorded simultaneously with fura-2 showed a continuous increase of the transient during the pulses at all membrane potentials. The amplitude of the calcium transients for the large pulses could not be followed with fura-2 due to saturation of the dye. 3. The signals obtained with both dyes were used to determine the kinetics of the calcium-fura-2 reaction inside the fibres. The mean values of the kinetic parameters were: the on rate constant (kon) = 5.1 x 10(8) M-1s-1, the off rate constant (koff) = 26 s-1, and koff/kon (KD) = 69.7 nM. 4. The fast phase of decay of the calcium transients after the pulses was studied from the records obtained with AP III. For depolarizing pulses of the same duration, the rate of decay of the transients after the pulse was slower the stronger the depolarization. For pulses to the same membrane potential, the rate of decay was slower the longer the pulse duration. Both stimulating patterns indicated saturation of the removal system in the muscle fibres due to occupancy of slowly equilibrating myoplasmic calcium binding sites by released calcium. 5. The fast phase of decay of the signals obtained with AP III was well fitted with a model of the system for removing calcium from the myofilament space. 6. The rate of calcium release (Rrel) from the sarcoplasmic reticulum was calculated once the removal system was characterized in the same fibre.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium-induced inactivation of calcium release from the sarcoplasmic reticulum of skeletal muscle

The ability of myofilament space Ca2+ to modulate Ca2+ release from the sarcoplasmic reticulum (SR) of skeletal muscle was investigated. Single fibers of the frog Rana pipiens belindieri were manually skinned (sarcolemma removed). Following a standard load and pre-incubation in varying myoplasmic Ca2+ concentrations, SR Ca2+ release was initiated by caffeine. Ca2+ release rates were calculated from the changes in absorbance of a Ca2+ sensitive dye, antipyrylazo III. An apparent dissociation constant (Kd) for dye-Ca2+ binding of 8000 microM 2 was determined by comparing the buffering action of the dye with that of ethylenebis(oxonitrilo)tetraacetate (EGTA) using the contractile proteins of the skinned fiber as a measure of free Ca2+. This value for Kd was used in the calculation of Ca2+ release rates. As the myoplasmic space Ca2+ was increased from pCa 7.4, Ca2+ release rates declined sharply such that at pCa 6.9 the calculated release rate was 72 +/- 3% (mean +/- SEM) of control (pCa 8.4). Further increases in myoplasmic Ca2+ from pCa 6.9 to pCa 6.1 did not result in a further decline in release rate. The effect of a decreased driving force on Ca2+ ions was investigated to determine whether it could account for the change in release rates observed. At pCa 6.9, where the greatest degree of inactivation occurred, the measured effects of a change in driving force could account for at most 40% of the observed inactivation. Varying concentrations of Ba2+ and Sr2+ in the myofilament space had no inactivating effect on the SR Ca2+ release rates. The ability of myofilament Ca2+ to inhibit SR Ca2+ release at concentrations normally encountered during muscle activation suggests a role for released Ca2+ as a modulator of the SR Ca2+ channel.

Calcium-dependent mechanical oscillations occur spontaneously in unstimulated mammalian cardiac tissues.

In quiescent rat ventricular myocardium, bathed in solution of 2 mM Ca++ or less, it has been previously demonstrated that spontaneous microscopic oscillatory cell motion is present and interacts with an incident laser beam to produce scattered light intensity fluctuations which can be monitored to quantify the underlying motion. The present study shows that scattered light intensity fluctuations are not present under any conditions in frog atrial or ventricular preparations, but do occur in each type of mammalian cardiac tissue studied in the unstimulated state. The magnitude of scattered light intensity fluctuations in mammalian tissues varies with species and cellular Ca++ loading. In some tissues, e.g., rabbit or ferret ventricle, either an increase in the Ca++ concentration in the perfusate [( Ca++]e), reduction of perfusate Na+ concentration [( Na+]e), or addition of cardiac glycosides was required to elicit scattered light intensity fluctuations; in other tissues, however, e.g., the canine Purkinje fiber, atria, and ventricle, and guinea pig atria, scattered light intensity fluctuations were present at 2 mM [Ca++]e in the absence of experimental Ca++ loading. Scattered light intensity fluctuations were not affected by LaCl3, or verapamil, and were reversibly abolished by caffeine. When the pCa in the myofilament space is kept constant in detergent "skinned" fibers, scattered light intensity fluctuations are not present during contractile activation. We conclude: that scattered light intensity fluctuations are due to spontaneous intracellular Ca++ oscillations that require a functional sarcoplasmic reticulum; that the potential to exhibit these oscillations is a fundamental property of mammalian excitable cardiac cells; and that, in many mammalian tissues, these oscillations are present in the unstimulated state, even in the absence of experimental perturbations to enhance cell Ca++ loading.

