Store-operated Ca2+ Entry In Muscle Research Articles

Tubular (T-) System SOCE-PMCA Balance Is Regulated by RyR Leak in Resting Skeletal Muscle

Skeletal muscle possesses a unique, rapidly activating store-operated Ca2+ entry (SOCE) mechanism. SOCE is activated when the activity of the ryanodine receptors increase, following action potential-induced Ca2+release or even increased RyR Ca2+ leak (Koenig et al 2018, Comm Biol; Cully et al 2018, PNAS). The role of SOCE in muscle remains poorly defined, but a role in calcium content regulation is likely, which means it must work in concert with fibre Ca2+ extrusion mechanisms for balance. Kinetics of Ca2+ movements in imaging experiments performed with high spatiotemporal resolution have shown that the t-system PMCA exists in the same microdomain as RyRs and the SOCE protein components; and all components are equally distributed throughout the muscle fibre (Cully et al 2012, AJP). We studied RyR mutant mice (RyR KI; Lopez et al 2018, BJA) and calsequestrin 1 null (CSQKO) mice (Paolini et al 2007, J.Physiol) to examine the regulatory relationship of RyR Ca2+ leak with SOCE and PMCA activity using mechanically skinned fibres. We determined that RyR Ca2+ leak increased in the order WT<RyR KI HET<RyR KI HOM<CSQKO. This leak caused a steady state reduction in SR calcium content, which we quantified in absolute terms. We found PMCA activity to be increased in same order as increased RyR Ca2+ leak, where CSQKO muscle showed the most rapid rate of Ca2+ extrusion when [Ca2+]t-sys was initially 0.1 mM and [Ca2+]cyto was varied between 28-1342 nM. The increase in Ca2+ extrusion was offset by increasing SOCE so that physiological, mM [Ca2+]t-sys levels were maintained. Our data conform with Cell Boundary Theorem (Rios, 2010, J.Physiol Sci), providing a platform to explain the increased [Ca2+]cyto in resting intact fibres (Lopez et al 2018) in the presence of increased RyR Ca2+ leak.

Calcium Entry Units: Exercise-Dependent Formation of New SR-TT Junctions Containing STIM1 and Orai1 in Muscle

Depletion of calcium (Ca2+) from intracellular stores (endoplasmic or sarcoplasmic reticulum, ER or SR respectively) triggers Ca2+ entry across the plasma membrane, a process known as capacitative Ca2+ entry or store-operated Ca2+ entry (SOCE). SOCE is mediated by the interaction between STIM1 (stromal interaction molecule-1), which functions as the Ca2+ sensor in the ER, and Ca2+ permeable Orai1 channels in the surface membrane. To date, the subcellular location for SOCE in skeletal fibers, likely important to prevent/delay fatigue during repetitive activity, has not been unequivocally identified.Using electron microscopy, we show that prolonged muscle activity (treadmill running in mice) drives the formation of previously unidentified intracellular junctions between the SR and extensions of the TT membrane, located within the I band between the Ca2+-release unit (CRUs, the sites of excitation-contraction coupling) and Z-line. Using immunohistochemistry and immunogold labeling, we also demonstrate that the exercise-dependent formation of these new junctions contain the molecular machinery known to mediate SOCE in muscle: STIM1 Ca2+ sensor proteins in the SR, already present in the I band in control conditions, and Ca2+-permeable Orai1 channels, which move into the I band with TTs during prolonged muscle activity.The activity-dependent formation of these unique SR-TT junctions reflects a striking and unexpected remodeling of the existing sarcotubular system at the I band of the sarcomere. We here name these junctions “Ca2+ Entry Units” (CEUs), and propose that these structural entities are the preferential site for SOCE, at least during repetitive activity, representing the ideal pathway to rapidly recover Ca2+ ions from the extracellular space and, hence, important to prevent/delay fatigue.

What Role for Store‐Operated Ca2+ Entry in Muscle?

Store-operated Ca²⁺ entry (SOCE) is a receptor-regulated Ca²⁺ entry pathway that is both ubiquitous and evolutionarily conserved. SOCE is activated by depletion of intracellular Ca²⁺ stores through receptor-mediated production of inositol 1,4,5-trisphosphate (IP₃). The depletion of endoplasmic reticulum (ER) Ca²⁺ is sensed by stromal interaction molecule 1 (STIM1). On store depletion, STIM1 aggregates and moves to areas where the ER comes close to the plasma membrane (PM; within 25 nm) to interact with Orai1 channels and activate Ca²⁺ entry. Ca²⁺ entry through store-operated Ca²⁺ (SOC) channels, originally thought to mediate the replenishment of Ca²⁺ stores, participate in active downstream signaling by coupling to the activation of enzymes and transcription factors that control a wide variety of long-term cell functions such as proliferation, growth, and migration. SOCE has also been proposed to contribute to short-term cellular responses such as muscle contractility. While there are significant STIM1/Orai1 protein levels and SOCE activity in adult skeletal muscle, the precise role of SOCE in skeletal muscle contractility is not clear. The dependence on SOCE during cardiac and smooth muscle contractility is even less certain. Here, we will hypothesize on the contribution of SOCE in muscle and its potential role in contractility and signaling.

