Stephen Hollingworth

Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements.

Binding of the fluorescent Ca2+ indicator dye fura-2 by intracellular constituents has been investigated by steady-state optical measurements. Fura-2s (a) fluorescence intensity, (b) fluorescence emission anisotropy, (c) fluorescence emission spectrum, and (d) absorbance spectra were measured in glass capillary tubes containing solutions of purified myoplasmic proteins; properties b and c were also measured in frog skeletal muscle fibers microinjected with fura-2. The results indicate that more than half, and possibly as much as 85%, of fura-2 molecules in myoplasm are in a protein-bound form, and that the binding changes many properties of the dye. For example, in vitro characterization of the Ca2+-dye reaction indicates that when fura-2 is bound to aldolase (a large and abundant myoplasmic protein), the dissociation constant of the dye for Ca2+ is three- to fourfold larger than that measured in the absence of protein. The problems raised by intracellular binding of fura-2 to cytoplasmic proteins may well apply to cells other than skeletal muscle fibers.

A small-molecule inhibitor of skeletal muscle myosin II

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosins interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.

Fura-2 calcium transients in frog skeletal muscle fibres.

1. Intact single twitch fibres from frog muscle were mounted at long sarcomere spacing (3.5‐4.2 microns) on an optical bench apparatus for the measurement of absorbance and fluorescence signals following the myoplasmic injection of either or both of the Ca2+ indicator dyes Fura‐2 and Antipyrylazo III. Dye‐related signals were measured at 16‐17 degrees C in fibres at rest and stimulated electrically to give a single action potential or brief train of action potentials. 2. The apparent diffusion constant of Fura‐2 in myoplasm, Dapp, was estimated from Fura‐2 fluorescence measured as a function of time and distance from the site of dye injection. On average (N = 7), Dapp was 0.36 x 10(‐6) cm2 s‐1, a value nearly 3‐fold smaller than expected if all the Fura‐2 was freely dissolved in the myoplasmic solution. The small value of Dapp is explained if approximately 60‐65% of the Fura‐2 molecules were bound to relatively immobile sites in myoplasm. 3. In resting fibres the fraction of Fura‐2 in the Ca2+‐bound form was estimated to be small, on average (N = 11) 0.06 of total dye. However, because of the large fraction of Fura‐2 not freely dissolved in myoplasm, and the indirect method employed for estimating Ca2+‐bound dye, calibration of the resting level of myoplasmic free Ca2+ ([Ca2+]) from the fraction of Ca2+‐bound dye was not considered reliable. 4. In response to a single action potential, large changes in Fura‐2 fluorescence (delta F) and absorbance (delta A) were detected, which had identical time courses. As expected, the directions of these transients corresponded to an increase in Ca2+‐dye complex. For wavelengths, lambda, between 380 and 460 nm, peak delta A(lambda) was closely similar to the Ca2+‐dye difference spectrum for Fura‐2 determined in in vitro calibrations. Beers law was used to calibrate the concentration of Ca2+‐dye complex formed during activity (delta[CaFura‐2]) from the delta A(lambda) signal. Peak delta[CaFura‐2] was found to vary between 0.01 and 0.4 mM, depending on the total concentration of injected Fura‐2 ([Fura‐2T]), which ranged as high as 0.9 mM. 5. In fibres in which peak delta[CaFura‐2] was less than 0.06 mM, delta[CaFura‐2] had a limiting minimal half‐width of 50‐60 ms. However, as peak delta[CaFura‐2] increased (up to 0.3‐0.4 mM), delta[CaFura‐2] half‐width became markedly prolonged (up to 150‐200 ms), indicative of a strong buffering action of large concentrations of Fura‐2 on the underlying [Ca2+] transient (delta[Ca2+]).(ABSTRACT TRUNCATED AT 400 WORDS)

Sarcoplasmic reticulum calcium release compared in slow- twitch and fast-twitch fibres of mouse muscle