Measurement and modification of free calcium transients in frog skeletal muscle fibres by a metallochromic indicator dye.

Myoplasmic free calcium transients were monitored with the metallochromic indicator dye Antipyrylazo III (AP III) in single frog skeletal muscle fibres cut at both ends, stretched so as to minimize or eliminate contractile filament overlap and voltage clamped using a double-Vaseline-gap system (approximately 6 degrees C). The dye entered the central fibre segment by diffusion from the solution applied to the two cut ends. The diffusion coefficient of AP III was about 20 times lower in the fibre than in solution. This very slow diffusion was not due to binding of dye since the ratio of bound to free dye obtained from analysis of the diffusion was only about 0.45. For a given depolarizing pulse, the ratio of dye-related absorbance changes delta A at 720 and 550 nm was the same as that produced on adding calcium to dye in calibrating solution, indicating that these signals were due to changes in myoplasmic calcium. The delta A signals at 700 or 720 nm were used to monitor transient changes in concentration of calcium-dye complex [CaD2] and of free calcium [Ca] in the myofilament space. By applying the same pulse at different times during dye entry, it was observed that increasing dye concentrations [D]T produced the following effects: (a) [CaD2] was increased; (b) [Ca] was decreased at early times during a pulse; (c) a declining phase of [Ca] observed at late times during pulses was decreased and finally reversed to a slow rising phase at high [D]T; (d) the decay of [Ca] after the pulse was slowed. Analyses of the effects of [D]T on (a) the magnitude of [CaD2] at a given early time during the calcium release produced by pulses to a given voltage and on (b) the time constant for [Ca] decay after a pulse were both consistent with a calcium: dye stoichiometry of 1:2 in the fibre as found in calibrating solution. Analysis of the effect of [D]T on the [Ca] decay time constants also revealed the presence of intrinsic rapidly equilibrating myoplasmic calcium binding sites and provided the basis for obtaining estimates of the combined concentration [Ca] of free calcium plus calcium bound to such sites. Unlike the estimates of [Ca], these estimates of [Ca] are independent of the value of the calcium-dye dissociation constant.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers.

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.

Activation of fast skeletal muscle: contributions of studies on skinned fibers.

The membrane potential of vertebrate twitch fibers closely controls Ca fluxes between intracellular compartments, which in turn control contraction. Recent work on intracellular Ca movement is reviewed in the general context of current efforts to synthesize physiological, biochemical, and structural observations on the contractile mechanism and its regulation, emphasizing the increasing role of functionally skinned fibers in this synthesis. Skinned fiber preparations, with removed or disrupted sarcolemma, bridge the gap between properties of isolated subsystems and their constrained operation in the intact fiber. Recent studies indicate that the surface action potential propagates along the transverse tubules, but not the sarcoplasmic reticulum (SR), which appears to be a distinct intracellular compartment. Voltage-dependent charge movements in the transverse tubules probably control Ca flux across the SR membranes. Current questions concern the mechanism of the signal that bridges the junctional gap between the two membrane systems, the mechanism and properties of the activated Ca efflux to the myofilament space, and the operation of the Ca pump of the SR during activation. New methods applied to intact fibers, cut fibers, skinned fibers, and subcellular systems are yielding the kind of information needed for a complete description of these central steps in excitation-contraction coupling and of Ca regulation of the myofilaments.

Chloride and osmotic contractures in skinned frog muscle fibers

Single contractures were elicited in segments of skinned frog muscle fibers when the segments were moved from relaxing-loading solutions to various test solutions. The effective test solutions produced an increase in the concentration of chloride ions in the myofilament space, [Cl] ms , and/or presumably caused the sarcoplasmic reticulum to undergo a change in volume. The contractures were quantified in terms of their maximum tension and time-integral. Two outer segments from each fiber underwent a contracture in a control solution (chloride ions were substituted for all of the methanesulfonate ions in the relaxing solution). The mean values of tension and area in the control contractures of each fiber were divided into the corresponding values from a test contracture obtained in the central segment of the same fiber. Test contractures obtained upon increasing [Cl] ms and increasing the product, [K] ms ×[Cl] ms , were compared to contractures that were obtained by increasing [Cl] ms while keeping [K] ms ×[Cl] ms constant. The former contractures were greater in magnitude for a given [Cl] ms . Whereas the former solutions may have caused an increase in the volume of the sarcoplasmic reticulum and altered the electrical potential across the membranes of the sarcoplasmic reticulum as well, only a change in potential was presumed to have occurred in the latter solutions. Other types of contractures were investigated to show that both swelling of the sarcoplasmic reticulum and changes in the electrical potential of its membranes can cause release of calcium ions and elicit contractures in skinned fibers.

