A quantitative description of tubular system Ca(2+) handling in fast- and slow-twitch muscle fibres

Abstract

Key points: Current methods do not allow a quantitative description of Ca(2+) movements across the tubular (t-) system membrane without isolating the membranes from their native skeletal muscle fibre. Here we present a fluorescence-based method that allows determination of the t-system [Ca(2+) ] transients and derivation of t-system Ca(2+) fluxes in mechanically skinned skeletal muscle fibres. Differences in t-system Ca(2+) -handling properties between fast- and slow-twitch fibres from rat muscle are resolved for the first time using this new technique. The method can be used to study Ca(2+) handling of the t-system and allows direct comparisons of t-system Ca(2+) transients and Ca(2+) fluxes between groups of fibres and fibres from different strains of animals.

Abstract: The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that maintains a large Ca(2+) gradient and exchanges Ca(2+) between the extracellular and intracellular environments. Little is known of the Ca(2+) -handling properties of the t-system as the small Ca(2+) fluxes conducted are difficult to resolve with conventional methods. To advance knowledge in this area we calibrated t-system-trapped rhod-5N inside skinned fibres from rat and [Ca(2+) ]t-sys , allowing confocal measurements of Ca(2+) -dependent changes in rhod-5N fluorescence during rapid changes in the intracellular ionic environment to be converted to [Ca(2+) ] transients in the t-system ([Ca(2+) ]t-sys (t)). Furthermore, t-system Ca(2+) -buffering power was determined so that t-system Ca(2+) fluxes could be derived from [Ca(2+) ]t-sys (t). With this new approach, we show that rapid depletion of sarcoplasmic reticulum (SR) Ca(2+) induced a robust store-operated Ca(2+) entry (SOCE) in fast- and slow-twitch fibres, reducing [Ca(2+) ]t-sys to < 0.1 mm. The rapid activation of SOCE upon Ca(2+) release was consistent with the presence of STIM1L in both fibre types. Abruptly introducing internal solutions with 1 mm Mg(2+) and [Ca(2+) ]cyto (28 nm-1.3 μm) to Ca(2+) -depleted fibres generated t-system Ca(2+) uptake rates dependent on [Ca(2+) ]cyto with [Ca(2+) ]t-sys reaching final plateaus in the millimolar range. For the same [Ca(2+) ]cyto , t-system Ca(2+) fluxes of fast-twitch fibres were greater than that in slow-twitch fibres. In addition, simultaneous imaging of t-system and SR Ca(2+) signals indicated that both membrane compartments accumulated Ca(2+) at similar rates and that SOCE was activated early during SR Ca(2+) depletion.

Figure 1. Changes in t‐system rhod‐5N fluorescence in a mechanically skinned soleus fibre of rat during unidirectional t‐system Ca²⁺ fluxes are resolved with confocal imaging in xyt mode

A, a selected xy image of a skinned soleus fibre with t‐system‐trapped rhod‐5N bathed in a standard internal solution containing 1.3 μm Ca²⁺ corresponding to the time marked 346 s in the experiment represented in B. The t‐system fluorescence signal was in equilibrium with the [Ca²⁺]_cyto. The white line in the lower half of the confocal image represents the line‐wise average of the t‐system rhod‐5N fluorescence along the y‐axis (long aspect of the fibre) within the borders of the preparation. A small degree of non‐uniformity along the preparation of t‐system fluorescence was observed, which can be attributed to non‐uniformities in [rhod‐5N]_t‐sys (x, y) that become evident when [Ca‐rhod‐5N]_t‐sys is high. B, selected xy images of t‐system rhod‐5N fluorescence emitted from a mechanically skinned soleus fibre during solution exchanges from releasing solution containing caffeine to standard solution containing 1.3 μm Ca²⁺ and back to releasing solution containing caffeine. The spatially averaged profile of the t‐system rhod‐5N fluorescence emitted from the preparation is shown in the plot. The vertical pale blue bars on the plot and oblique line marked with ‘artefact’ above the images indicate the timing of solution changes in the experiment. The presence of caffeine or 1.3 μm Ca²⁺ is indicated above the images and plot. Note that the y‐axis of the image also represents time as the scanning laser passes along the long aspect of the fibre (Launikonis & Ríos, 2007). This is best observed from the solution exchange that induces movement of the fibre, as seen in the images marked 356.6 and 357.4 s (‘artefact’).