Experiments were carried out to compare the amplitude and time course of Ca2+ release from the sarcoplasmic reticulum (SR) in intact slow‐twitch and fast‐twitch mouse fibres. Individual fibres within small bundles were injected with furaptra, a low‐affinity, rapidly responding Ca2+ indicator. In response to a single action potential at 16 °C, the peak amplitude and half‐duration of the change in myoplasmic free [Ca2+] (Δ[Ca2+]) differed significantly between fibre types (slow‐twitch: peak amplitude, 9.4 ± 1.0 μM (mean ± S.E.M.); half‐duration, 7.7 ± 0.6 ms; fast‐twitch: peak amplitude 18.5 ± 0.5 μM; half‐duration, 4.9 ± 0.3 ms). SR Ca2+ release was estimated from Δ[Ca2+] with a computational model that calculated Ca2+ binding to the major myoplasmic Ca2+ buffers (troponin, ATP and parvalbumin); buffer concentrations and reaction rate constants were adjusted to reflect fibre‐type differences. In response to an action potential, the total concentration of released Ca2+ (Δ[CaT]) and the peak rate of Ca2+ release ((d/dt)Δ[CaT]) differed about 3‐fold between the fibre types (slow‐twitch: Δ[CaT], 127 ± 7 μM; (d/dt)Δ[CaT], 70 ± 6 μM ms−1; fast‐twitch: Δ[CaT], 346 ± 6 μM; (d/dt)Δ[CaT], 212 ± 4 μM ms−1). In contrast, the half‐duration of (d/dt)Δ[CaT] was very similar in the two fibre types (slow‐twitch, 1.8 ± 0.1 ms; fast‐twitch, 1.6 ± 0.0 ms). When fibres were stimulated with a 5‐shock train at 67 Hz, the peaks of (d/dt)Δ[CaT] in response to the second and subsequent shocks were much smaller than that due to the first shock; the later peaks, expressed as a fraction of the amplitude of the first peak, were similar in the two fibre types (slow‐twitch, 0.2–0.3; fast‐twitch, 0.1–0.3). The results support the conclusion that individual SR Ca2+ release units function similarly in slow‐twitch and fast‐twitch mammalian fibres.

Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicators dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.

Intracellular calcium movements during excitation–contraction coupling in mammalian slow-twitch and fast-twitch muscle fibers

In skeletal muscle fibers, action potentials elicit contractions by releasing calcium ions (Ca2+) from the sarcoplasmic reticulum. Experiments on individual mouse muscle fibers micro-injected with a rapidly responding fluorescent Ca2+ indicator dye reveal that the amount of Ca2+ released is three- to fourfold larger in fast-twitch fibers than in slow-twitch fibers, and the proportion of the released Ca2+ that binds to troponin to activate contraction is substantially smaller.

Sarcomeric Ca2+ gradients during activation of frog skeletal muscle fibres imaged with confocal and two-photon microscopy.

1 Intra‐sarcomeric gradients of [Ca2+] during activation of action potential stimulated frog single fibres were investigated with the Ca2+ indicator fluo‐3 and confocal and two‐photon microscopy. The object of these experiments was to look for evidence of extra‐junctional Ca2+ release and examine the microscopic diffusion of Ca2+ within the sarcomere. 2 By exploiting the spatial periodicity of sarcomeres within the fibre, we could achieve a high effective line‐scanning rate (≈8000 lines s−1), although the laser scanning microscope was limited to < 1000 lines s−1. At this high time resolution, the time course of fluorescence changes was very different at the z‐ and m‐lines, with a significant delay (≈1 ms; 22 °C) between the rise of fluorescence at the z‐line and the m‐line. 3 To calculate the expected fluorescence changes, we used a multi‐compartment model of Ca2+ movements in the half‐sarcomere in which Ca2+ release was restricted to triadic junctions (located at z‐lines). Optical blurring by the microscope was simulated to generate fluorescence signals which could be compared directly to experimental data. The model which reproduced our experimental findings most accurately included Ca2+ binding by ATP, as well as indicator binding to immobile sarcomeric proteins. After taking sarcomeric misregistration within the fibre into account, there was very good agreement between the model and experimental results. 4 We conclude that there is no experimental evidence for Ca2+ release at locations other than at z‐lines. In addition, our calculations support the conclusion that rapidly diffusing Ca2+ buffers (such as ATP) are important in shaping the Ca2+ transient and that the details of intracellular indicator binding need to be considered to explain correctly the time course of fluorescence change in the fibre.

Excitation-contraction coupling in intact frog skeletal muscle fibers injected with mmolar concentrations of fura-2

Experiments were carried out to test the hypothesis that mM concentrations of fura-2, a high-affinity Ca2+ buffer, inhibit the release of Ca2+ from the sarcoplasmic reticulum (SR) of skeletal muscle fibers. Intact twitch fibers from frog muscle, stretched to a long sarcomere length and pressure-injected with fura-2, were activated by an action potential. Fura-2s absorbance and fluorescence signals were measured at different distances from the site of fura-2 injection; thus, the myoplasmic free Ca2+ transient (delta [Ca2+]) and the amount and rate of SR Ca2+ release could be estimated at different myoplasmic concentrations of fura-2 ([fura-2T]). At [fura-2T] = 2-3 mM, the amplitude and half-width of delta [Ca2+] were reduced to approximately 25% of the values measured at [fura-2T] less than 0.15 mM, whereas the amount and rate of SR Ca2+ release were enhanced by approximately 50% (n = 5; 16 degrees C). Similar results were observed in experiments carried out at low temperature (n = 2; 8.5-10.5 degrees C). The finding of an enhanced rate of Ca2+ release at 2-3 mM [fura-2T] is opposite to that reported by Jacquemond et al. (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophys. J. 60:867-873) from analogous experiments carried out on cut fibers. In two experiments involving the injection of larger amounts of fura-2, reductions in SR Ca2+ release were observed; however, we were unable to decide whether these reductions were due to [fura-2T] or to some nonspecific effect of the injection itself. These experiments do, however, suggest that if large [fura-2T] inhibits SR Ca2+ release in intact fibers, [fura-2T] must exceed 6 mM to produce an effect comparable to that reported by Jacquemond et al. in cut fibers. Our clear experimental result that 2-3 mM [fura-2T] enhances SR Ca2+ release supports the proposal that delta [Ca2+] triggered by an action potential normally feeds back to inhibit further release of Ca2+ from the SR (Baylor, S.M., and S. Hollingworth. 1988. J. Physiol. [Lond.]. 403:151-192). Our results provide no support for the hypothesis that Ca(2+)-induced Ca2+ release plays a significant role in excitation-contraction coupling in amphibian skeletal muscle.

