Store-Operated Calcium Entry in Skeletal Muscle: What Makes It Different?

Abstract

Current knowledge on store-operated Ca²⁺ entry (SOCE) regarding its localization, kinetics, and regulation is mostly derived from studies performed in non-excitable cells. After a long time of relative disinterest in skeletal muscle SOCE, this mechanism is now recognized as an essential contributor to muscle physiology, as highlighted by the muscle pathologies that are associated with mutations in the SOCE molecules STIM1 and Orai1. This review mainly focuses on the peculiar aspects of skeletal muscle SOCE that differentiate it from its counterpart found in non-excitable cells. This includes questions about SOCE localization and the movement of respective proteins in the highly organized skeletal muscle fibers, as well as the diversity of expressed STIM isoforms and their differential expression between muscle fiber types. The emerging evidence of a phasic SOCE, which is activated during EC coupling, and its physiological implication is described as well. The specific issues related to the use of SOCE modulators in skeletal muscles are discussed. This review highlights the complexity of SOCE activation and its regulation in skeletal muscle, with an emphasis on the most recent findings and the aim to reach a current picture of this mesmerizing phenomenon.

Keywords: Ca2+ entry sites; Orai; SOCE pharmacology; STIM; phasic SOCE; skeletal muscle; store-operated Ca2+ entry.

Figure 1

Schematic representation of the main components of the triad and the contractile apparatus. The thick and thin lines represent the myosin and actin filaments, respectively. AP—action potential, mg29—mitsugumin 29, JPH—junctophilin, RyR1—ryanodine receptor 1, DHPR—dihydropyridine receptor, CASQ1—calsequestrin 1, PMCA—plasma membrane Ca²⁺ ATPase, SERCA—sarco-endoplasmic reticulum Ca²⁺ ATPase.

Figure 2

Schematic representation of possible sites of SOCE in skeletal muscle. (A) SOCE at the triad is well supported and the most described in the literature. STIM and Orai are expressed in the triad membranes and respective SOCE was confirmed. (B) SOCE at the CEU is well described as a newly formed structure that is induced by strenuous exercise. STIM and Orai are expressed at the CEUs but the respective SOCE Ca²⁺-flux has not yet been demonstrated to date. (C) STIM at the lSR moves to the triad upon activation or activates Orai at the PM. It was also reported to modulate the activity of the SERCA pump. (D) Localization at the NMJ remains speculative for the time being.

Figure 3

Two fluorescence-based techniques to measure SOCE in skinned skeletal muscle fibers. (A) Schematic representation of the different protocol steps to perform measurements of either chronic SOCE (steps 1–3) or phasic SOCE (steps 1–5). (1) Isolation of an EDL muscle and preparation of small muscle bundles under paraffin oil. (2) Incubation of muscle fibers with a low-affinity Ca²⁺-sensitive dye (e.g., Rhod-5N). (3) Skinning of individual fibers under paraffin oil traps Rhod-5N in the sealed t-system (to report [Ca²⁺]_tsys) and opens access to the cytoplasm. (4) Transfer of a skinned muscle fiber with Rhod-5N trapped in the t-system to an experimental chamber filled with a physiological salt solution mimicking the muscle cytoplasm. Addition of a high-affinity Ca²⁺-indicator, e.g., Fluo-4, to enable the measurement of [Ca²⁺]_cyto. (5) Mounting of the preparation on the stage of a confocal microscope that is able to perform fast image acquisition, e.g., being equipped with a resonant scanner, or a spinning disk system, and electrical field stimulation (EFS) via two platinum electrodes that are immersed in the bath solution positioned in parallel to the fibers’ long axis. (B) Measurement of chronic SOCE in a skinned fiber following steps 1–3 (e.g., [97]). Typical recording of [Ca²⁺]_t-sys derived from the calibrated fluorescence of Rhod-5N. The fiber is bathed in an internal salt solution containing 1 mM free Mg²⁺ and 67 nM free Ca²⁺; then, chronic SOCE is induced through direct activation of the RyR1 by exposing the fiber to a “release” solution containing 0 mM free Mg²⁺ and 30 mM added caffeine. Activation of chronic SOCE is seen as a steep depletion of [Ca²⁺]_t-sys. The depletion is fully reversible as the t-system reloads with Ca²⁺ upon restoration of physiological [Ca²⁺]_cyto and [Mg²⁺]_cyto. The sequence of depletion and re-uptake can be repeated at different [Ca²⁺]_cyto values. (C) Measurement of phasic SOCE in a skinned fiber following steps 1–5 in A. Typical phasic SOCE recording, consisting of the imaging of [Ca²⁺]_t-sys (left axis) and [Ca²⁺]_cyto (right axis) over time, as derived from a rat skinned EDL fiber. EFS triggers APs in the sealed t-system and concomitant SR Ca²⁺ release with respective [Ca²⁺]_cyto transients. Note that the high EGTA buffering leads to very sharp [Ca²⁺]_cyto transients, which are undersampled by the employed sampling rate, which results in a pseudo-modulation of [Ca²⁺]_cyto transient amplitudes. Phasic SOCE manifests as rapid depletion of [Ca²⁺]_t-sys upon each induced AP (with every EFS pulse). As for the measurements of chronic SOCE, [Ca²⁺]_t-sys recovers after the cessation of EFS due to the function of NCX and/or PMCA. Parts of the figure have been taken and modified from Koenig et al. [39]), published under CC BY 4.0 license.

Figure 4

Proposed model of pSOCE activation following AP-evoked Ca²⁺ release in skeletal muscle. (i) At rest, some STIM1–Orai1 complexes are prearranged at the junctional membranes close to the position of the RyRs. (ii) During AP-evoked Ca²⁺ release from the SR, Ca²⁺ depletion within the SR occurs locally and is restricted to a nanodomain behind the RyR channels, which are presumably shaped by the network of CASQ. (iii) The local depletion of Ca²⁺ in a spatially restricted domain behind the RyRs allows for rapid dissociation of Ca²⁺ from STIM1 to activate SOCE.