Review
Calcium indicators and calcium signalling in skeletal muscle fibres during excitation–contraction coupling

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Abstract

During excitation–contraction coupling in skeletal muscle, calcium ions are released into the myoplasm by the sarcoplasmic reticulum (SR) in response to depolarization of the fibre’s exterior membranes. Ca2+ then diffuses to the thin filaments, where Ca2+ binds to the Ca2+ regulatory sites on troponin to activate muscle contraction. Quantitative studies of these events in intact muscle preparations have relied heavily on Ca2+-indicator dyes to measure the change in the spatially-averaged myoplasmic free Ca2+ concentration (Δ[Ca2+]) that results from the release of SR Ca2+. In normal fibres stimulated by an action potential, Δ[Ca2+] is large and brief, requiring that an accurate measurement of Δ[Ca2+] be made with a low-affinity rapidly-responding indicator. Some low-affinity Ca2+ indicators monitor Δ[Ca2+] much more accurately than others, however, as reviewed here in measurements in frog twitch fibres with sixteen low-affinity indicators. This article also examines measurements and simulations of Δ[Ca2+] in mouse fast-twitch fibres. The simulations use a multi-compartment model of the sarcomere that takes into account Ca2+’s release from the SR, its diffusion and binding within the myoplasm, and its re-sequestration by the SR Ca2+ pump. The simulations are quantitatively consistent with the measurements and appear to provide a satisfactory picture of the underlying Ca2+ movements.

Introduction

A change in the cytosolic Ca2+ concentration (Δ[Ca2+]) controls important electrical and metabolic events in many types of cells. For example, in striated muscle, an increase in [Ca2+] activates the cells’ contractile machinery. Due to its widespread importance, intracellular Ca2+ signalling has been intensively studied in many laboratories. An important methodology in these studies has been the intracellular use of fluorescent Ca2+ indicator dyes. This review focuses on issues that arise when the experimental goal is to make a quantitatively accurate measurement of Δ[Ca2+] in vertebrate skeletal muscle fibres with a Ca2+ indicator dye. These issues likely apply to measurements of Δ[Ca2+] in a number of other cell types.

Section snippets

Background on studies of Δ[Ca2+] in skeletal muscle fibres

In skeletal muscle fibres stimulated electrically, Δ[Ca2+] arises primarily from the release of Ca2+ by the sarcoplasmic reticulum (SR) in response to depolarization of the transverse tubular (T-tubular) membranes. The release takes place at the triadic junctions (Franzini-Armstrong, 1970), which are specialized locations within each sarcomere, where the T-tubular and SR membranes come into close apposition (Fig. 1). Once released, Ca2+ diffuses within the myoplasm while also complexing with

The family of low-affinity Ca2+ indicators

All of the indicator dyes discussed in this article are thought to react with Ca2+ according to a 1:1 binding reaction

Ca2++DKD,CaCaDD and CaD represent the Ca2+-free and Ca2+-bound forms of the indicator, respectively; KD,Ca denotes the dissociation constant of the indicator for Ca2+, which is equal to the ratio of the reverse (koff) and forward (kon) rate constants of the reaction. With many of the indicators, the metal binding site can also react with Mg2+ with an appreciable affinity; in

Advantages of low-affinity Ca2+ indicators

To estimate Δ[Ca2+] with an indicator dye, intracellular measurements are usually made either of the indicator’s FR and activity-related ΔF, or AR (resting absorbance) and activity-related ΔA. Calibration of the optical measurements in units of Δ[Ca2+] is, in principle, a two-step process: (i) conversion of the measurements to ΔfCaD (the change in the fraction of the indicator in the Ca2+-bound form); (ii) conversion of ΔfCaD to Δ[Ca2+]. For these steps, it is often assumed that the indicator’s

Complications due to the intracellular environment

Accurate estimation of ΔfCaD and Δ[Ca2+] (and, in the case of high-affinity indicators, fR and [Ca2+]R) may be difficult in muscle fibres because, with most indicators, a substantial fraction of the indicator binds to intracellular constituents, including soluble and structural proteins (Baker et al., 1994, Baylor et al., 1986, Beeler et al., 1980, Harkins et al., 1993, Hirota et al., 1989, Hollingworth et al., 2009, Hove-Madsen and Bers, 1992, Konishi et al., 1988, Konishi et al., 1991,

Multi-compartment model of Ca2+ movements within the sarcomere

As mentioned in Section 4, the myoplasmic Ca2+ transient elicited by an action potential in skeletal muscle fibres varies widely in amplitude and time course in different regions of the sarcomere (see also Section 13). This occurs because (i) the sites of SR Ca2+ release within the myoplasm are restricted to the locations of the triadic junctions, and (ii) the ability of Ca2+ to spread within the sarcomere is limited by the binding of Ca2+ to its buffers and by the rates of diffusion of Ca2+