Simulation of Ca2+ movements within the sarcomere of fast-twitch mouse fibers stimulated by action potentials.

Ca2+ release from the sarcoplasmic reticulum (SR) of skeletal muscle takes place at the triadic junctions; following release, Ca2+ spreads within the sarcomere by diffusion. Here, we report multicompartment simulations of changes in sarcomeric Ca2+ evoked by action potentials (APs) in fast-twitch fibers of adult mice. The simulations include Ca2+ complexation reactions with ATP, troponin, parvalbumin, and the SR Ca2+ pump, as well as Ca2+ transport by the pump. Results are compared with spatially averaged Ca2+ transients measured in mouse fibers with furaptra, a low-affinity, rapidly responding Ca2+ indicator. The furaptra ΔfCaD signal (change in the fraction of the indicator in the Ca2+-bound form) evoked by one AP is well simulated under the assumption that SR Ca2+ release has a peak of 200–225 μM/ms and a FDHM of ∼1.6 ms (16°C). ΔfCaD elicited by a five-shock, 67-Hz train of APs is well simulated under the assumption that in response to APs 2–5, Ca2+ release decreases progressively from 0.25 to 0.15 times that elicited by the first AP, a reduction likely due to Ca2+ inactivation of Ca2+ release. Recovery from inactivation was studied with a two-AP protocol; the amplitude of the second release recovered to >0.9 times that of the first with a rate constant of 7 s−1. An obvious feature of ΔfCaD during a five-shock train is a progressive decline in the rate of decay from the individual peaks of ΔfCaD. According to the simulations, this decline is due to a reduction in available Ca2+ binding sites on troponin and parvalbumin. The effects of sarcomere length, the location of the triadic junctions, resting [Ca2+], the parvalbumin concentration, and possible uptake of Ca2+ by mitochondria were also investigated. Overall, the simulations indicate that this reaction-diffusion model, which was originally developed for Ca2+ sparks in frog fibers, works well when adapted to mouse fast-twitch fibers stimulated by APs.

Comparison of Simulated and Measured Calcium Sparks in Intact Skeletal Muscle Fibers of the Frog

Calcium sparks in frog intact skeletal muscle fibers were modeled as stereotypical events that arise from a constant efflux of Ca2+ from a point source for a fixed period of time (e.g., 2.5 pA of Ca2+ current for 4.6 ms; 18°C). The model calculates the local changes in the concentrations of free Ca2+ and of Ca2+ bound to the major intrinsic myoplasmic Ca2+ buffers (troponin, ATP, parvalbumin, and the SR Ca2+ pump) and to the Ca2+ indicator (fluo-3). A distinctive feature of the model is the inclusion of a binding reaction between fluo-3 and myoplasmic proteins, a process that strongly affects fluo-3′s Ca2+-reaction kinetics, its apparent diffusion constant, and hence the morphology of sparks. ΔF/F (the change in fluo-3′s fluorescence divided by its resting fluorescence) was estimated from the calculated changes in fluo-3 convolved with the microscope point-spread function. To facilitate comparisons with measured sparks, noise and other sources of variability were included in a random repetitive fashion to generate a large number of simulated sparks that could be analyzed in the same way as the measured sparks. In the initial simulations, the binding of Ca2+ to the two regulatory sites on troponin was assumed to follow identical and independent binding reactions. These simulations failed to accurately predict the falling phase of the measured sparks. A second set of simulations, which incorporated the idea of positive cooperativity in the binding of Ca2+ to troponin, produced reasonable agreement with the measurements. Under the assumption that the single channel Ca2+ current of a ryanodine receptor (RYR) is 0.5–2 pA, the results suggest that 1–5 active RYRs generate an average Ca2+ spark in a frog intact muscle fiber.