Figure 2. In situ and in vitro calibration of rhod‐5N, fluo‐5N and [Ca²⁺]_t‐sys

A, rat skinned fast‐twitch fibre with t‐system‐trapped rhod‐5N was continuously imaged and exposed to a cycle of a standard solution containing 67 nm Ca²⁺ with 50 mm EGTA and releasing solution containing 50 mm EGTA, 30 mm caffeine and 10 μm Mg²⁺. After the third exposure to 67 nm Ca²⁺ the fibre was exposed to ionomycin and 2 mm Ca²⁺, causing the t‐system fluorescence to increase. The fibre was then successively exposed to 5, 8, 0 and 0.316 mm Ca²⁺ to calibrate t‐system rhod‐5N fluorescence and [Ca²⁺]_t‐sys. The interval of solution change is indicated by the light blue bars and blue, red and green lines above the trace. B, in vitro calibration of rhod‐5N in a physiological solution with 0 (control) and 15 mg ml⁻¹ BSA and in situ calibration. C, in vitro calibration of fluo‐5N in a physiological solution with 0 (control), 10, 15 and 50 mg ml⁻¹ BSA and in situ calibration. Hill curves were fitted to the data points in B and C. The in situ calibrations showed a K _D,Ca of approximately 0.80 and 0.35 mm for rhod‐5N and fluo‐5N, respectively.

Figure 3. Changes in t‐system rhod‐5N fluorescence signal and [Ca²⁺]_t‐sys during chronic depletion of SR Ca²⁺ and abrupt changes in [Ca²⁺]_cyto

A, rat slow‐twitch skinned fibre with t‐system‐trapped rhod‐5N was continuously imaged during exposure to a cycle of internal solutions containing 30 mm caffeine with no added Ca²⁺ and standard internal solution containing [Ca²⁺] at 28, 67, 1342 and 28 nm. All solutions contained 50 mm EGTA. Caffeine depleted the [Ca²⁺]_t‐sys consistently to the same low level. Note the profile of the rising phase of the rhod‐5N fluorescence transient was dependent on the [Ca²⁺]. F _max and F _min of t‐system rhod‐5N fluorescence were determined in the presence of ionophores and 5 mm and 0 Ca²⁺, respectively. The right y‐axis shows the [Ca²⁺]_t‐sys determined from the rhod‐5N fluorescence signals (Fig. 1, see text). The interval of solution change is indicated by the vertical light blue bars and horizontal blue and green lines above the trace. B, the steady‐state [Ca²⁺]_t‐sys in fast‐ and slow‐twitch fibres. Note the values at 0 [Ca²⁺]_cyto are in the presence of caffeine, to represent a Ca²⁺‐depleted t‐system. Values in brackets represent numbers of fibres for fast‐ and slow‐twitch fibres, respectively. The [Ca²⁺]_t‐sys values in the presence of [Ca²⁺]_cyto of 28–1342 nm were compared (one‐way ANOVA with Tukey's multi‐comparison test), were P < 0.05 for both fast‐ and slow‐twitch fibres. In fast‐twitch fibres, significant differences were found between [Ca²⁺]_t‐sys at [Ca²⁺]_cyto values of 28 and 1342 nm, and 28 and 200 nm (P < 0.02 in both cases). In slow‐twitch fibres, significant differences were found between [Ca²⁺]_t‐sys at [Ca²⁺]_cyto values of 28 and 67 nm (P < 0.04); 28 and 200 nm (P < 0.001); 28 and 1342 nm (P < 0.0001); 67 and 1342 nm (P < 0.003); and 200 and 1342 nm (P < 0.02). The difference between [Ca²⁺]_t‐sys at 67 and 200 nm [Ca²⁺]_cyto was not statistically significant in slow‐twitch fibres. Significant differences between fast‐ and slow‐twitch fibres were not found.

