Store-operated Ca2+ entry is activated by every action potential in skeletal muscle

Abstract

Store-operated calcium (Ca²⁺) entry (SOCE) in skeletal muscle is rapidly activated across the tubular system during direct activation of Ca²⁺ release. The tubular system is the invagination of the plasma membrane that forms junctions with the sarcoplasmic reticulum (SR) where STIM1, Orai1 and ryanodine receptors are found. The physiological activation of SOCE in muscle is not defined, thus clouding its physiological role. Here we show that the magnitude of a phasic tubular system Ca²⁺ influx is dependent on SR Ca²⁺ depletion magnitude, and define this as SOCE. Consistent with SOCE, the influx was resistant to nifedipine and BayK8644, and silenced by inhibition of SR Ca²⁺ release during excitation. The SOCE transient was shaped by action potential frequency and SR Ca²⁺ pump activity. Our results show that SOCE in skeletal muscle acts as an immediate counter-flux to Ca²⁺ loss across the tubular system during excitation-contraction coupling.

Fig. 1

Identifying extracellular Ca²⁺ influx across the t‐system membrane activated by single-action potentials. a Schematic representation of the individual protocol steps of the technique. (i) isolation of a single fibre of rat EDL muscle, (ii) incubation of the fibre with the Ca²⁺‐sensitive dye Rhod‐5N, (iii) Mechanical removal of the sarcolemma (skinning) and resulting trapping of the dye within the sealed t‐system, (iv) incubation of the fibre in a physiological salt solution containing the Ca²⁺‐sensitive dye Fluo‐4, (v) electrical field stimulation via platinum electrodes in parallel to the fibre’s long axis, and simultaneous confocal imaging of both dye fluorescence within the region of interest (ROI). b Typical recordings of [Ca²⁺]_t‐sys (left axes) and [Ca²⁺]_cyto (right axes) over time as derived from skinned rat EDL fibres during electrical stimulation. xyt image series of the fluorescent signals of rhod‐5N and fluo‐4 trapped in the t‐system and loaded into the cytosol, respectively, were spatially averaged and calibrated (Methods section). Electrical stimulation at 1, 2 and 5 Hz as indicated was started shortly after beginning of the recording and was continued until a new steady state in the t‐system was reached, unambiguously identifiable by the termination of cytosolic transients. Note that the employed sampling rate of 55 frames s⁻¹ is below the Nyquist criterion, which results in an apparent modulation of cytosolic Ca²⁺ transient amplitudes. Green lines represent mono‐exponential fits to the decaying phase of [Ca²⁺]_t‐sys upon stimulation. c A summary of the respective rate constants as derived from the exponential fits for all tested frequencies. Data are derived from 10 fibres and given as mean ± SEM. EDL, extensor digitorum longus

Fig. 2

The phasic Ca²⁺ influx is resistant to the DHPR antagonist and agonist, nifedipine and BayK8644, respectively. Depletion of [Ca²⁺]_t-sys during 2 Hz stimulation of a skinned fibre under control conditions (a), and in the presence of either 10 µM nifedipine (b) or 10 µM BayK8644 (c). An exponential curve was fitted to the declining phase of the [Ca²⁺]_t-sys in each case. The mean rate constant and [Ca²⁺]_t-sys steady-state lower plateau levels fitted from four fibres as presented as box and whisker plots in d. One-way ANOVA revealed no statistical significance for comparing the rate constants (p = 0.8128) and the steady-state levels (p = 0.4412), respectively

Fig. 3

Action potential-activated extracellular Ca²⁺ influx is store‐operated. Representative traces of [Ca²⁺]_cyto and [Ca²⁺]_t‐sys under control conditions (a) or in the presence of either 10 µM tetracaine (b) or 30 µM tetracaine (c). Green lines represent mono‐exponential fits to the decay in the [Ca²⁺]_t‐sys signal during 2 Hz stimulation. d The mean [Ca²⁺]_t-sys steady-state lower plateaus at the end of the 2 Hz stimulation are presented as box and whisker plots. Data under control conditions (n = 7), and in the presence of 10 µM (n = 4) and 30 µM tetracaine (n = 5) were analysed with One-way ANOVA and Tukey’s post hoc test. ***statistical significance with p < 0.0001. Abbreviations on figure: 10 µM tetracaine, 10 T; 30 µM tetracaine, 30 T

Fig. 4

Store dependence of Ca²⁺ influx during electrical stimulation. A summary of the amount of Ca²⁺ entering the cytoplasm from the t‐system in response to the total Ca²⁺ released from the SR following a single-action potential (black line). The ratio of Ca²⁺ entering the cytoplasm from the t‐system and the SR is shown as grey line. Mean ± SEM from six fibres

Fig. 5

Tonic and phasic SOCE are observed under SR Ca²⁺ pump inhibition. Representative traces of [Ca²⁺]_cyto and [Ca²⁺]_t‐sys at rest and during 2 Hz field stimulation in the absence (a, top trace) and presence (a, lower trace) of 100 µM CPA. Note that the addition of CPA caused the [Ca²⁺]_t‐sys to decline to a new steady‐state level. This new, lower level did not affect the phasic activation of SOCE upon electrical stimulation in the presence of CPA. b Summary of [Ca²⁺]_t‐sys steady‐state plateau levels before and after 2 Hz electrical stimulation in the absence and presence of CPA. Result from 6 fibres is displayed as a box and whisker plot. One-way ANOVA and Tukey’s post hoc test. *indicates statistical significance with p < 0.001

Fig. 6

SOCE is an immediate counter‐flux isolated by cytosolic Ca²⁺ buffering. a [Ca²⁺]_t‐sys and cytoplasmic Ca²⁺ transients in a skinned fibre in the presence of 0.2 mM EGTA during field stimulation of 2 Hz. Note the absence of the phasic [Ca²⁺]_t‐sys depletion observed under these conditions, in contrast to cytosolic Ca²⁺‐buffering with 10 mM EGTA (Fig. 1). b Summary of steady‐state [Ca²⁺]_t‐sys in skinned fibres following 2 Hz field stimulation in the presence of 0.2 and 10 mM EGTA. Result from 4 fibres is presented as a box and whisker plot. Paired two-tailed Student’s t-test, ***indicating statistical significance with p = 0.0005.