Properties of chloride-stimulated 45Ca flux in skinned muscle fibers.

Isometric force and 45Ca loss from fiber to bath were measured simultaneously in skinned fibers from frog muscle at 19 degrees C. In unstimulated fibers, 45Ca efflux from the sarcoplasmic reticulum (SR) was very slow, with little or no dependence on EGTA (0.1-5 mM) or Mg++ (20 micrometer-1.3 mM). Stimulation by high [Cl] at 0.11 mM Mg++ caused rapid force transients (duration approximately 10 s) and 45Ca release. This response was followed for 55 s, with 5 mM EGTA added to chelate myofilament space (MFS) Ca either (a) after relaxation, (b) near the peak of the force spike, or (c) before or with the stimulus. When EGTA was present during Cl application, stimulation of 45Ca release was undetectable. Analysis of the time-course of tracer loss during the three protocols showed that when EGTA was absent, 16% of the fiber tracer was released from the SR within approximately 3 s, and 70% of the tracer still in the MFS near the peak of the force spike was subsequently reaccumulated. The results suggest that (a) the Cl response is highly Ca-dependent; (b) stimulation increases 45Ca efflux from the SR at least 100-200-fold; and (c) the rate of reaccumulation is much slower than the influx predicted from published data on resting fibers, raising the possibility that depolarization inhibits active Ca transport by the SR.

Regulation by magnesium of intracellular calcium movement in skinned muscle fibers.

The effect of Mg on Ca movement between the sarcoplasmic reticulum (SR) and myofilament space (MFS) was studied in skinned muscle fibers by using isometric force as an indicator of MFS Ca. In Ca-loaded fibers at 20 degrees C, the large force spike induced by Ca in 1 mM Mg (5 mM ATP) was strongly inhibited in 3 mM Mg, and force development was extremely slow. After a brief Ca stimulus in 1 mM Mg, relaxation in Ca-free solution was significantly faster in 3 mM Mg. These changes were due to altered Ca movements, since the effect of 3 mM Mg on steady force in CaEGTA solutions was small. Changes in Mg alone induced force transients apparently due to altered Ca movement. In relaxed fibers, decreasing the Mg to 0.25 mM caused phasic force development. In contracting fibers in Ca solutions, increasing the Mg caused a large transient relaxation. The effects of increased Mg were antagonized by 0.5 mM Cd, an inhibitor of the SR Ca transport system. The results indicate that active Ca uptake by the SR in situ is stimulated by Mg, and that it can affect local MFS [Ca++] in the presence of a substantial Ca source. These results provide evidence that an increased rate of Ca uptake in 3 mM Mg could account for inhibition of the large force spike associated with Ca-induced Ca release in skinned fibers.

Human Skeletal Muscle: Properties of the "Chemically Skinned" Fiber

A "skinning%" procedure is described for irreversibly disrupting the sarcolemmal membrane of human skeletal muscle and allowing calcium and other diffusible solutes (such as adenosine triphosphate) access to the myofilament space. Single skinned fibers give isometric tensions of about 1.5 kilograms per square centimeter when exposed to ionized calcium event after 1 to 2 weeks of storage at 5 degrees C. For up to 5 days the preparation will sequester and, under appropriate conditions (anion substitution, caffeine addition, or magnesium withdrawal), release calciumn. The regulation of intracellular calcium distribution and the calcium-induced activation of the contractile proteins are discussed and related to the morphology of humnan fibers and to similar processes occurring on other muscle preparations.

Intracellular calcium movements in skinned muscle fibres.

1. Force development and (45)Ca movement between the internal membranes and the myofilament space were measured in segments of skinned muscle fibres.2. At 20 degrees C calcium ions in the myofilament space of calcium loaded fibres caused the internal membranes to release a substantial amount of calcium which produced a large force spike. Calcium produced a similar force spike in fibres containing only the physiological level of calcium when the temperature was lowered to about 6 degrees C.3. The force spike at 6 degrees C was inhibited when the magnesium concentration in the bathing solution was increased from 1 to 6 mM. The pattern of force development in preparations transferred directly from oil to a free calcium solution indicated that the normal concentration of Mg(2+) in the myofilament space of intact fibres is of the order of 10(-3)M.4. A possible role for the regenerative calcium release mechanism in the physiological activation of muscle fibres is suggested.