Simulated responses of Ca2+ indicators having different Ca2+ affinities

As discussed in Section 4, the larger the value of an indicator’s koff and hence KD,Ca, the more reliable is the indicator’s ability to track the time course of Δ[Ca2+]. This expectation is confirmed in the simulations in Fig. 5. For these simulations, the value of [DT] was reduced from 100 μM (the standard value; Table 3) to 1 μM so that the small buffering effect of the indicator on Δ[Ca2+] would be negligible. The SR Ca2+ release waveform elicited by an action potential (see legend of Fig. 5

Fibre techniques and experimental procedures

The experimental examples discussed below are drawn from intact fibre experiments on frog and mouse muscle carried out in our laboratory. The general procedures have been described (Baylor and Hollingworth, 1988, Baylor and Hollingworth, 2003, Hollingworth et al., 1996, Konishi et al., 1991, Zhao et al., 1996) as well as the procedures for recording Ca2+-related signals from two indicators in the same fibre in response to the same Δ[Ca2+] (Hollingworth et al., 2009, Konishi et al., 1991, Zhao

Simultaneous comparison of myoplasmic Ca2+ signals from PDAA and furaptra

Fig. 6 compares ΔfCaD signals from PDAA and furaptra in a frog twitch fibre that was injected with both indicators and stimulated to give an action potential. The measurements were made from the same fibre region at essentially identical times, so that the measurements reflect the indicators’ responses to the same Δ[Ca2+]. The values of FDHM of ΔfCaD are 6.3 ms for PDAA and 9.3 ms for furaptra. These are similar to the simulated values of ΔfCaD in Fig. 5 at KD,Ca = 1000 and 50 μM (for

Comparison of Ca2+ signals from furaptra and other low-affinity fluorescent indicators

Comparisons of the time course of furaptra’s ΔF with that of the ΔA or ΔF of other Ca2+ indicators give information about the ability of the other indicators to monitor the time course of Δ[Ca2+]. Comparative information of this type has been obtained in frog intact fibres for all of the indicators in Table 1 except for DMPDAA, which has only been used in cut fibres. However, the similarity of the FDHM of DMPDAA’s ΔA signal elicited by an action potential (9.0 ms at 16 °C; Table 2) to that of

Possible interference from [Mg2+] and Δ[Mg2+]

Of the low-affinity fluorescent indicators available for studies of Δ[Ca2+] in skeletal muscle fibres, the previous section indicates that furaptra, mag-fura-5, mag-indo-1 and mag-fluo-4 are the indicators of choice. Because all of these are tri-carboxylate indicators whose values of KD,Mg are in the millimolar range (column 3 of Table 1), a drawback that applies to all is that they have a non-negligible sensitivity to Mg2+. [Mg2+]R is therefore expected to affect FR and hence the conversion of

Studies of Δ[Ca2+] in mouse fast-twitch fibres

In recent years, Ca2+ indicator dyes have been widely used to study excitation–contraction coupling in mammalian skeletal muscle, particularly of rodents (e.g., Baylor and Hollingworth, 2003, Baylor and Hollingworth, 2007, Calderon et al., 2009, Calderon et al., 2010, Capote et al., 2005, Caputo et al., 2004, Carroll et al., 1995, Carroll et al., 1997, Chin and Allen, 1996, Collet et al., 1999, Delbono and Stefani, 1993, Garcia and Schneider, 1993, Gomez et al., 2006, Head, 1993, Hollingworth

Simulations of Δ[Ca2+] in mouse fast-twitch fibres

To estimate Δ[Ca2+] and the associated intra-sarcomeric Ca2+ movements from measurements like those in Fig. 9, the compartment model shown in Fig. 3 was adapted to mouse fast-twitch fibres. To do so, two changes were made to the geometry of the model. First, the radius of the myofibril was reduced from 0.5 to 0.375 μm, to reflect the somewhat smaller average size of the myofibrils in mouse fibres vs. frog fibres (Goldspink, 1970, Peachey and Eisenberg, 1978). Second, the location of Ca2+

Conclusions

This article has examined a number of issues that affect the accuracy of interpretations of optical signals from Ca2+ indicator dyes introduced into the cytosol. Many of these issues have not reached the level of major concern in most previous studies of intracellular Ca2+ signalling with fluorescent Ca2+ indicators. The reasons for this are several-fold. First, many studies seek a qualitative answer to the biological question of interest – for example, is Δ[Ca2+] important in the process under

Acknowledgements

This work was supported by grants from the National Institutes of Health (GM 86167) and the Muscular Dystrophy Association. We thank Dr. Simona Boncompagni for carrying out an electron microscopy analysis of fibres from the region of the EDL muscle that we have studied physiologically and Drs. Masato Konishi and Lawrence C. Rome for comments on the manuscript.

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