Molecular Mechanism of Store Operated Ca2+ Entry in Adult Mammalian Skeletal Muscle

In skeletal muscle, store-operated Ca2+ entry (SOCE) is a trans-sarcolemmal Ca2+ influx pathway activated when sarcoplasmic reticulum (SR) Ca2+ stores are depleted. However, the mechanism of activation and physiological role of SOCE in adult skeletal muscle remains largely unknown. We recently reported that both STIM1 and Orai1 proteins are required for SOCE in skeletal myotubes. Here we investigated the mechanism of SOCE in adult skeletal muscles using mouse FDB fibers following electroporation of either cherry-tagged dnOrai1 cDNA or STIM1 siRNAs. Using a Mn2+ quench assay, we found that thapsigargin-induced SR Ca2+ store depletion activates a Ca2+ influx pathway in adult FDB fibers that is inhibited by: 1. multiple SOCE channel blockers (La3+, BTP-2 or SK&F96365) (3321±598 counts/sec, n=16 and 477±274 counts/sec, n=4 in the absence and presence of La3+, respectively), 2. expression of cherry-tagged dnOrai1 (571±130 counts/sec, n=12), or 3. STIM1 knockdown (7565±1590 counts/sec, n=3 and 217±247 counts/sec, n=5 in control and after STIM1 knockdown, respectively). To further assess the role of SOCE in muscle, we generated skeletal muscle-specific HA-tagged dominant negative Orai1 transgenic mice (HSAdnOrai) using a transgene driven by the human skeletal muscle actin (HSA) promoter (provided by Dr. J. Molkentin). HSAdnOrai mice survive beyond weaning and develop/breed normally. Western blot analysis using an HA antibody confirmed dnOrai1 transgene expression in skeletal muscle, but not in heart, lung, brain, spleen kidney, or liver. Primary myotubes derived from HSAdnOrai mice show significantly decreased SOCE following store depletion as assessed in Mn2+ quench (>92%) and Ca2+ influx (>95%) assays These results demonstrate that STIM1-Orai1 coupling mediates SOCE in adult skeletal muscle and that HSAdnOrai transgenic mice are a valuable tool for future studies of the physiological role of SOCE in skeletal muscle.

The Role of Store-Operated Calcium Entry in Store Repletion During Repetitive High Frequency Tetanic Stimulation of Single Skeletal Muscle Fibers

Store-operated Ca2+ Entry (SOCE) involves a trans-sarcolemmal Ca2+ influx mechanism triggered by Ca2+ store depletion. Recently, we demonstrated that SOCE activation in skeletal myotubes involves a functional coupling between STIM1 Ca2+ sensor proteins in the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the sarcolemma. However, the physiological role of SOCE in muscle remains unknown. Here, we monitored myoplasmic Ca2+ transients in mag-fluo-4 loaded mouse flexor digitorum brevis fibres during repetitive high frequency tetanic stimulation (60 consecutive 500ms, 50Hz stimulation trains every 2.5s). In normal Ringer's solution, tetanic Ca2+ transient amplitude decays in three phases: an initial rapid phase (trains 1-10), a second phase of maintained amplitude (trains 10-40), and a final phase of decay (trains 40-60). The maintained phase corresponds to a slightly elevated tail transient integral during each interpulse interval, consistent with activation of Ca2+ influx between tetani. Addition of 0.5mM CdCl2 plus 0.2mM LaCl3 did not alter the initial or final phases of Ca2+ transient decay, but significantly (p<0.01) compromised both the maintained Ca2+ transient (4±3% reduction from trains 10 to 40 in normal Ringer versus 30±3% reduction with Cd/La) and the increase in tail transient integral (which decreased 21±7% with Cd/La) observed during the second phase. Similar results were obtained following addition of either BTP-2 or SKF96365, two known SOCE inhibitors, consistent with SOCE mediating store repletion during the secondary phase of maintained release. Together, these results suggest that repetitive high frequency tetanic stimulation activate a SOCE flux used to replenish SR Ca2+ stores required to maintain subsequent Ca2+ release. Current experiments are testing the validity of this assertion using molecular interventions (transient STIM1 knockdown and dnOrai1 expression) to more selectively inhibit SOCE.