Figure 4. Rhod‐5N does not significantly buffer Ca²⁺ inside the t‐system

A, the 10–90 % rise time of Ca²⁺‐depleted fibres exposed to 1.3 μm [Ca²⁺]_cyto isolated from bundles exposed to different concentrations of rhod‐5N in physiological solution. B, the fast tau (time constant) component of the exponential function fitted to the [Ca²⁺]_t‐sys (t) of Ca²⁺‐depleted fibres after exposure to 28 nm [Ca²⁺]_cyto. The three groups of fibres were isolated from bundles exposed to different concentrations of rhod‐5N in physiological solution, as indicated. The results are not statistically different in A and B (one‐way ANOVA with Tukey's multi‐comparison test).

Figure 5. [Ca²⁺]_t‐sys (t) and t‐system Ca²⁺ fluxes during SR Ca²⁺ depletion and at different [Ca²⁺]_cyto

A–C, selected sections where t‐system rhod‐5N fluorescence signal (t) from a fast‐twitch fibre has been converted to [Ca²⁺]_t‐sys (t). The horizontal lines at the top and vertical pale blue bars indicate the solutions bathing the fibre and the exchanges between caffeine and internal solution to/from 28 (A), 67 (B) and 1342 (C) nm Ca²⁺. The open circles indicate the [Ca²⁺]_t‐sys value derived from individual images in the xyt series (see Methods). The t‐system Ca²⁺ flux (in red) has been derived from [Ca²⁺]_t‐sys (t) assuming a B _t‐sys of 1 and (SA:Vol)_t‐sys of 32 nm (see text). D, the peak t‐system Ca²⁺‐uptake fluxes for fast‐ and slow‐twitch fibres are shown. The result is from 28 and 18 fibres, respectively. Data points are means ± SEM, with errors shown when they are bigger than the symbol. Data were fitted by Hill curves, with values for B _max, K _D,Ca and Hill coefficient of 42.4 ± 17.5 nmol m⁻² s⁻¹, 502.5 ± 561.9 nm and 0.87 ± 0.35 for fast‐twitch fibres and 5.02 ± 1.05 nmol m⁻² s⁻¹, 77.9 ± 34.7 nm and 2.05 ± 1.57 for slow‐twitch fibres.

Figure 6. SOCE and STIM1 isoforms in fast‐ and slow‐twitch muscle

A, t‐system rhod‐5N fluorescence (t) during the exchange of standard internal solution containing 200 nm Ca²⁺ for one containing 1 mm tetracaine and no added Ca²⁺. B, scatter plot of peak store‐dependent Ca²⁺ fluxes in fast‐ and slow‐twitch fibres. The data points were fitted by linear regression, where m = −12.3 ± 2.2 and −9.9 ± 2.8, c = 4.57 ± 3.1 and 0.45 ± 5.0, and r ² = 0.48 and 0.35 for slow‐ and fast‐twitch fibres respectively. Results are from more than 20 fibres in both cases. C, increasing amounts of total protein (3 to 24 μg) from rat EDL and soleus (SOL) skeletal muscle were separated on 4–12% Criterion SDS‐PAGE gels and probed for STIM1 with an antibody that detects both STIM1L (∼110 kDa) and STIM1S (∼90 kDa). Myosin heavy chain (MHC) in the post‐transferred coomassie blue gel is also shown (top panel) and is indicative of the relative amount of protein loaded. Plots show the average relative amounts of STIM1L and STIM1S in EDL and SOL muscles (n = 7 each), expressed relative to the amount of the given STIM1 isoform in EDL muscles on a given gel. *P < 0.05, paired Student's two‐tailed t test.

Figure 7. Simultaneous tracking of t‐system and SR Ca²⁺

A skinned fibre preparation with t‐system‐trapped rhod‐5N and SR‐trapped fluo‐5N was exposed to 1.3 μm [Ca²⁺]_cyto, followed by the releasing solution and then returned to 1.3 μm [Ca²⁺]_cyto. The continuously imaged signals from the t‐system and SR showed very similar transients during this protocol. The interval of solution change is indicated by the vertical light blue bars and the horizontal lines above the trace indicate solution composition.